Safety and efficacy of undenatured type II collagen in the

Transcription

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rNr J cLlN pHARl\tRESXxil(3t4)
(2002)
101-1.10
E F F E C T SO F O R A L L YA D M I N I S T E R E D
U N D E N A T U R ETDY P EI I
COLLAGENAGAINSTAHTHRITICINFI-AMMATORY
DISEASES:
A MECI-{ANISTIC
EXPLORATION
BAGCHI D.,1M!S[,{ER8.,2tsAcCtjt M.,3KOT|{AR|S.C..3DOWNS8 . W . , 3
F A F A R DR . D . , 'P R E U S SH , G . 4
s,
1 ) D e p a r t m e notf P h a r m a c yS c r e n c e sS, c h o o lo f p h a r m a c ya n d H e a l t hp r o f e s s i o n Creighton
University
MedicalCenter.Nebraska.USA.
2) E-CAPS,lnc.and HammerNutritjonLimiied,Washlnctton,
USA
3) lnterHealth
Research
Center,California,
USA.
4) Departmentof Physrology,
Medicineand Pathology,
GeorgetownUniversity
MedicalCenter,Washington
D C ,U S A
Summary: Afthritisafflictsapproximately
43 miltionAmericansor approximately
16 6% of the US popua1n.
Thetwo mosl comrnonand bestknowntypeso{ arthritisare osteoarthritis
pA) andrheumatorcl
arthritis(RA) A
:;ignificantamoun[ol scientificresearchhasbeen cionein ailemptsto explalnwhat initiatest'ormsof afthritis,
how it is prornoredat'tdperpetuatedand ttow to effectivelyinteuenein the diseaseprocess andpromote carillageremodeling.Cunentpharmacologicalstrategtes
matnlyaddressimmune suppression
and antiinflamma,
tory mechanismsand have had limitedsuccess. Recent researchprovidesevidencethat alterattops
tn ne
three'dirnenstonal
configuratian
ot glycoproterns
are responsible
for the recognitionlresponse
signalingthal calalyzesT-cellattack.Oraladministration
of autoantigens
has been shownta sfppress avarietyof experimental,
ly induced atttotmmunepathologies,includlngantigen-inducedRA.The interactior-t
gut assaclated
betvveen
lymphoidlasue rn the duodenttmanct epitopesof orallyadrninistered
unclenatured
typell collagenfacilitaies
oral toleranceto the antigenand stems systemicT-cellattackon joint caftilage.PreviousstudTeshave srtav,tn
that smalldoses of orallyadmrrtistered
undenaturedt'ypell chicken collageneffectrvelydeactrvate
klller T-ceil
aftack.A novelglycosylated
undenaturedfypell collagenmaierial(UC-ll)wasdevelopedto preseNebialaqical
activity.Thepresenceof activeepilopesin the LJC-llcollagenis confirmedby an enzyme-linkedimmunoscrbent assaytestand distinqulshesthisformiromhvdrolyzedor denaturedcollagen.Oral intakeof smal arncuns
of glyccsylatedUC-llpresentsactiveepiiopes,with the correctthree-dimensranal
structures,
tc peyerr patcn
es, whichrr-tfluences
the signahngrequiredfor the developmentof immune tolerapce.llC-ll has demonstrated
A d d r e s fso r c o r r e s p o n c l e nDc eB. a g c h iP, h . D ,F . A C N . C . N S , l v l . A l . C h E ,S c h o ooi i p b a r m a c y a n H
d e a t t hp r o t e s s i o n s ,
crerglrtonUirrversity
Medrcaicenter,25oocaliforniapiaza,omaha, NE 68178,usl..
0251-1649t2AA2l3l
4 00101+9 $025O/O
e-,2002BicscienceEd printlnc
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the abilifyto induce tolerance,effectivelyreducingjoint pain and swelilngin RA subjects.A pilot study was conducted for 42 daysto evaluatethe efficacyof UC-il (10 mglday)in fivefemalesubTecls(58-78years)suffering
from significantjarnt pain. Significantpain reduction including morning sliffness,sllffnessfollowingpeiods of
rest,painlhal worsenswith useof the affectedjoint and lossofjoint rangeof mottonand functronwas observed.
Thus,UC-llmay seNeas a noveltherapeutictool in joint inflammatory
conditionsand symptomsof OA and RA.
lntroduction
i m m u n i t ys t i l l p r e s e n l su n r e s o l v e d
c h a l l e n g e st h a t
may requirea paradigmshift in researchfor the
Arthritisrepresentsa group of debilitatingdiseases of the joints,bones,tendons,musclesand
eventually
organs.lt afflictsapproximately
43 milljon
Americans,imposinga cost in excessof $65 billion
annually (1) The two most common types are
(OA)and rheumatoid
osteoarthritis
arthritis(RA),tra-
r l ua vr iuerl n
u
vn
Pm
, ent
nf
pffociirre
vu
^r{n
rnn
al lu sdlu
rhn
rnnino
tr rgldPluJ.
It has been prooosedthat mechanismsinvolved
in host defense,protectionand maintenance
of self{orcesin which tolerance
integrityare counteracting
mechanismsefficiently
suppressimmuneattackon
perspecAn evolutionary
selfto a requiredthreshold.
-U ll it l+u i/ ^ -l .d1l l ty, , .u -v 1l l l^l U{ ;U^ ^ . - 1d^J^ or Vn Uo _ r e l a i o d ' , u r ovav ur u_ra nudr _r tun .r.u' d, l
tiveallegesthata tendencytowardautoimmunemal(1, functionshouldtheoretically
arthritisand "autoimmune"
be higherduringyears
arlhritis,respectrvely
proinflammatory
2). However,
responsehas beenidenti- when youngrmmunesysternsare aggressrvely
fiedto be a commonmediatorin bothtypesof arthri- t e c t i n g t h e r e p r o d u c t i v ep o t e n t i a lo f t h e h o s t .
Misrecognition
of self would be a predictabledefitis(1 2).
U n d e r s t a n d i nogl R Ap a t h o g e n e shi sa sc h a n g e d ciencvof the svstcm18) Autoimmunedisordersare
with advancoverthe years.RA is characterized
by attacko{ kjller less prevalentin the young,increasing
T-cellson type ll jointcollagen,whjchresultsin dam- i n n a n e : n r - 1 d e r : l i n p o I r o n 1 6 f i , ' r - l i ' / t r n n t a n l l a l
a g et o c a r t i l a g el o, i n ts w e l l i n gp,a i na n di n f l a m m a t i o n l n d o o d i h o r o i c r n l o r r r o l g l j g p l S h i p b e t W e e n a d V a n C (3-6) The body'sattemptsto remodeljoinl cartilage ing age and an increasedincidenceof arlhritis.To
are outpacedby immunemediatedaflacKon and explainthis,one rationaletheorrzes
that RA disease
degradation
of jointcartilage(3-6).Collectively,
functionof the imthese must resultfrom a deteriorating
eventshavebeenclraracterized
as an out-of-control mune system,whichprovidesidealconditionsfor a
ar llnimmt rntr rCsnnnsc f3\ Fxtpnqrrra roqeernlr has
(B) Decreasedrecognibreakdown.n self-lolerance
A,,nr-:îr'
ovnlnrarj
iho
m r r l i i,{-: -n-a.l-ou' 1
u
y I i 1 i l l { c s ô{t .I ê^c^L^ n) g
ntilon,
t i o na n d u p - r e g u l a t esde l f - a t t a ci ks a l o g i c acl o n c l u ^m
rFennnqo
and
e n m n.ayn-c,.,+i î lnt u r y/ rh o
i l^rêût . si i lî l i l c rr î l- e u n a s i o n c o n s i s t e nwl i l h a n a g e - r e r a l edde c l i n ei n i r n n i s m si n a n e f f o r tl o u n d e r s t a n dm. a n a g ea n d m a i n - m r r n o o f { i n i o n e r r/ Q \ H n u i e v e r o v n l q n a t i n n' 'e 'r -aen*2' r J. l n g
" a u t o T e a c t r vo' tfyt 'h e , m m u n es y s r e mi n R A d r s e a s e
tein immrnp a.nmnattrnn.p Roqea'r-h in tranqneniC
micepointsto the possibility
favor
thatB-lymphocytes
and
an emphasison functional
flawsin surveillance,
i m m u n o g l o b u l i on us t s i d et h e j o i n ti n d i r e c t lpyr o v o k e recognitionand response(and their symptomatic
R
, .A.
102
n:lhnnonoqic
r-..
r.z. r*: :*
'rta
- 3 i l - rt êr da +Li ;' ,Ln t v Tet -^cael l t i 'rnenc^ e
ptor ln
qnll
the joint (7), However,our understanding
of auto-
-^-ir^--^r;^^^\
I I lql lllE)LALlul
l)l
n
^nsqirilnr
"l q- t+r lf t,r^l - *t lLl A
u , I tr hrou F u J r t v r , , r y
+hat etrUC-
t u r a lr l a w si r r m m u n es y s t e mc o m p l e x e sa,n d p o s s i -
Eftectsof undenaturecJ
type ll cotlagenagainstanhriticdlseases
.i^^, '^LrD>uE),
tdentification
markersmay be missingfrom the joint
collagenand immunecomplexes.This perspective
providesinsightintohow the immunesystemincurs
a lossof self-tolerance
and exploresthe possibility
of
flawsin glycosylation/galactosylation
Thisphenomenon is at the rool of impairedimmunological
recognichallengeof explaining
how arthriticprocessesorigi- tion and responseactivitiesfor the hyper-autoreacnateand progress(2) Most of the past and current ttve immuneseltdestruction
of joint collagenin the
work on rheumatoiddiseasesexaminestrategiesto p a t h o g e n e s o
i sf R A ( 1 9 , 2 0 ) . H e n c e ,a l t e r a l t o nisn
intervene
or halt"out of control"immunologic
and/or glycosylation/
galactosylation
arehallmarkcharacterinflamrnatoryevents associatedwith autoimmune isticso1 FA Thrsalso providesa possibleexplanaparadigmproposesthat tion as to who orallyingestednativetype ll collagen
disease(10).Thetraditional
RAis an immunological
disorderfor an as yet uniden- produces tolerance,down-regulating
autoimmune
tifiedarthrogenic
antigen.Variousimmunological
fac- aggression(3,4).
torsare involved,
suchas CD4-inducer
lymphocytes,
lmpaired galactosylationaffects glycoprotein
C D 4 c e l l s , m a c r o p h a g e sn, e u t r o p h i l a
s n d t u m o r synthesis,alteringthe requisitethree-dimensional
necrosrsfactor-(9, 10).Thrsconventional
view has conformations
of glycoproteins
suchas type ll collanrnn'
'l^n
^h-'-^^^l^î^-l
lh^'^î^^
+h-r
f
t h e l o s so f s e l f - r e c o g n i t i o n
l _ r u u u u e up i l i l i l r i l u u t o g r c alti l e r a p r e sU r a t r a v o r g e n a n d l g G ,p r o d u c i n g
manipulation
of cyclooxygenase-2
eventsand immune Langand Yeaman(20)demonstrated
that removalof
suppression,
withlessthanidealresults.
Almostall of carbohydrate
moietiesfrom antigensresultedrn a
the biomolecules
responsibie
for innateand adaptive l o s so f a n l i b o d yt r i n d i n gI.n R A p a t i e n t sd, e c r e a s e d
immuneresponseare glycoproteins
(11) However, levelsof p1-4galactosyltransferase
activityin periphlittleattentionis directedat the possibilitythat im- eral blood B- and T-lymphocytes
correlateswith the
pairedglycosylation
aflectsthe configuration
of gly- decreasedgalactosylation
of serumlgG (13).
c o p r o t e i n si n, c l u d i n lgg Ga n d t y p el l c o l l a g e nT h e s e
l m m u n o g r o b u l ia
n rse b y d e f i n r r i ognl y c o p r o r e i n
m a y a l t e rr e c o g n i t i oann d r e s p o n s e
s i g r a l i n go u r i n g moleculesproducedby plasrnacellsin responseto
immune surveillance,
incitingattackon the body's a n r m m u n o g e w
n ,h i c hf u n c t i o na s a n t i b o d j e(s1 1 ) .I n
o w nj o i n tc o l l a g e n( 1I - 1 9 ) .
R A , i m m u n ec o m p i e x e st h a l c o n s i s te x c l u s i v e loyf
Tnis view sLggeststlrat the lerm hyperreactive i m m u n o g l o b ual irnep r e s e n ti ,n d i c a t i nagr o i ea s b o t h
"immuneabnormality"
may be a misnomerfor RA,as antibodyand antigen.Both cartilageand immune
t h e i m m u n e s y s t e m t s b e h a v r n ga p p r o p r r a t e l y systemcomplexesare, for the most part, made of
a g a i n s th o s t t i s s u e su t t r m a l e i yd e n r i f i eads f o r e r g n glycoprotein
structures
in whichglycoprotein
synthepathogenicantjgens(i0) Alteredglycosylation
could q i q r o n r r i r p e lUh ras nI tas.uoseJcJ. or \l/ y c> .ur uh -n{-t rdnl -u^ d- ^t î u u^ u^ t' * Î i^Jr E^ nt Et i t l
p r o d u c ea n u m b e ro { i d e n t i l i c a t reornr o r sr e s p o n s ' b l e glycosylation
(16). lmpairedglycosylation/galactosyf, ov r, u
r rvn - rroun! ,r u ,rql r: li il n n' ! j Jc co il l{--a: rl rt e
nccinililia.
l
a
l
i
o
n
on
u kr \ .
Ârmrnr nu ni r v tr hi ,ac n
i n t e r s e c al st a n u m b e ro f j u n c t u r ecso n t r i b u t , n g
vuJJruil[tsJ
are misidentification
of type ll jorntcollagenas anti- to the rnitiation,
promotionand progresston
stagesof
genic by aberrantlgG, possiblebrndingof hypo- R A ( 1 1 - 1 9 )
celactoc\/laipd lnG urith Ceftain fheumatoid faCtOrS
Comparisonsof the /V-glycosylated
pattern of
l e a d i n gt o s i g n i f i c a nl e
t v e l so f i m m u n ec o m p l e x e s serumlgG isolatedfrom healthyindividuals
withthat
characteristico' RA and/or approprrareglycomrc of RA patientsdernonstrates
that differencesobhh,
vry
tha
trrs
t..^^+
rorgYt
m
r r .r \o/ y
h
uo
u
o
u ii il n
u lr nv n
! ,ireu: ul r
a
u2
u rr 2
J rl \y/rar tre.
Recentstrategies
for therapeutic
managemenl
of RA,
therefore,focus on methodsof inhibitingsymptom
manifestation
to reducethe severityof the end-stage
o f t h i sd i s e a s e( 1 0 ) .
Ftiologicaland therapeuticresearch'aces the
103
Ranchi
)
pt el
servedin BA patientsare due to changesin the rela- g e n s i n t e r a c tw i t h g u l - a s s o c i a t e dl y m p h t i s s u e
tive extentof glycosylation
comparedwith normal / G A l T r r e s , r l i n n i r - 2 1 1o p l l 1 o l i ,n - n n q i t p ^ + { - . : t O r a l
i n d i v i d u a l Isn. R A ,a n i n c r e a s endu m b e ro f o l i g o s a c - .t nu llrsr l' l- 4r .G; n rr U I , U| , -oriL- l -y J^ mr ' ^l dl ll l U. l -nJc-eJc Jn rf nV [' i)eu unr cyrrrLl :. rupua l ur rrnll'-r - c h a r i d e s t r u c t u r e sl a c k t h e t e r m r n a lg a l a c t o s e n r t r r o e l l r r r r o l l n n l l r n o n , , r\ lulul . -- i ll l1\ , |hdr rs Iur em l^ inlno r t s t'râl nl.e o I I S
r e s i d u e( 1 9 ) .I h i s s u g g e s t tsh a tR A m a y b e a g l y c o - e f { p c l: v e r r e q sj n I r r n i n . rn f I I - c ^ l l . a t ' A . 1 ., n t v n o l l i O t n I
sylation
d i s e a s er.e f l e c t r ncgh a n g e si n r h ei n l r a c e l l u - c o l l a g e r ,i n d u c i n g; m m u n o l o g i c ah ly p o r e s p o n s i v e l a r p r o c e s s r n go, r p o s t - s e c r e t o rdye g r a d a t i o no f
n e s s ,a n d r e d u c i n gp a n a n d i n f l a m m a t i o(n3 - 6 ) I n
,V-linked
(12 19) Otherresearch
oligosaccharides
has contrast,whrle denaturedcollagenmay provde a
reporteda decreasein galactoseresrduesin the n u t r i t i o n saol u r c eo f s u b s t r a t teo r l o i n tc a r t r l a gsey n oligosacchar.de
charnsof the seurn lgG of RA pa
t h e s i sr,e s e a r cdhe m o n s t r a t et hsa i t c o e s n c t i n d u c e
tients,whichwas presumedto alfectthethree-dimerr- i m m u n o l o g i c ahl y p o r e s p o n s i v e n easns d h a s n c t
sionalstructure
of the CH"domain.Galactosedeplet- demonstrated
an effecton reducingoain and inflanred lgG reducedClq bindingand Fc receptorbirrding, m a t i o n( 6 ) .A l t l o u g ht h e s a m ea m i n oo c i d sa r e p r e which impliesan imporlantbrological
functionoi the sentrn both forms,the tertiaryand quaternarystrucglyconutrient
moietyof lgG (16) Rademacher
el a/. tures in the denaturedform may be completely
(17)clearlydemonstrated
thatgalactose-deficient
lgG destroyedand the galaclosemoiety is degraded
glycoformsare directlyassociated
with pathogenicity ( F i g 1 ) n o t a l l o w i n ge p i t o p e r e c o g n i t i o ni n t h e
i n c o l l a g e ni n d u c e dr h e u m a t o i da r t h r i t i si n m i c e . Peyer'spatch (3, 10,22).Furthermore,
the hydrolyzNonpathogenic
autoantibocjies
weremadepathogen- ed or denatured
formrnaybe pharmacologically
inefic by alteringtlreirgiycosylalion
feclivebecauseol the lossof conformatiorl.
state(17).
Interestlmmunization
with undenatured
type Il collagerr : n a l ' i t h a a f f a ^ i . n r n r r 1 r n l n 1 2 1 4 o r l ^ n n l : ^ r r o r r i . [ g
(antigen)has been shown [o inducearthritis(21) c o n f i n e dt o R A d i s e a s eas l o n e ,b u l c o n f e ra p p r e c i a However,orally ingestedundenaturednativeanti- b l e b e n e f i t si n s o m e c a s e so f O A a s w e I A p r l o t
104
F i g . 1 E l e c l r o lnl r r c r o g r a l ){ lm
t a g n i f i c a t i xo n
5 0 0 0 0 )0 1u n c i e n a t u r et ycpl el l c Dl a E e i vr 5 d e n a t u r etdy p el l c o l l a g e nU n c l eaf l u f e dt y p el l c o l
i a g e n( o ni e l t )s h o w s n t a c ti e r t i a r a
y n d q u a t e r n a rgyl y c o p r o t er n t o g f l layl l o w r n fgo re l l t t o p er e c o g n i i t oann d h y p o r e s p o | t s i vmen r u n es t i l n u
l a t i o nD
. e r l a t u r etdy p el l c o l l a g e n( o nl r g h t c) o n t a i n n
s o tertlara
y n d q L r a l e t n agr iyv c o p r o tIe r n l e c n t y .
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r yv r o e r c el h a t 1 0 ' r g / d a y Materialsand methods
s r u o yp r o v i d e sp r e l i m r n a e
o f a c o m m e r c i a le n z y m e - l i n k e di m m u n o s o r b e n l
a s s a y( E L I S Av;e r i i i e d
u n d e n a t u r egdi y c o s y l a l erdy p e
Chemicals.
Pepsin(Catalog
nlmberI U B 3 4 23 1)
l l c o l l a g e n ( U C - l l l n t e r H e a l t hN u t r a c e u t i c a l lsn - w a s p u r c h a s e df r o m W o r t h i n g t o nB i o c h e m i c a l
c o r p o r a t e dB, e n i c i a C
, A U S A ) a d m i n i s t e r eodr a l l y Corporation(Freelrold,
NJ, USA).Unlessotherwise
{r ;r 'v, û r e e l r r e o e l rauor nr JaUnrn / y n
h
o ,/ r^r . iunL r f o r r r n
J rr r r l n
u rf
b yr i
-2uA/ u
stated,all other chemtcalswere purchasedirom
r ja
u it n
tt
wornen,aged 58-78yearsold, for 42 days.Twoof the SigmaChemicalCo (St Louis,l'/O USA)
w o m e nw e r ep r e v r o d s ldyi a g n o s e dw ' l h O A a n d l h e
r e m a r n i ntgh r e ee x h i b i t e sdi r n i l asr y m p t o m sb u t h a d
U C - l l U C - l l w a s o b r t a r n e df r o m I n t e r H e a l t h
no clinicaldiagnosis.Therewere no adverseetfects Nutraceutrcals.
Thepresenceof glycosyaled "active"
associatedwiththe rntakeof UC-ll(Tablel)
epitopesin the UC-llcollagenmatrixwas confirmeC
P e y e sr p a l c h e sa r er e , a t i v el ay r g ea g g r e g a t eos1 by a validatedELISAtest. Furthermore,
electron
lymphtissuelocatedin the GALTof the smallintes
microscopicanalysisof UC-ll was conductedto
" d o m e "e p i t h e l i u m
t i n e ( 1 0 ,2 2 ) .T h e o v e r l y i n g
c o n - d e m o n s l r a t e t h e c o n f o r m a l r n n a l i n l c n r i t i in r t h p l r r n l e
tains large numbersot intraepithelial
lymphocytes. helicalstructure.
Sn.yrc n{ lhe eniihelr:l nollq h;rrp r-nmr rley micrniOldS
F o r e l e c t r o n m i c r o s c o p i ca n a l y s t s ,a s m a l l
in their surfaces,known as M-cells.M-celis are amountof UC-lipowderwas fixedwithKarnovsky
fixirnportantin the lransferof antigen from the gut a t i v el o r2 h . r i n s e dw i t hc a c o d y l a roeu l l e rl o r2 0 m i n ,
l u m e nt o t h eP e y e r ' p
s a t c h( 1 0 ) .P e y e r ' ps a t c h e st h e n placedin 1% osmiumtetroxrdetor 2 h, rinsedwrrn
facilitate
the generation
of an rmmuneresponsewith- distilled
waterfor 1 minand placedovernightin 0 5%
i n t h e m u c o s aA
. n a n t i g e ni n t h e P e y e r ' sp a t c hs t i m - uranyiacelate.The sample was then dried using
u i a t e sB - c e l pl r e c u r s o rasn d m e m o r yc e l l s( 1 0 ) .C e l l s ethanoland placedinto propyleneoxidefor 30 min
pass to the mesentericlymph nodes where the a n d f i n a l l yp l a c e di n 5 0 . 5 0p r o p y l e n o
exide:SPURR
i n t m u n er e s p o n s ei l n e e d e d i, s a m p l i i i e dA. c t i v a l e d (embeddingmaterial)tor 2 h and then lnto 100%
l\/rnrrhon\/tpq n:qc inin the blood Stream via the thOSPURRovernightlt was then placed into a 7A 'F
racic duct. Oral loleranceoccurs only alterthe cor- oven overnight.A section was taken using ultra
r e c tt h r e e - d i m e n s i o n
an
l { o r m a t i oonf U C - l la n t i g e n microtome,stainedwith uranylacetatefor 4 rrtn.
co
i s r d e n t i L eads n o n p a t h o g e n(r1c0 , 2 2 ) .
rinsedwithdistilled
water,stainedwithleadcitratefor
Tabf e I Measurenenl
ol pait) level following a 42-day sludv ol onl administra[ion ot' undenatLtred type ll cailagen (UC-ll)
R e d u c t r oinn P a i n
I
2
3
4
5
Week 1
Week2
Week3
Week4
Week5
Week6
Week7
l%)
3
5
5
6
7
3
5
5
6
B
3
5
4
5
5
3
5
4
5
5
3
5
3
3
4
3
2
3
2
3
3
2
5
?
1
0
22
27
22
34
A d n r i n l s t e r e dd o s e A s i n g l e , d a i y o r a l d o s e o f 1 0 m g g l y c o s y l a t e d u n c l e n a t u r e dt y p e l l c o l l a g e n { U C , l l ) . P a i n i n c l e x i 0 = u n b e a r a r l t e
1 = tolerable.
r05
Dqvul
rl u.
Yr dl.
2 m i n a n d r i n s e da g a i nw i t hd i s t i i l ew
d a i e ra n d o r r e o .
The transmissionelectron microscopeprocedure
was conductedin an Elr/JEOLi 00CX(Peabody,
MA
USA) An electronmicrographof undenatured
type rr
collagenys. denaturedtype ll collagenis shown jn
Frg. 1. Undenatured
type ll collagen(on left)shows
intact tertiaryand quaternaryglycoproteinintegrity
allowingfor epitoperecognrtion
and hyporesponsive
immunestimulation.
Denaturedtype ll collagen(on
right)containsno tertiaryor quaternaryglycoprotein
integrityEpitopesof healthyundenatured
type ll collagencontainthe correctcompositionand structural
conformationof galactose-dependent
glycoprotern,
as evrdencedby ELISAanalysis(Fig.2)
IUD
materiai(insolublecoilagen)and the supernatant
(solublecollagen)were coliectedand anaiyzedfor
n a i r v el y o eI l c o l l a g e u
n s i n ga c o m m e r c i a lal yv a i l a o i e
CaptureELISAkit suppliedby ChondrexLLC (Redmond, WA, USA) The quantityof UC-il (mg96)was
determinedrn bothsupernatant
solubieiype ll cot,ag e n a n d i n s o l u b liey p e l l c o l l a g e nf o l l o w i n g
incubation for0, 15 30, 60 and 90 min at 32 .C and pH 2 O
Pilot stuoy to evaluatethe efficacyoi uC-ll rn
humansurbTecfs.
An open labelpilotsludy was pertormedin five lrurnansubjects(womenaged SB-78
years)sutteringfrom significarrt
loint pain, using a
commerciaiELISA-verified
undenatured
type ll coliagen (UC-lllnterHealth
Nutraceuticals)
To be eligibie,
Time-dosemeasurements
of UC-ll activilyin sim- patients had to meet the American
College of
ulated human gastric fluid. Five samples of UC-ll Rheumatology
crileriaPatientswereexcludedfront
wereanalyzedfor collagenactivityviaELISAarralysis.
the studyit they had myocardialinsutficiency,
renal
Samplesweredigestedin pepsin,simulatingan arti(serumcreatine> 2 0 mg/dl), disturinsufficiency
ficialstomach.The pepsinsolutionwas made using
banceof liverfunction,alkalinephosohatase> 300
995 ml distilledwater,3.73g KCI 4 g HCtand 30 mg
units/liter,
serumglutamicoxaloacetic
transamjnase
pepsin.Fivecollagensamplesof j4,7 g each were
> 50 units/liter,
or bilirubin> 1 5 mg/dl) malignancy
incubated
l n d i v i d u a lfioyr 0 , 1 5 3 0 6 0 a n d 9 0 n r i ni n
or a considerably
reducedgeneralstateof heaithas
1 0 0 m l p e p s i ns o l u t i o na t 3 2 . C a n d p H 2 . 0 . T h e
d
e
t
e
r
m
i
n
e
d
b
y
t
h
e p h y s i c i a nT h e f i v e s u b j e c t s
A ;r^g
^ ê+s; û- o p
s a s s t o p p e db y i n c r e a s i ntgh e p H
u
l rouesw
e n r o l l e d i n t h i s s t u d y p r e s e n t e da h i s t o r y o i
t o 6 . 0 u s i n g O5 M N a O H s o l u t i o nB o t h t t r e s o l i d
osteoarthritis
more lhan rheumatoid
symptomolog.l,
Thesesublectsreportedeariymornng srtffness,
strffness followingperiodsof rest pain that worsened
with useof the affecteojointand lossof jorntrangeof
motionand function.Weatlrer
changesfrornwarmto
cold cr cry to morstwere also repcrtedas parns e r er e q . L i r ei d
e n h a n c i nfga c l o r sA l l p a r i e n tw
o sign
an informedi:attentconsentfornrpriorto participat i o n .T h e s u be c t s w e r ea l s o g i v e na q u e s t i o n n a i r e
witlr detailprotocolprocedures,
possiblerisks and
benefits,etc. Twoof the five suojectswho suffereo
from osteoarthritis
symptomswere clinicallydiagncsed3 yearsplor to particjpation
in this stucly.The
r e m a i n i n gt h r e es u b j e c t sr e p o r t e ds i m ia r s y m p t o F i g . 2 U n d e n a t u r e di y p e l l c o l L a g e nt f l p e h e l i x m o i e c u l e e x h i b t i n g
epilope posltr0ns.
mology.Measurements
includedweeklydlary-format
Effectsof undenaturedlpe ll collagenagainstarthriticdlseases
o D s e r v a t i o na sn o q u a l r t a r i v{ e e d o a c kE. a c hs u b j e c l mentationof UC-llis shownin Tabiel Subject1 per^ î^^l^
^"^
^-,1,, i^^^
^{ 1n -^
c e i v e dn o r e d u c t i o inn h e rp a i ns t a t u st h r o u g h o ut ht e
r" ê^ c^ ie. , r^ v, e o
a l l yo o s e o
l l u r l l g Iul\a, - llllû' n a n
a
s r n q r eo
r a .o
- r' -r -r i+J t.y. --L+U^l l-r-d û iL l ^ , r;u^r . .LÛ b e d t i m ef o r 4 2 c o n s e c u l i v e open labeltrial.Subject2 percerved
g
a reductionin
iJr
l r lrJr c,
Fa
eU
e n l ocur rUhl isovett rvrv; rLcr d
aq
od
tt nU lr o: rteU lt hl leUi rr l r A
pa
pain duringthe srxthweek of the study,wlrileunder
u rqJnU
\ 'rl "r 'v^ e
Je o
r nk g
u
L
p a i nl e v e o
l n a s c a l eo f 1 - 1 0 w
, i t ha s c o r e0 11 0 r e p - t h e s es a m ec o r o i t i o n sS u b j e c t s3 z a n d 5 r e p o r r e d
r e s e n t i n g" u n b e a r a o r ea"n d a s c o r eo f 1 d c n o t i n g a r e d u c t i oinn t h e i rp a r ni e v e d
l u r i n gt h et h i r dw e e ko f
"tolerable,"
fol' t r e a t m e n tT. h u s ,a t r i a ld o s e o f 1 0 m g U C - l lw a s
priorto participation
and immediately
assocrated
wilh a -26",Lreductionin perceivedpain
lowingcompletionof 7 days of treatment
a s i n c i i c a t ebdy f o u ro f t h e f i v es u b j e c t s( 2 2 % , 2 2 % ,
22/",34"/",Tablel) Furthermore,
no sideeffectswere
associatedwith UC-ll treatmerrt.In essence,treatBesults
m e n tw i t ha d a i l yo r a ld o s e o f 1 0 m g U C - l lw a s w e l l
reduciionin loint
Time'dosemeasurements
of UC ll activityin gas- tolerated
and Droduceda srgnificant
tric fluid.Followingingestion,the UC-ll glycoprotein painsymptoms.
. o s e -a n d
e n c o u n t e rhsy d r o c h l o rar cc i da n d p e p s i nD
+
; - ^ i ^ ^ ^ ^ r!u-v .r . -r L+^- i u+u r,u,J - r ; ^w^ e r e c o n d u c t e dt o d e t e r _
Lil rrc-uE|Jgr
rninewhetherthesemonomerswere stillin the triple D i s c u s s i o n
helicalform, whrchwe confirmedby ELISAassay.
F i r r rr r c i d p m n n q l r 2 t c q t h e t i m e - d O S e m e a s u f e m e n t s
FpitoperecognrlronLpiropes(anlrgen
c oere'Trr
o f U C - l la c t i v i t iyn s i r n u l a t ehdu m a ng a s t r i cf l u i da t 3 2 nants)are structuracomponentsof an antigenmolo C a n d p H 2 0 F i g u r e3 c l e a r l ye x h i b i t st h e U C - l l eculeresponsibie
witlrl-cell
for its specificinteraction
a c t v r t yi n s u p e r n a t a nsto l u b l et y p e l l c o l l a g e na n d antibodymoleculeselicitedby the same or related
ype I
insoubletype ll collagenovera periodof time (0 90 a n t i g e n( 2 3 ) .E p i t o p e o
s f h e a l t h yu n d e n a l u r et d
rnirr)Thus,these resultsdemonstratethat following c o l l a g e nc o n t a i nt h e c o r r e c ct o m p o s r t i cann d s l r u c giycocro
incubation
509'.of tura conformation
of UC-ilfor 90 min,approximately
of galactose-dependent
t e i n ,a s e v i d e n c e b
s o l u b l eU C - l li s a v a i l a b lteo t h e e p i t o p e s .
d y E L I S Aa n a l y s i Q
s a) ftg.2). A
novel giycosylated
u n d e n a t u r e dt y p e l l c o l l a g e n
material(UC-ll)was developed[o preserve
Pilot study to evaluate the efficacy of UC'll in
brological
humansubiecfs.An open labelpilotstudywas con- actvity Tlre presenceof glycosylated"active"epiducted in five female subjects(aged 58-78years) t o p e si n t h e U C - l lc o l l a g e nm a t r x i s c o n f i r m e d
by a
denronstrating
the symptornsof signifcantlointpatn validaledELISAtesl arrddisttngurslres
thisformtrom
T h e s es u b l e c t rse c e r v ead s i n g l eo r a ld a i l yd o s eo f 1 0 h y d r o l y z e dd, e n a t u r e da g a l a c l o s y l a t e c oi l l a g e n
m g U C - l lo n a n e m p t ys t o m a c hp r i o rt o b e d t i m ef o r (25) Oral intakeof 10 rng of this form of UC'll prezl9 nqpqonl rtirre d.ayc. All q hrpr-*q rpttrrj fhp ' rCS6eCs e n t sa c t i v ee p i t o p e sc, o n s i s t r nogf c o n f o r m a t i o n a i l y
glycosyiated
t r v ep a r nl e v e lo n a s c a l eo f 1 - 1 0( a s c o r eo f 1 Or e p - correcttlrree-dirnensioral
slructLrres,
to
--^-^r^^
n a q n e n n i n . r. Jr I n a q a n r t r
ICJCI
Peyer'spatchesin the GAL| (22, 26) Followng nlll lg
lUlr
tu
Of l
Ul lugd
dULE
UUJUUI
-hpir
donn'rnn "rnlp'ano"l
The q rhipc-s rater^j
nan
g e s t i o n ,U C - l l c o l l a g e n g l y c o p r o t e i ne n c o u r r t e r s
^
.
.
|
a
^
r
^
^
^
n
l
r
^
a
t
i
n
n
:
n
d
a
1
t
r
q v t r r r J U , u rc l t L d t u u > E d l J l l t L d i t u r I o r r u u u t I i nt 9n ll tt e
r al_
h y d r o c h l o r ia
ccid and pepsin Dose- and t meI e r l o r l c e e v e r y 7 o a y s . N / e a s L t ' e t l l e no l' r l a i r l e v e ] d e p e n d e rsr t u d i e ss h o wt l r e s em o n o m e r a
s r es t r l il n
i n t h e s e h u m a n s u b j e c t s f o l l o w i n g 4 2 - d a y s u p p l e - t l r e i trr , p l eh e l i c af,o " ' r .( F ' g 3 ) a ' r c trr a v eol o w nt o I n e
u
/ .
r | u
107
R:nnlrl
)
at al
r-r Solubletype ll collagen
+
Insolubletype ll collagen
=
o ^
5B
'oE
?( Ua 0
C O
- o
l
100
BO
60
40
20
0
Time (min)
immunosorbent
mcasurementsof
Fig. 3 Time-dosemeasurements
of Lrndenatured
type ll collagenactlvityin gaslrictluid.Enzyme-linked
u n d e r ] a t u r ei ydp e l l c o l l a g e ne p i t o p e s .
n urhichthev hind Pensin rlnes not
Pnvor'q n2l.hpq
b r e a kd o w n t l r e t r i p l eh e l i c a cl o n f i g u r a t i oonf t h e s e
m o n o m e T sd u e t o b i o c h e mc a l l i r n i t a t i o n s ,o t h e
a c l r v es i l e sa l w a y sr e m a i ni r r t a c tw. h i c hi s c o n f i r m e d
h , , LrLrr urnc n o-r^r-o rr y.o,r ^J . i ÎDc ^l J ^J ^ i nw i l ln o tc l e a v eb o n d sc o n uy
l a r nn g l h e a m i n o a c r d sv a l i n e .a l a n i n eo r g l y c i n e
( 2 7 ) .f h e a m i n oa c i d c o m p o s i t i o o
n l n a t i v et y p e I l
cnllrnon
iq he:riihr
dictrilrr rtnd
rarilh nhrnrno
(tA
)O\
o r i c r. r p q t h 2 l n e n q i n w i l l
This o,vcine-ricn senren.F
not cleave the nativecollagenconfiguration(27).
,--r;^^
^{
^nllrnorl
thp inlrct
nr rrin-r linoclinr
^îl^-.^^
f;llril r2 .^rnbi-
-^-,
raron oT coilagen monomers, sugars ano letopeptirJpci
hro:l"q
rlnrarrr inln
mnn.mar.
nnllrnan
n-r,-
t i r - J p s/ q m : l l e r o l r , c n n o n l i r ^ l pl n t t q t c y n n s l r n a o d n i o n p n r r n n p q /\ J!UO, ' l. O
-.
2
F, vl ^F rnu o
C n
or EIJTLUIJEJ
V r rl tr nr a
u r l. h o r h : r r r J Ir rl rr vo il u
P rn
r ul gi J^
b o r n d t o c o l l a g e nm o l e c u l e sa r e s u s c e p r i b l teo
arrrJ nc-
nnncir-
r-lpavpd
in tlrp n. rl rlr rinn
, "' / "
/311 Tl'tq el .\^/q thtr nnllarrer
dinosilOn
llip o l1pliy fnrtraltnn
tO
l n O q o r q l . r - t l t l l \ /a y l n q 1 t . 1 a r - l d : i : n r - r l c ^ r , \ / 6 o ^ i + ^ n 6 S O f
lrro nnll:n rn nl\/.nnrntpi6
s o q 1 l 1 i 1 ni n ^ r o : l a r
raiillr :rrrJ
rlro
Thoso
reennrrilinn
enilnneq
hii
nnqrrrrrpiri
ntr,onno
r a l o r yi e s o o n. s es. : -l-g- l :n- na r r nr Or n t
I n e n ro f l o i e r a r r c(e1 0 3 2 )
Protlerlv
r0B
âl
Tt - u
Eil
ol\/cnqvl2ttr.J
nrnlira.rl
vruilrsrat
û nr L
..
o-
Parror
e
fhF
er-irnnoq
h'
u
Trlui "n n' - J
/1?,
" ' . -r"n-n,r a- i g u -
immr
Iu ul f--[- l l cu nu lLl reqovegnr
p rr rocutl
I ir n rt E
r a r i l n t l r o P ou ryi uo ir ' c r n r l n h o cr u J iI n rl tl ']nc
|,uLut
l y m p h o i dt i s s u e so f t h e d u o d e n u m t, r i g g e r i n gt h e
complexserjesof immunologiceventsthat, in the
case of RA, down-regulate
the body's attackon its
o w n t y p el l j o i n tc o l l a g e nT.h i sr e s e a r c d
hemonstrate d t h a tP L G Aw a s w e l t o l e r a t e a
d g a i n s ct o l l a g e nl l i n d u c e da r t h r i t i sT h e s ea c t i v ee p i t o p e sm e e tc o n f o r ga
l yl m a t i o n asi p e c icf a t l o n so f t h e t h r e e - d i m e n s i o n
c o p r o t e i snt r u c t u r erse q u j r ebdy i n r m u n es u r v e i i l a n c e
t o s i g n a ia p p r o v a al n i t o l e r a n c eA. n tg e n e p l t o p e
glycosylat
on has been shownto play an impoftant
r o l e i n T - c e lrl e c o g n i t r oann d B c e l l r e s p o r r s i v e n e s s
( 2 1, 3 4 , 3 5 ) T h i sr e c o g n i t r oann d a p p r o v ael f { e c t i v e l\/ i r.nc n{'tlrp
ln-rF.
':-ocj ,mm
r r rI Lpu a l 9 6 r n o u?L1l q1u r| \. k h
Uy
l r o p - ing T-cellmediatedinflamr,rat
on, pa n and swelling.
, _ t_l r ,e . _n_o_r r .o _
U C - l l h a s d e m o n s t r a t eidt s a b i l i t yt o i n d u c et o l e r nid
nnt
lrrn' r-r
a r 1 c ee. f f e c t i v e irey d J C i n .go i n lp a ' r a n c S W enl lg i n
hrrhr I
o
n
i
u | ! Jr 'nt rurl oJ c_ r
RA subjects(3-6)
rrrFl-l fnr
-lir-l mn.,,fia.t
utu
I rvu
nelnhac
( 2 1 ) .F u r t h e r m o rK
e ,i me l a / ( 3 3 )d e m o n s t r a t etdh a t
a s i n g l eo r a la d m i n i s t r a t i o n
f poly(lactic-co'glycclic
a c i d ) ( P L G A ) n a n o p a r t i c l e si n d u c e d t o i e r a n c e
a g a n s lc o l l o g c lnl , n d u c e ad ' l r rlri s n I n c e p a r l . c l e s
o f P l G A w e r ee v i d e n rn l h e P e y e r ' sp a l c . l e so f a n i
m a l s f o r 1 4 d a y sf r o m o r i g r n af le e d i n g( 3 3 ) H y p o responsiveness
resultswhen epitopesof ingested
F f f C f - ' l S O f ut ,l t tl dv ae nl atiut t u tt raau. . t Lh yt npaw tl lt eaJa,t tl a
aa
g au nt
A:^- ^^^. fti r(' r i: lt t, -L l L u
t aa n
j azti ,n c tt J . a
/)ud)c]5
Thescienceof glycobioiogy
is rapidlyexpanding, r s i e r e u
d n d e n a t u r etdy p el l c o l l a g e n
e t f e c r i v eol ye a c u n c a p p i n ge n o r m o u sr e s e a r c ho p p o r t u n i t r easn d tivatekillerT-cellattack on type ll joint collagenrn
p r o m i s i n gt h e r a p e u t rtco o l s ( 11 ) . l t p r o v i d e sn e w humans(3, 22). Our pilotstudyexhrbited
the efficacy
insightsinto diseaseinitiation,
promotionand pro- o f U C l l ( 1 0m g r d s y li n e f f e c t i v erl ye d u c i n g
l o i n tp a i n
g r e s s i o n ,e s p e c i a l l yr e g a r d i n ga u t o j m m u n ed i s - a n d s w e l l i n g
r n h u m a ns u b j e c t sw i t h o uat n y a d v e r s e
e a s e s s r c h a s F A ( 1 2 ) A p r e p o n d e r a n coef t h ee v i - e f f e c t s .U C - l l c o n t a i n s c o n J o r m a t i o n a lcl yo r r e c I
dence suggeststhat all autoimrnune
diseasescan "active"epitopes requiredto interactwith Peyer's
b e t r a c e db a c kl o e r r o r sa l s o m ej u n c l u r eo f b i o r d e n - palchesjn the GALTand terminateantigenicsignall i t i c a l i o nr e
, c o g n i r i oann d r e s o o n s se i g n a l i n gP.r o p e r ing of a pathogenicnature,characteristic
of RA (10).
glycosylation
glyco- Thisapproachprovidesnew insightsintothe etiology
is requiredfor glycoconjugation,
molecularinterconversions,
biotransformations,
and o f a u t o i m r n u n ei n f l a m m a t o r d
y i s e a s e sa n d t h e i r
g l y c c p r o t e rann d g l y c o l i p isdy n l h e s i(s1l , 1 2 )
amelioration
with safeand effectivetreatments.
g a l a c l o s y l a l i oa nl l c r st h e r e q u i In RA rmpa,red
srte three-dimensjonal
conformationsof glycopro,
t e i n s ,i n c l u d i n cge r t a i ni m m u n ef a c l o r ss, u c ha s l g G Acknowledgment
a n d p o s s r b ye v e n l y p e l l c o l r a g e np, r o d l c i n gt h e
T h ea u t h o r st h a n kM s K r i s t i n S
loss of self,identity
(12) Alterations
in glycosylation
e t r o n gf o r t e c h n i
a n d o f g a l a c l o s yslt r u c t u r easr eh a l l m a rckh a r a c l e r i s - cal assistance.
tics of RA.Thisloss of self-identification
altersrecogn t t i o na n dr e s p o n s e
s i g n a l i ndgu r i n gi m m u n es u r v e i l lance,incitingattackon the body'sownjointcollagen Fleferences
(r3 1B)
(1)Trentham
D . E, H a l p n e rA . D . ,T r e n t h a m
R E , e t a l U . s eo /
Other autoimmunedisordershave also been
undenaturedlype ll callagen in the trcatrr)enlol theumatatdannftts
associatedwith faulty glycosylation(12 17) This
c l i n P r a c A i l e r t. v l e d, 2 , 2 5 4 , z a u .
i m p l i e st h a tc e r t a i na u t o i m m u ndei s e a s e m
s a yr e s u l t
( 2 )H e l m i c C
k G . ,L a w r e n c R
e . C, P o l l a r dR A . ,e t a l A r l h r i taj sn o
when naturally
occurringbiomolecules
are identified olher rheunatic conditians: Who is alfected now, will be aftected
as rorergn
p a l l r o g e n iacn l i g e n sd, u e l o t h e , ra l l e r e d / a t e r ?A r t h r i t iCs a r eF e s , B , 2 0 3 ,1 9 9 5 .
( 3 ) T r e n t h a mD . E D y n e s i u s - T r e n t h R
am
. A . ,O r a v E J . , e t a l
compositionand structural
conformation.
As a result,
Eflectsol aral adminis|€ton oi type ll collagen on rheuntatoidafthna p o r : p r i a t ei m n u n o t o g ; c aal l a l n s a r e g e n e r a t e d
lls. Scrence261 1727,1993.
a n d a g g r e s s i v ed e fe n s e t a c t i c s a r e e r n p l o y e d
(4) BarnettlML., CombitchrD.,Trer-rtham
D E A pilat trialal oral
a g a i n stth e h o s t ' so w n t i s s u e s( 1 5 ) .
lype ll collagen n tlte trcatment ol pvenile rheumatoid arthnils.
Fecently,safe and effectrve
alternattves
to tradi- A l l ^ . i l r sF n e u m .3, 9 6 2 3 , ' 9 9 6 .
( 5 ) B a r n e tltv ,Ll . K r e r n eJr . l V . .S t C l a i rE . W ,e t a l T r e a l m e n
o tf
tronalmodels of diseasemanagementhave been
rheumatoidanhritiswith aral type ll collagen.Besu/rso/ a multicerrcr
u s e di n R A ( 3 6 ) .O r a la d m i n i s t r a t i o fna u t o a n t i g e r r sdouble-blind,piaceba-cantralleC
lrial Arthntis Bheurn, 41 29O,
h a s b e e ns h o w nt o s u p p r e s a
s v a r i e t yo i e r p e r i m e n - 19 9 8 .
( 6 ) N a g l e r - A n d e r s oCn. , B o b e r L A . , R o b r n s o nN l . E . ,e t a l
t a l l yi n d u c e da u t o t m m u ndei s e a s e si,n c l u d i n ga n t i g e n - r n d u c eR
d A ( 3 - 6 3 3 ) A s o u r u n d e r s t a n d i ror fg Supp/ession af type ll coilagen-incluced afthrilis by tntragastrrc
ol saluble type ll callagen. proc Nail. Acad. Sci., 93,
g l y c o b i o c h e n i s rj rnyc r e a s e se.x p l a n a t i o nr es g a r d i n g aciministration
7443,1986.
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ot:/
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I rt
\ldtovrvrytd'
1 tt
Lt 4ta j'
'ndttâd
c
A'thttt:'
rn'j
-J
l m m u n o l . 4 4 , 3 8 1, 1 9 9 6
(26) Terato K., Hasty KA,
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. c t . .& . 1 2 { ) / , 1 9 6 !
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(29) Trelstad R L., Kang A.H., lgarashi S /so/aron af lwa disttnct
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( 3 0 ) H e r b a g e D . , B o u i l l e tJ . , B e r n e n g o J C . B i a c h e m i c a i a n d
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ta.-d
tn ).
p\\r/r/e
.,rp(
^t
.^ntr)ô
| ^lt-nan
I ^r,lr
I
Q,^l
r t
"-.- _ .-.-. _.tetn..
A L r t o i m m u1n6. 1 5 1 2
, 001.
2
7
3
,
r
5
5
1
,
1
9
9
8
.
( 2 1 )C o f t l r a yA . , B a c k l u n dJ , B r o d d e f a lJk , e t a l .E p i t o p eg l y (35) Back und J , Carlsen S., Hoger T PreCamtnanl selectian af
cosylatianplaysa criticalrole for T cell recognttonaf lype ll callagen
f , p//s spec//lc lo'llte glLo t.atcd caltoqeF t,u- ll sDn)ps t2A3 270t
;n callaoet,-indt[^dailfuits E.rr J lmmrr"o . 28, 2r80. 1998
in humanized lransgenc mrce and tn il)eumatct.l arlhrilis. Proc. Nail
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Acad Sci ,99, 9960 2002
ment n earlyrheumatotdarthritisAthrLtis
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(23)BurkhardtH KolierT.,EngsrronlA , eI a Epttope.tpecitic
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110
. . T H R I T I S& R H E U M A T I S M
( a t . 1 9 , N o . 4 , A p r i l 1 9 9 6 ,p p 6 2 3 4 2 8
@ lg{fu AmericanCollegeof Rheumatology
623
A PILOT TRIAL OF ORAL TYPE II COLLAGEN IN THE TREATMENT
OF JUVENILE RHEUMATOID ARTHRITIS
MARTHA L. BARNETT, DANIEL COMBITCHI, ANdDAVID
E. TRENTHAM
objective.To evaluatethe efficacyof orar chicken
type II collagen (ccII) in the treatment of juvenile
rheumatoidarthritis (JRA).
Methods, Ten patients with active JRA were
treated with CCII for 12 weeks.Efficacyparamerers,
which included swollen and tender joint- count and
score, grip strength, S0-footwalking time, duration of
morning stiffness, and patient and physician global
scoresof diseaseseverity,were assessed
monthly.
Results.Alr patientscompretedthe fulr courseof
therapy. Eight patientshad reductionsin both swollen
and tender joint countsafter 3 months of ccIL The
rneanchangestrom baselinein swoilenand tenderjoint
countsfor the 8 respondersat the end of the studywere
-6Lvo and -54 Eo respectivery.
'
Mean varuesfor other
efficacy parameters also showed improvement from
baseline.There were no adverseeventsthat were consideredto be treatmentrelated.
Conclusion. Oral CCII may be a safe and effec_
tive therapyfor JRA, and its usein this disease
warrants
further in vestigation.
Juvenile rheumatoidarthritis (JRA) affecrs an
estimated65,000-70,000
childrenin the US (l). While
it has
suggested
thar
JRA has a better piognosis
!l"n
than adult rheumatoidarthritis(Ray (2), more
recent
data show that -45% of chilcirenwith JRA have
active
diseaseat 10-yearfollowup (3).
current treatmentoptions for JRA are often
unsatisfactory,becauseof both limited
efficacvand
Supportedby a grant from the Robert Wood
Johnson,Jr.
c-haritabletrust and bv fiIH
d;i M0i-nn-oio:z ro the Beth rsrael
HospitalGenerarcrinilar i;r;;;ir'crnr.r,
Dr, Barnert,swork was
supportedin parr by a-pfizerfeilowship
in tr" gJh ir;;.I Hospitar_
Harvard/M]T Hearih s. i."..r r" o'i.Ji.,notogy
cr i nicar In vesrigaror
Training program.
Martha L. Barnett,MD, DanielCombitchi.
BA, David E.
Trentham,MD: Berh trruei H;ilJloston,
Massachusetrs.
Address reprint requestito Martha r-. gu;erl,
r,to, piui_
sion of Rheumarology,
Beth f r*rf i"rpiral, 330BrookiineAvenue,
B o s r o n ,M A 0 2 ? 1 5 .
Sub_mitted
for publicationJune 30, 1995;acceptecJ
..:_
in revtsed
form Ocrober30, 1995.
concernabout toxicity. Theseincludeantiinffammatory agentssuch as aspirin,naproxen,tolmetin, or
ibuprofen,antimalariar-agents,
gord. and methotrexate, as well as physical therapy. In a minority of
patients,rapidly progressivediseaseis refractory to
thesetherapiesand leadsto permanentjoint destruction with physical incapacitation.systlmic corticosteroidsare relativelycontraindicated
in the treatment
of JRA, exceptin patientswith severeporyarthritisor
severesystemicdiseasethat has failed to respondto
more conservativetreatment.In addition to multiple
other toxicities,growth suppressionis a maior deterrent to the use of steroidsin the treatmentor rna. a
multicenter study of D-penicilamine and hydroxychloroquinein the treatmentof severeJRA showed
that, when given in conjunctionwith a nonsteroidal
antiinflammatorydrug (NSAID), neither agent was
superiorto placebo(4). Methotrexatehasbeen shown
to be an effectivetreatmentof refractory JRA (5), but
parentsand physiciansarikeremain concerned
about
possiblelong-termsideeffects.The toxic-to-therapeutic ratio of cytotoxic agents,such as cyclophospha_
mide, is even more narrow. Moreover, reports
of
malignancyeither during or after therapy wit^himmunosuppressive
drugshaveprecludedtheirusein all but
the most severelyill patients.
The evidencethat sensitizedr ceilsparticipate
in provokinginflammationin RA and otherrheumatic
diseases(6) providesdirectionto the searchfor
treatment modalitiesbased on specific immunosuppres_
sion, which would be both highry effectiveand minimally toxic. The ability to ind'ce antigen-specific
peripheralimmunetoreranceby oral administraiion
of
antigenshas beenrecognizedfor some rime (7).
It is
presumedthat the physiorogicinteraction proteins
of
with the gut immune systemhas evolved to prevent
s y s t e m i ci m m u n e r e s p o n s e st o i n g e s t e dp i o t e i n s .
Hypersensitivityreactionsto food proteins ur. .ur.,
and the mechanism
of orartoleranceis basedon this
unique immunologicsystem, Given in low doses.
624
BARI\TETTET AI
orally administered
antigens
induceactiveimmunosuppression,whereashigh antigendoses lead to clonal
anergy(8).
Oral administration
of type II collagenhasbeen
shownto amelioratearthritisin two animalmodelsof
RA, one inducedby immunizationwith type II collag e n( 9 . 1 0 a
) n d t h eo t h e ri n d u c e db y F r e u n d ' sc o m p l e t e
a d j u v a n (t l l ) . I n a c l d i t i o na, p l a c e b o - c o n t r o l l epdh,a s e
II study in 60 adrrltswith severe,active RA demonstraLedsignificant(P < 0.03)improvementin tender
and swollenjoint counts after 3 months of treatment
(12).A multicenter,double-biind,dose-ranging
study
of oral chickentype II collagen(CCil) in adult RA has
recently been completed(Barnett ML et al: manuscript in preparation),The presentopen study of oral
CCil in the treatmcntof JRA was undertakenbasedon
theseeariierresults.
PATXEI{TS AF{D IVTET'F{ODS
A iotal of 10 patientswith JRA were enrolledin the
study. To be eiigible,patientshad to meet the American
Coliegeof Rheumatologycriteriafor JRA (13).In addition.
patientshad to be betweenthe agesof B and 14yearsandhad
to have active arthritis,as defineCby the presenceof a3
s wollenj o i n ts a n d = 6 te n d e jro i n ts . Pa ti entsw i ti r any onset
subtypewere eligibleprovidedthat they had the required
numberof inflamedjoints at the time of enrollment.Thus, a
patientwho had involvementof <4 joints within the first 6
monthsof disease(and who wouid thereforebe classifiedas
having pauciarticularonset)would nonethelessbe eligible
for enrollmentin this studyprovidedtherewere =3 swollen
and >5 tenderjoints at the time of studyentry. Patientswere
excludedif they were unableto discontinuetreatmentwith
disease-modifying
antirheumaticdrugs (DMARDs), if they
had structura.i
damageto thejoints that was not considered
to be amenableto physicalrehabilitationif inflammation
were to subside,or if they had seriousconcurrentmedical
problems.
Dunng the courseof the trial, patientswere permitt ed t o c o n ti n u etre a tm e n w
t i rh N S AID s or l ow -dosecorri cosleroids(no morethanthe equivalentof l0 mg prednisone/
day ) , pr o v i d e dth a t th e d o s e sre ma i n edstabl eduri ng the
lr eat m e npt e ri o d .In c re a s eisn N S AID o r predni sone
dosage
or init ia ti o no f a n y o th e ra n ti rh e u m a titcherapyrepresented
pr ot oc o Iv i o l a ti o n s P
. a ti e n tsw e re re q ui redto di sconti nue
DMARDs at the startof the trial, with no mandatedwashout
per iod.
Patientswho met all entry criteriawere enrolledand
begant r e a tme nw
t i th C C II fo r a 3 -m o n t hperi od.A l l pati ents
and t hei r p a re n tsw e rere q u i re dtr: s i g nan i nformedconsent
f br m det a i l i n gp ro to c o lp ro c e d u re sp, o ssi bl eri sks and benef it s ,et c . T re a tm e nct o n s i s te o
d f 1 0 0td d ay oi C C II for the
first monthand 500pgldaythereiifter.CCII was providedas
a liquid s u s p e n s i oinn 0 .l l v l a c e ti ca c i d at 4' C and addedro
c old or a n g ej u i c e i mme d i a tc l yp ri o r to i ngesti on.D osesancl
t ec hniq u ew e re th e s a m ea s th o s eu s e di n the previ oustri al
i n a d u l t s ( 1 2 ) . P a t i e n t sw e r e r e q u i r e d t o r e t u r n f o r m o n t h l
v i . s i t s ,a t w h i c h t i m e s a f ' e t ya n d e f f i c a c y m e a s u r e m e n t sw e r
o b t a i n e d .P a t i e n t sw h o e x h i b i t e da n i n i t i a l p o s i t i v e r e s p o n s
b u t s u b s e q u e nw
t o r s e n i n ga f t e r t h e i n i t i a i 3 - m o n t h t r e a t m e r
period were considered for fur-thertreatment with the stud
m e d i c a t i o n ,o n a c a s e - b y - c a s eb a s i s .
Clinical effi.cacywas assessedby ascertaining painfi
a n d s w o l l e nj o i n t c o u n t s a n d j o i n t s c o r e s a c c o r d i n g t o l h
method of Weinblatt et al (14), evaluating a total of 5
diarthrodial joints for pain/tenderness and 52 joints fc
swelling;-durationof morning stiffness,grip strength, 50-ioc
w a l k i n g t i m e , a n d p a t i e n t i p a r e n ta n d p h y s i c i a ng l o b a l s c o r e
o f d i s e a s ea c t i v i [ y a t e a c h v i s i t . L a b o r a t o r y d a t a . i n c l u d i n
c o m p l e t eb l o o d c e l l c o u n t ( C B C ) , e r y t h r o c y t e s e d i r . r e n t a r i o
rate (ESR), rheumatoid factor (RF) level, and serum Ig(
a n t i b o d i e st o t y p e I I c o l l a g e n( 1 2 ) , w e r e r e c o r d e d a t b a s e l i n
and afier 3 months of therapv.
R,ESULTS
All l0 patientswho enrolled and began stud
medicationcompletedthe full 3 monthsof treatment
Therewere5 girlsand 5 boys,with a meanageof 10.
yearsand a meandiseasedurationof 4.3 y'ears.Th
diseaseonset type was polyarticularin 3 patients
pauciarticular
in 3 patients,and systemicin 4 patient:
F o u r p a t i e n t sh a d p r e v i o u s l y b e e n t r e a t e d r v i t
DMARDs, and t had beentreatedby his parents',,rit
a varietyof herbalrnedications.
Patient6 discontinue
azathioprineI day prior to beginning therapy rvit
CCII, but no otherpatientswere takingDMARDs a
the time of enrollment.Six of the l0 patientsreceive
concomitantstabiedosesof biSAIDs and/oriow-dos
prednisone
duringthe study period (aiongw'ith acet
aminophen
in 1); I patientcontinuedto take acetamir
ophen,and 3 patientstook no concomitantrnedica
tionsfor theirarthritis.Eightof the i0 patientswerei
SteinbrockerfunctionalclassII (15) at stuciyentr).
and the rernaining2 patients(patients2 and9) were i
class III. HLA typing was not performed.Demc
graphic and clinical featuresof the patientsare pre
s e n t e di n T a b l e1 .
Eight patientshad reductionsin both swolle:
and tenderjoint counts after receivingCCII frrr
months.The meanchangesfrom baselinein swolle:
and tenderjoint countsfor the 8 responders
at the en,
o f t h e s t u d yw e r e - 6 1 % a n d - 5 4 % o ,r e s p e c t i v e l yS. i .
patientshad >33% reduction in both swollen &rr
t e n d e r j o i nct o u n t s .I n d i v i d u apl a t i e n tv a l u e sf o r s w o l
len and tenderjoint counts at baselineirnd after
monthsof therapyare shownin Figure l. The time t,
tr,
onsetof responsefor the l0 patientswas 'r:rriable.
p a t i e n tl , a l m o s a
t l l o f t h e i m p r o v e m e nw
t a sa c h i e v e ,
within I monthof the initiationof treatment.but th
I
,.*
{ onAr TypE rr coLLAcEN rN JRA
TabJe l.
Demographic and clinicalfeatures of
Patient
Yearsof
JRA
Age/sex
I
tz/f
2
I
r0
t2/F
t4lM
9/F
J
/
.+
5
6
7
8
9
0 .5
11/M
t|/M
t3lM
9/F
JO
I 0/M
Poly
Systemic
Pauci
Systemic
2
5.5
9
Poly
.t)l
6/l
t h e patientswith juvenilerheumatojd
arthritis(JRA)*
Onset
subtype
Pauci
Systemic
Pauci
Poly
Systemic
J
1
625
Prior
DMARDs
nrrrx,u-q
l
SSZ
MTX, AZA, AUR
MTX
Concomitant
medication
Napr. 350mg rwicea day
Nlp.. 250mg twice a day,
Pred,2.5 msldav
I b u . 1 , 8 0 0m s / = d a v '
Pred. 2 mgtdiy
Ibu. 800mg/day
Pred, 5 rng tw-icea day,
acetaminophen
Acetaminophen
* Poly: polyarticular;Napr.
= naproxen;
MTX : methotreilfi HC..Q= hydroxychloroquine;
: pauciarticuiar;
pauci
Pred.= prednisonei
SSZ;,"ff"r"i"?ir.l iUr. = IDuproIen:
AZA = azathioprine;
AUR = auranofin.
been rrearedwirh herbal remediesprior
to the initiation of chicken t1,pe
Ii collagen
,t::T:_nad
!r
vgrrlr9llL.
responseoccurredmore srowryin
other patients.
-or on
average for all 10 patients, tire percentus.
totar
improvernent in swoten and tender
counts
achievedafter onry 1 month of treatment:"irt
was 35vo and
49Vo,respectively.
Swollenand tenderjoint scoresdecreased
frorn
baselinein 9 of the 10patlents.
The meanreductions
fgr a! 10 subjectsin swoilenand
tenderjoint scores
after 3 months of therapywere 43Vo
andslVo, respectively. Theseresultsare-shown
in Figure Z.
.. .. M.un valtresfor morning stiffnessand 50-foot
walking time showed improvement
from baseiine.
Clinical.efficacyresults foi these parameters
are presented in Figure 3. Although giip
strengih is nor
consideredto be a reriabremiasure-in
child-ren,mean
valuesin right and ieft grip srrengrh
for th; i0;;ients
did show a slight improvement
from baseline to 3
months (data not shown).In addition,
mean patient
and physiciangJobalassessment
scoresalsoimproved
comparedwith baseline.One patient (patieni'+;
t uO
total resolution_of
arthritisby the end of treatrnentand
has subsequentry
beenabreto discontinuealr medications with no return of symptoms
during a lL_month
followup period. No significanttrends
in any hematologic parameters,includingCBC
and ESR, *ri" noted
the study. NoL. ofthe parienrs,.rrrJposirive
9"r1rg
for RF or collagenantibodi.spiior
ro or on compretion
of treatment.
CCII was well tolerated.Mi]d, transient
skin
rasheswere noredin 4 patienrs
duringrhe ;;;t; in 3,
the rash did not seem to be
related to tha study
medication,and in no instancedid
the .uuh prompt
A)rs
B)
40
t
c
36
40
30
a a
E.
gl
5 2 5
t a o
:t
45
1 a
t t
Z basellne
I month 3
Zero
Swoilon
Jolnts
o
rt
20
15
l0
4
5
6
pailsnt#
7
8
9
0
A
5
6
7
I
I
1 0
psilent #
Figure l. Swollen and
tenderjoint countsfor individualpatients.
The numberof swollen(A) and t e n d e r
at baselineand after
(B)joints for each individual patient
3 monthsof treatmentwith chicken
type II col.lagen
is shown.
626
tsARhIETTET AI
A)
B)
70
60
o u
o
fi so
H 50
6
o 40
o
3 0
I
5
6
c
30
?0
20
1
?
3
4
5
6
7
I
9
't
10
2
3
4
p s l t e n t#
Figure
2. Swollen and tender joint scores for individual
and after 3 months of treatment
with chicken
5
6
7
I
9
10
pstlenl $
p a t i e n t s , S w o l l e n ( A ) a n d t e n d e r ( B ) j o i n t s c o r e sf o r e a c h i n d i v i d u a l p a r i e n r a r b a s e i r n r
type II collagen are shown.
interuption of therapy.In 1 patient,an erythematous,
pruritic rash was presenton the legs at the time of
study entry. This rash appearedto worsenduring the
first month of the trial, but it then resolvedwithout
specifictherapy while the patient continued to take
CCil. Two other patientsreportedtransienterythernatousrashes(not observedby the investigator)which
were believecito be related to new soap or new
iaundry detergent.Facial flushing,which occurred20
rninutesafter ingestionof CCII and lastedl-2 hours,
was noted by 1 patient during the initial 2 weeks of
treatment,but subsequently
resolvedspontaneously.
Patient6 had a history o{ chronic hepatitisC at
the time of study entry. During the secondmonth of
the trial period, the findings on routine blood tests
performedby his personaiphysicianwere notablefor
A)
elevatedtransaminase
levels. F{is only symptornii
that time was an increasein fatigue.One week iater
when his transaminase
levelswere found to have i-iser
further, he underwenta liver biopsy. This reveale,
miid chronicactiveliepatitissimilarto that exhibitei
on a previousbiopsy performedin 199i. and it wa:
decidedthat his dosageof oral corticosteroids
shoulc
be increased.
Repeatliver functiontests(LFTs) per
formed the day prior to the initiation of high-dosi
prednisonetreatrnentdemonstratedspontaneous
im.
provementin histransaminase
valuesto <5096of their
peak levels,but this testresult becameavailableonll
after the patienthad taken one 20-mgdose of prei
nisone.Thepatientdiscontinued
prednisone
high-dose
afterthis singledose,andhis LFT findingsrerurnedrc
normal within 1 week and subsequentlvremainec
cf,
360
^
300
q
;
E
a
c
240
o
o
q
o
c
qt
|80
g
q
T
,
o)
=
o
lo
E
i 0
ti0
,l,E
bsso{lne
monlh
3
b a c e l l fl €
month 3
F i g u r e 3 . S e c o n d a r y e f f i c a c y p a r a m e r e r s ( A , m o r n i n g s t i f f n e s s ; B , S 0 - f o o t w a l k i n g t i m e ) a t b a s e l i n e a n d a f t e r 3 m o n t h s o f t r e a r m e n r with
c h i c k e n t y p e I I c o l l a g e n .I n d i v i d u a l p a t i e n t n u m b e r s a r e s h o w n o n t h e g r a p h s n e x t t o t h e i r r e s p e c t i v ep l o t m a r k e r s .
{'ofal
TYPE II COLLAGEN IN JRA
normalfor the durationof the study.At no time during
this period was his CCII therapyinterrupted,and this
transient rise in LFT valueswas not believedto be
relatedto the study medication.Of note, since the
cohclusionof the trial, the patient has had another
sirnilar episode of transienttransaminitiswhile not
taking CCII.
After conclusionof the study protocol, a second 3-month course of CCII was requestedfor and
providedto 4 patients(patients1,2,7, and 8). patient
4 was examined14 monthsafter study completion,at
which time it was confirmedthat she remainedcompletely free of any symptoms of arthritis with no
rnedications,had no tenderor swollenjoints, and had
normal laboratoryvalues.
627
ter the fed autoantigen
at other sites.This phenomenon of bystandersuppression
has beendemonstrated
i n e x p e r i m e n t a la u t o i m m u n ee n c e p h a l o m y e l i t i s
(EAE), a cell-mediatedautoimmune diseaserhat
servesas an animalmodelfor multiplesclerosis.EAE
can be inducedby immunizationwith myelin basic
protein (MBP) or proteolipidprorein (PLP). Oral administrationof MBP hasbeenshownto suppressboth
MBP- and PlP-induced EAE (ZZ). Similarly, oral
administrationof type II collagenhas been shown to
ameliorateRA inducedin animal modelsby immunizationwith either Freund'scompleteadjuvant(11),
CCil (9,10),or methylalied
bovineserumalbumin(23).
Thus, it may not be necessaryto identify the target
autoantigenfor a givendisease.it is necessaryonly to
orally administera proteinwhich is presentat the site
of
inflammationand which is capable of inducing
DISCUSSION
regulatory celis to secrete suppressivecytokines.
Oral tolerization is a well-recognLzed
phenomThese findings have important impiications for the
enon in which the oral administrationof antigeninuse of oral tolerance as a therapeuticapproach for
duces peripheral immunetoleranceto the fed antigen
the treatment of T cell-mediatedinflammatory auto(7). The utiiity of oral tolerization as a trearment
immunediseases
in humansin which the inciting autornodaiityfor a varietyof autoimmunediseases,
includantigenis unknownor in which there is autoreactivity
ing RA (12), multiple sclerosis(16), type I diaberes
to multipie autoantigens
in the target tissue.
mellitus(17),and uveitis(18),is currentlyunderactive
Alternatively, a dominantpathway for oral tolinvestigation.To date, no significantadverseevents
erancemay involve T cell anergrzation(24,25).In this
have been noted in any animalor hurnanstudy of oral
case, the induction of oral tolerancewould be pretolerance, and the simpiicity and apparentsafety of
sumedto resultin diseasesuppressiononly when the
this forrn of treatmentmake it extremelyappealingin
fed antigen is also the target autoantigen for the
thesechronic, disabiingdiseases.
diseaseunderstudy.The demonstration
of a sustained
Basedon resultsof anirnalstudies,the mecharemissionof arthritis in 1 of our 10 patients might
nism responsiblefor oral toierancevaries depending
arguably be more consistentwith this 1atter view,
on the dose of fed antigen,with low dosesinducing
basedon the longevityof her response.However, this
active suppressionand high dosesresultingin cionar
would imply that type II collagenwas the diseaseanergy (8). The regulatorycells that orchestrateactive
specificautoantigenin her case, and while collagen
suppressionact via the secretionof suppressivecytoreactivity can be demonstrated
in somepatients with
kines, such as transforminggrowth factor p andinterRA, it is unknownwhetherthis is actuallyinvolved in
leukin-4 (19), Experimentsin animals support the
the primary pathogenesisof the diseaseor merely
notion of the generationof regulatorylymphocytesin
reflectstissuedegradation.
Peyer's patcheswhich subsequently
migrateto mesThe presentstudy demonstrates
that oral CCII
enteric lymph nodes and spleen (20).secretion of
may be a safeand effectivefcrrmof treatmentfor JRA.
regulatorycytokinesby thesecells in vitro is depenThe most remarkableimprovementsin clinical paramdent on antigen-specificstimulationwith the fed antietersof arthritiswerenotedin parientsI and 4, both of
gen (21).Thus, it is presumedthat active'suppression whom were girls
with reiativelvrecent onset of disof inflammationby theseregulatorylymphoCytesreease.Patient t had polyarticularonset, whereas paquires further migration of these celis to a local mitient 4 had systemicfeaturesof fever and rash in
croenvironment,wherethe fed antigenis present.
additionto polyarticularjoint
involvementat onset.Of
Becausethe regulatorycellsgeneratedby oral
note, of the 3 boys with pauciarticular
onset of distolerization are primed in an antigen-specific
manner
ease, 2 experiencedminimai, if any, benefit from
but suppressin a non-antigen,specific
manner,they
collagen(patients5 and7).As mentionedabove,HLA
mediate"bystandersuppression"
whenthey encountyping was not performed,but it would be of inrerest
628
BARI.{ETT ET AL
to know whetherthesepatientswere HLA-827 positive. If this werethe case,it rnightsuggestthat type II
collagen is ineffectivein the treatrnentof juvenile
spondylarthropathies.
In an open-labelstudy, one must always be
concernedaboutthe contributionof the placeboeffect,
and this may be even rnoretrue in a pediatricpopulation. Theretbre,conciusionsregardingefficacybased
on this pilot trial wouldbe premature,but nonetheless,
thesepreliminarydata support the assertionthat further study of oral CCII in the treatmentof JRA is
warranted.The observationthat 1 patientachieveda
completeremissionof her arthritisis especiallycompelling in this regardand is similar to the experience
observedin a minorityof adultstreatedwith CCII (12).
More importantly,as pertainsto this pilot study, oral
CCII appearsto be extrernelywell tolerated in this
pediatricpatientpopulation.The only adverseevent
noted duringthe study that was beiievedto be related
to the study medicationwas transientfaciai flushing,
which occurredin 1 patientfor -2 weeksafter collagen treatmentwas begun.The elevatedtransarninase
leveis noted in patient 6 during the secondmonth of
the study resolved without interruption of collagen
therapyand were believedto be relatedto his underlying chronic hepatitisC. The combinationof favorable safetydata and promisingclinical resuits in this
pilot trial stronglyindicatethat thereshouldbe further
studiesof this novel therapeuticagentin the treatment
oi JRA.
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Int. J. Med. Sci. 2009, 6
312
International Journal of Medical Sciences
Research Paper
2009; 6(6):312-321
© Ivyspring International Publisher. All rights reserved
Safety and efficacy of undenatured type II collagen in the treatment of
osteoarthritis of the knee: a clinical trial
David C. Crowley1, Francis C. Lau2, Prachi Sharma1, Malkanthi Evans1, Najla Guthrie1, Manashi Bagchi2,
Debasis Bagchi2,3, Dipak K. Dey4, Siba P. Raychaudhuri 5,6,
1. KGK Synergize Incorporated, London, ON, Canada
2. Department of Research and Development, InterHealth Research Center, Benicia, CA, USA
3. Department of Pharmacology and Pharmaceutical Sciences, University of Houston College of Pharmacy, Houston, TX,
USA
4. Department of Statistics, University of Connecticut, Storrs, CT, USA
5. Department of Medicine, Division of Rheumatology, Allergy and Immunology, School of Medicine, University of
California Davis, Davis, CA, USA
6. VA Medical Center Sacramento, Hospital Way, Mather, CA, USA
Correspondence to: Siba P. Raychaudhuri, [email protected]
Received: 2009.07.14; Accepted: 2009.10.08; Published: 2009.10.09
Abstract
Previous studies have shown that undenatured type II collagen (UC-II) is effective in the
treatment of rheumatoid arthritis, and preliminary human and animal trials have shown it to
be effective in treating osteoarthritis (OA). The present clinical trial evaluated the safety and
efficacy of UC-II as compared to a combination of glucosamine and chondroitin (G+C) in the
treatment of OA of the knee. The results indicate that UC-II treatment was more efficacious
resulting in a significant reduction in all assessments from the baseline at 90 days; whereas,
this effect was not observed in G+C treatment group. Specifically, although both treatments
reduced the Western Ontario McMaster Osteoarthritis Index (WOMAC) score, treatment
with UC-II reduced the WOMAC score by 33% as compared to 14% in G+C treated group
after 90 days. Similar results were obtained for visual analog scale (VAS) scores. Although
both the treatments reduced the VAS score, UC-II treatment decreased VAS score by 40%
after 90 days as compared to 15.4% in G+C treated group. The Lequesne’s functional index
was used to determine the effect of different treatments on pain during daily activities.
Treatment with UC-II reduced Lequesne’s functional index score by 20% as compared to 6%
in G+C treated group at the end of 90-day treatment. Thus, UC-II treated subjects showed
significant enhancement in daily activities suggesting an improvement in their quality of life.
Key words: undenatured type II collagen, osteoarthritis, glucosamine, chondroitin, WOMAC, visual analog scale, Lequesne’s Functional Index
INTRODUCTION
Arthritis afflicts approximately 43 million
Americans or approximately 16.6% of the US population. The two most common types of arthritis are osteoarthritis (OA) and rheumatoid arthritis (RA). OA
of the knee and hip is a growing health concern and is
the most common forms of arthritis (1-3). Pain and
disease can range from very mild to very severe (3).
Patients with OA have pain that typically worsens
with weight bearing, including walking and standing,
and improves with rest (4). Other symptoms include
morning stiffness and gelling of the involved joint
after periods of inactivity. Currently, OA affects
http://www.medsci.org
313
nearly 21 million people in the United States, accounting for 25% of visits to primary care physicians,
and half of all Non-Steroidal Anti-Inflammatory
Drugs (NSAID) prescriptions. The diverse clinical
patterns of OA are observed in approximately 10% of
people older than 60 years thus compromising the
quality of life of millions of Americans. In addition,
OA costs the North American economy approximately $60 billion per year.
Current treatment of OA includes exercise,
heat/cold therapy, joint protection, weight loss,
physiotherapy/occupational therapy and medications (3-5). The most common medications include
acetaminophen and NSAIDs. Although these drugs
are effective for reducing pain associated with OA,
they do not reverse the disease. In addition, there are
considerable side effects associated with the use of
these drugs. As a result, OA sufferers have turned to
natural nutraceuticals to ease their pain and discomfort. These products are commonly used because they
are well tolerated and considered safe. Nutraceuticals
are defined as functional foods, natural products, or
parts of food that provide medicinal, therapeutic, or
health benefits, including the prevention or treatment
of disease. Currently, glucosamine and chondroitin
are the two most commonly used nutraceuticals in
humans as well as in animals to alleviate pain associated with arthritis (6). However, recent randomized
controlled trials and meta-analysis of these supplements have shown only small-to-moderate symptomatic efficacy in human OA (7). An emerging novel
nutraceutical ingredient known as UC-II has received
considerable attention in the treatment of OA. UC-II is
a novel undenatured type II collagen derived from
chicken sternum cartilage. Previous studies have
shown that undenatured type II collagen is effective
in the treatment of RA (8-11), and preliminary human
(12) and animal (13) trials have shown it to be effective
in treating OA. Obese-arthritic dogs given 4 mg or 40
mg daily dose of UC-II for 90 days showed significant
declines in overall pain, pain during limb manipulation and lameness after physical exertion (14). Greater
improvement was observed with the 40 mg dose. No
adverse effects or significant changes in serum chemistry were noted. Following UC-II
withdrawal for a period of 30 days,
Visit 1
Figure 1. UC-II clinical study design.
The study was a two-site, randomized,
double-blind study conducted in London, Ontario and Corunna, Ontario,
Canada.
Physical
assessment,
medical
history, clinical
assessments
and blood tests
as indicated
Screening
all dogs experienced a relapse of overall pain, exercise-associated lameness and pain upon limb manipulation. Studies have also shown that small doses
of orally administered undenatured type II chicken
collagen inhibit killer T-cell attack (15). The present
clinical trial evaluated the safety and efficacy of UC-II
in the treatment of the knee in OA patients.
Materials and Methods
Study Design
This clinical trial (Human Clinical Trial Approval #06UOHI) was managed by KGK Synergize
Inc. (London, ON, Canada). The study was conducted
at two sites: 1) KGK Synergize Inc., and 2) Corunna
Medical Research (Corunna, ON, Canada). Figure 1
illustrates the study design while Table 1 lists the
procedures and observations at each time point.
Briefly, at screening (Visit 1) the consent form
was discussed, signed and a complete physical examination was performed. Activity level, diet history,
medication/supplement use and medical history
were recorded. The VAS score, the WOMAC Index
and Lequesne scores were obtained. Urine was collected for a pregnancy test for women of childbearing
potential. A blood sample was taken for determination of uric acid, CBC count and differentiation, albumin, total protein, sodium, potassium, chloride,
BUN, creatinine, ALT, AST, bilirubin, erythrocyte
sedimentation rate (ESR) and rheumatoid factor.
Upon review of blood test results, eligible subjects
were instructed to get an X-ray of the affected knees to
confirm diagnosis. A total of 52 subjects were recruited using the inclusion and exclusion criteria outlined in Table 2. At the first treatment visit (Visit 2),
selected subjects were randomly assigned to receive
UC-II (n = 26) or glucosamine HCl plus chondroitin
sulfate (n = 26, G+C). On each test day (day 0, 30, 60,
90), subjects were required to come to the clinic for
clinical assessment. The clinical assessments included
WOMAC, Lequesne’s functional index and 100-mm
VAS pain scores. A subject treatment diary was completed by each patient throughout the study period to
determine side effects, medication use, and product
compliance.
Visit 2
Visit 3
Visit 4
Visit 5
Randomization
Clinical
assessments as
indicated; first
dose in clinic
Clinical
assessments as
indicated
Clinical
assessments as
indicated
Clinical
assessments
as indicated
0
30
60
Treatment Period (Days)
90
314
Table 1. Schedule of observations and procedures
Procedure
Visit 1
Screening
X
Visit 2
Day 0
Visit 3
Day 30
Visit 4
Day 60
Visit 5
Day 90
Review inclusion/exclusion
X
X
X
X
X
Medical history including activity level and diet history
X
Physical examination
X
Biometric measurements:
Weight, height*, heart rate and blood pressure.
Urine pregnancy test
X
X
X
X
X
X
X
X
X
Informed consent
X
Concomitant medications
X
Blood samples:
Uric acid, CBC count and differentiation, albumin, total
protein, sodium, potassium, chloride, BUN, creatinine,
ALT, AST, bilirubin, ESR, rheumatoid factor
WOMAC, VAS and Lequesne scores
X
X
X-ray
X
Randomization
X
X
X
X
X†
X†
X
X
Blood sample: ALT, AST, bilirubin, albumin.
Knee flexion, Time to walk 50m, Swelling in the knee joint,
Time for climbing 10 steps
Physician's Global Assessment
X
X
X
X
X
X
X
X
Subject's Global Assessment
X
X
X
X
Investigational Product dispensed
X
X
X
Subject Treatment Diary dispensed
X
X
X
Investigational Product returned
Compliance calculated
Subject Treatment Diary returned
X
X
X
X
X
X
Adverse Events
X
X
X
* height was only measured at visit 1
†
If acetaminophen use was greater than 2 g/day for more than 7 days
Table 2. Inclusion and exclusion criteria
Inclusion Criteria
Males and females 40-75 years old
Females of childbearing potential must agree to use a medically approved form of birth control and have a negative urine pregnancy test
result
Unilateral or bilateral OA of the knee for greater than 3 months (American College of Rheumatology criteria) confirmed by radiologist's
report, i.e. X-rays showing osteophytes, joint space narrowing or subchondral bone sclerosis (eburnation)
Erythrocyte sedimentation rate (ESR) < 40 mm/hr
Moderate OA as indicated by Lequesne’s functional index score of 4.5-7.5 after 7 day withdrawal of usual medications
Able to walk
Availability for duration of study period (3-4 months)
Subject using other therapies for OA, such as exercise, heat/cold therapy, joint protection and physiotherapy/occupational therapy agrees
to continue these therapies as normal avoiding changes in frequency or intensity and to record therapies in the study diary
Subject agrees not to start any new therapies for OA during the course of the study
Able to give informed consent
Exclusion Criteria
History of underlying inflammatory arthropathy; septic arthritis; inflammatory joint disease; gout; pseudogout; Paget's disease; joint fracture; acromegaly; fibromyalgia; Wilson's disease; ochronosis; haemochromatosis; heritable arthritic disorder or collagen gene mutations or
rheumatoid arthritis
History of asthma, history of diabetes (Type I or Type II)
Hyperuricemia (urate, males > 480 umol/L, females > 450 umol/L)
Expectation of surgery in the next 4 months
Recent injury in the area affected by OA of the knee, i.e. meniscal tear (past 4 months)
Cartilage reconstruction procedure in the target knee
Severe OA as indicated by Lequesne’s functional index score of 8 or greater, after 7 day withdrawal of usual medications
Intra-articular corticosteroid injections in the target knee within the last 3 months
Viscous injections in the target knee within the last 6 months
Hypersensitivity to NSAIDs
Abnormal liver or kidney function tests (ALT or AST > 2 times the upper limit of normal; elevated creatinine, males > 125 umol/L, females >
110 umol/L)
315
Abnormal findings on complete blood count
History of coagulopathies, history of peptic ulceration and upper GI hemorrhage
Uncontrolled hypertension
History of congestive heart failure, history of allergic reaction to chicken and/or eggs
History of allergic reaction to local anesthetic or to any ingredients in the test product including shellfish
Hyperkalemia (potassium > 6.2 mmol/L)
Anticipated problems with product consumption
History of cancer as well as gastrointestinal, renal, hepatic, cardiovascular, hematological, or neurological disorders
High alcohol intake (>2 standard drinks per day)
Pregnant, breastfeeding or planning to become pregnant during the study
History of psychiatric disorder that may impair the ability of subjects to provide written informed consent
Use of other natural health products, including glucosamine and chondroitin, one month prior to study and during the study, other than
multivitamin and mineral supplements containing vitamins and minerals as the sole medicinal ingredients
Use of concomitant prohibited medication (narcotics, oral NSAIDs, topical NSAIDs) within four weeks of randomization
Use of acetaminophen or ibuprofen within 7 days of randomization
Subject is unwilling to stop taking pain medication other than the study medication (for arthritis or other types of pain) or is unwilling to
stop taking other medications for the treatment of OA
Any other condition that, in the opinion of the investigator, would adversely affect the subject's ability to complete the study or its measures
Supplements
Each UC-II (InterHealth Nutraceuticals, Inc.,
Benicia, CA) capsule contained 20 mg UC-II standardized to 5 mg of bioactive undenatured type II
collagen. Subjects in the UC-II group were instructed
to take two “sugar pills” in the morning to protect
blinding and two UC-II capsules in the evening accounting for a daily dose of 40 mg UC-II containing 10
mg of bioactive undenatured type II collagen.
Each G+C capsule contains 375 mg of
glucosamine HCl (USP Grade) and 300 mg of chondroitin sulfate (USP Grade). The subjects were instructed to take two G+C capsules in the morning and
two in the evening for a daily dose of 1500 mg glucosamine and 1200 mg chondroitin.
Removal of Patients from Therapy or Assessment
The criteria for removal of patients from the
study included:
Adverse events
For any adverse event, patients were examined
and appropriately managed or the patients would be
referred to another medical professional for proper
evaluation and treatment. If medical problems were
attributed to the trial compounds, then the trial drugs
were discontinued and the toxicities were reported.
Personal reasons
As stated in the Consent Form, subjects were
able to withdraw from the study for any reason at any
time.
Clinical judgment of physician
Subjects were withdrawn from the study
(without penalty) if, in the opinion of the treating
physician, it was not in the patient’s best interest to
continue. For instance, if during the course of the
study a patient became pregnant, she would be withdrawn from the study because it was not known how
the study compounds/medications might affect an
unborn child.
Protocol violation
Any subject found to have entered this study in
violation of the protocol or failed to follow the study
protocol were discontinued from the study at the
discretion of the Principal Investigator. Subjects were
withdrawn for protocol non-compliance if they adhered to the dosing schedule less than 75% of the
time.
Method of assigning patients to treatment groups
Patients were assigned to treatment groups (order of treatments) using computer-generated randomization tables. Patients were not stratified or assigned using any other specific method and were not
randomized after stratification or blocking procedures.
Selection of doses in the study
The justification for the daily dose of 40 mg
UC-II in capsules (providing 10 mg of undenatured
collagen II) is based on efficacy demonstrated in earlier studies (8,9).
Blinding
In order to protect blinding, subjects were
given bottles containing product labeled with “AM”
or “PM” to distinguish the time in which treatment
was to be taken. Each bottle contained descriptions of
all potential products to ensure blinding was protected. Additionally, each bottle was labeled with a
randomization number. In the event that an adverse
effect was considered serious and related to the investigational product, the blind would be broken for
that individual subject.
Neither the patient, nor investigator, nor research staff, were aware which test compound the
subject was assigned. Interim analysis was performed
in order to write a preliminary report and thus preliminary unblinding occurred by an individual unrelated to the study conduct. Personnel related to
analysis, statistics, and report writing remained
blinded.
Prior and concomitant therapy
Uses of medications such as narcotics, oral
NSAIDS, topical NSAIDS within four weeks of randomization and during the study, were not allowed.
Treatment compliance
Compliance was assessed by capsule count at
visits 3, 4, and 5 and review of subject diary.
Efficacy and Safety Variables
Efficacy and safety measurements assessed
Adverse events
During the study, subjects recorded adverse
effects in their subject diary. At each visit, the subjects
were asked if they experienced problems or difficulties. Any adverse events were documented and recorded in the study record and was classified according to the description, duration, severity, frequency, and outcome. The investigator assessed the
adverse events and decided causality. Classifications
were as per the Coding Symbol Thesaurus of Adverse
Reaction Terms (COSTART) U.S. Food and Drug
Administration (16).
Blood tests
Blood samples were taken from all subjects
during screening (visit 1) and at end of study (visit 5).
Blood samples (approximately 15 ml) were taken from
subjects at day 30 and day 60 (visits 3 and 4) for the
determination of ALT, AST, bilirubin, and albumin if
the subjects had been taking acetaminophen greater
than 2 g/day for more than 7 days. All blood samples
were analyzed by MDS Laboratory Services (London,
Ontario, Canada).
Appropriateness of Measurements
The efficacy and safety assessments used in this
study were standard for OA and are widely used and
recognized as reliable, accurate, and relevant.
WOMAC scores were determined, at screening,
and baseline, as well as at days 30, 60 and 90 as described in Bellamy et al (17). Other objectives also
performed at days 0, 30, 60 and 90 included determination of Lequesne’s functional index, VAS pain
scores, knee flexion, time to walk 50 m, time to climb
316
10 steps, physician’s and subject’s global assessment.
The Lequesne’s functional index is described in Lequesne et al. (18).
Statistical Methods
Sample size of 25 subjects per group was based
on the subject number used in Braham et al. (1). To
compare UC-II with G+C group, a linear contrast was
included in the analysis of variance. Data missing
subsequent to 30 days were imputed using the
last-observation-carried forward technique. Furthermore, comparisons between the UC-II and G+C
groups were made at each visit using analysis of
variance, using the baseline visit as a covariate. SAS
version 9.1 has been used to perform the statistical
analysis. Probability values less than 0.05 were considered statistically significant for between-group
comparisons.
Results
Baseline Statistics and Compliance of Trial Subjects
Demographic and baseline characteristics of patients are summarized in Table 3. Overall, the patient
profiles with respect to age, sex, height, weight, blood
pressure, heart beat and target knee were similar between both treatment groups. Table 4 shows treatment compliance of the trial patients. There were no
significant interaction terms or between-group differences for compliances. When compliances were
compared at each visit, there were no overall between-group differences among the two treatment
groups.
Table 3. Demographic and baseline characteristics of the
trial subjects
UC-II (N=26)
G + C (N=26)
Age (years)
58.9 ± 9.79
58.7 ± 10.3
Sex: male/female (%)
13/26 (50%)
17/26 (65%)
Height (cm)
167.7 ± 9.90
167.0 ± 8.73
Weight (kg)
84.3 ± 17.4
86.6 ± 21.0
Systolic Blood Pressure
(mm)
Diastolic Blood Pressure
(mm)
Heart Rate (bpm)
128.2 ± 9.36
126.3 ± 12.5
81.9 ± 7.43
79.7 ± 8.60
68.2 ± 7.72
67.4 ± 8.47
Left; n (%)
16 (61.5%)
13 (50%)
Right; n (%)
10 (38.5%)
13 (50%)
Target knee
Where applicable, values are expressed as mean ± SD
317
Table 4. Treatment compliance as assessed during specified visits
UC-II
G+C
Visit 3
[25] 90.5 ± 19.2
[25] 93.6 ± 11.5
Visit 4
[24] 93.2 ± 9.66
[26] 94.5 ± 11.8
compared to 14% in (G+C)-treated groups after 90
days. Within-group analysis indicated that treatment
with UC-II for 90 days significantly (p<0.05) improved WOMAC scores at all treatment time points
measured. In contrast, subjects received G+C did not
show any statistical significant change in WOMAC
scores at Day 90 of treatment (Fig. 2).
Visit 5
[23] 98.5 ± 5.15
[26] 93.3 ± 11.0
VAS Score
Visit 3
[25] 88.1 ± 18.7
[25] 92.5 ± 12.5
Visit 4
[24] 92.8 ± 8.97
[26] 91.6 ± 12.3
Visit 5
[22] 95.3 ± 9.92
[26] 89.7 ± 12.6
Visit
Treatment Group
AM Capsule Compliance
PM Capsule Compliance
There were no significant interaction terms and between-group
differences for compliances. When compliances were compared at
each visit, there were no overall between-group differences among
the five treatment groups. Values are expressed as [n] mean ± SD.
WOMAC Score
The interaction between visit and treatment was
significant in UC-II treated group for "pain walking
on flat surface" (p=0.034), "difficulty walking on flat
surface" (p=0.038) and "performing heavy domestic
duties" (p=0.031) as compared to G+C treated group.
There was evidence that UC-II treatment has a significant effect for “ascending stairs” (p=0.013) as
compared to G+C treatment. Additionally, when
groups were compared at each visit, UC-II was significantly better than G+C for “ascending stairs at 30
days and 60 days” (p=0.019 & 0.040 respectively), “at
night while in bed” (p=0.015) at 60 days and difficulty
walking on flat surface at 90 days (p=0.035). There
were no further statistically significant differences for
any other individual WOMAC components or summary scores. Treatment with UC-II was most effective
and reduced the WOMAC scores by 33%
The interaction between visit and treatment was
non-significant for all VAS components and summary
scores. However there was evidence that UC-II
treatment had a significant effect for “pain during
climbing up and down stairs”, “night pain” and
“resting pain” (p=0.035, 0.030 and 0.024 respectively).
When groups were compared at each visit, UC-II was
significantly better than G+C for “night pain”
(p=0.040) and “resting pain” (p=0.020) at 60 days and
“pain during climbing up and down stairs” (p=0.014)
and “resting pain” at 90 days (p=0.034). There were no
between-group differences for any of the VAS components or summary scores.
Although both the
treatments reduced the VAS score, UC-II was found to
be more effective with a 40% decrease after 90 days of
treatment compared to a 15% decrease in G+C treated
groups.
Within-group analysis indicated that subjects on
UC-II showed a significant reduction in total VAS
scores at Day 60 and Day 90 as compared to baseline.
However, subjects on G+C showed a significant reduction in total VAS scores at Day 30 and no significant difference was observed at either Day 60 or Day
90 as compared to baseline (Fig. 3).
120
Figure 2. Changes in WOMAC scores at Day
90 from baseline. WOMAC scores from each
treatment group were compared to baseline
value at specified time points. Each bar presents
mean ± SEM. *p<0.05, **p<0.005 indicate significantly different from baseline.
Relative WOMAC Scores (% of baseline)
UC-II
100
**
**
*
**
80
G+C
**
60
40
20
0
0
30
60
90
Treatment Duration (days)
318
Relative VAS Scores (% of baseline)
120
UC-II
**
100
G+C
**
80
**
60
40
20
0
0
30
60
90
Figure 3. Changes in VAS score at Day 90 from baseline. VAS scores from each treatment group were compared to
baseline value at specified time points. Each bar presents mean ± SEM. **p<0.05 indicates significantly different from baseline.
Adverse Events
Lequesne Score
Figure 4. Changes in Lequesne’s functional index at Day
90 from baseline. Lequesne’s functional index from each
treatment group was compared to baseline value at
specified time points. Each bar presents mean ± SEM.
*p<0.05 indicates significantly different from baseline.
Relative Lequesne's Index (% of baseline)
The Lequesne’s functional index was used to
determine the effect of different treatments on pain
during daily activities. The interaction between visit
and treatment was non-significant for all Lequesne’s
components and summary scores. Furthermore, there
were no between-group differences for any of the
Lequesne’s components or summary scores. However
there was evidence that visit has a significant effect in
UC-II treated group for “pain while up from sitting”
and “maximum distance walked” (p=0.036 and 0.002
respectively) as compared to G+C treated group.
There was as a strong trend toward UC-II efficacy.
UC-II treatment effectively reduced Lequesne’s functional index score by 20.1% as compared to 5.9 % by
G+C treatment.
Within-group analysis suggested that subjects
on UC-II demonstrated a significant reduction in total
Lequesne’s index of severity score from baseline to
Day 90, whereas no significant difference from
baseline was observed for subjects on G+C at any
treatment time points evaluated (Fig. 4).
Adverse effects that occurred during the 90-day
trial period are summarized in Table 5. Overall, there
were 58 adverse events noted in the subjects receiving
G+C treatment, whereas, only 35 adverse events were
observed in UC-II group. In terms of severity, 60% of
mild and 38% of moderate adverse events were experienced by subjects on G+C in comparison to 43%
and 54% by subjects on UC-II. In relationship to test
product a higher number of subjects (23%) on G+C
demonstrated adverse events possibly related to
product as compared to 11.4% of subjects on UC-II.
For UC-II the possible adverse events related to
products were constipation and headaches (intermittently). For G+C the possible adverse events related to
products were bloating, stomach pain, rash, water
retention (edema around eyes and scars), hives on
face and chest, and headache. However, there was no
significant difference in the occurrence of adverse
effects between the two treatment groups.
120
UC-II
G+C
100
*
80
60
40
20
0
0
30
60
90
Rescue Medication
A greater percentage of subjects used rescue
medication while on G+C as compared to UC-II at
every time point assessed. From baseline to Day 30 a
total of 8 subjects (33.3%) on UC-II used rescue
medication as compared to 23 subjects (88.5%) on
319
G+C. From Day 30 to Day 60, 13 subjects (54.2%) on
UC-II used rescue medication as compared to 21 subjects (80.8%) on G+C. Fourteen subjects (63.6%) on
UC-II used rescue medication as compared to 19 subjects (79.2%) on G+C from Day 60 to Day 90.
Table 5. Summary of analysis of adverse events in all subjects
Treatment Group
UC-II (n=26)
G + C (n=26)
Mild
Moderate
Severe
Relationship to Test Article (n)
15
19
1
35
22
1
Not related
Unlikely
Possible
Probable
Most Probable
Body System (n)
17
14
4
0
0
20
30
8
0
0
Pain
Gastrointestinal
Musculoskeletal/Soft Tissue
Neurology
Pulmonary / Upper Respiratory
Hemorrhage/Bleeding
Blood/Bone Marrow
Dermatology/Skin
Allergy / Immunology
Infection
Lymphatics
Hepatobilary / Pancreatic
Renal / Genitoruinary
Constitutional Symptoms
Syndromes
Auditory/Ear
Ocular / Visual
Metabolic / Laboratory
Total Number of Adverse Events Experienced During Study (n)
Total Number of Subjects Experiencing Adverse Events: n (%)
10
5
7
0
2
2
2
2
0
1
0
0
0
2
1
0
0
1
35
16/26 (61.5%)
17
15
5
2
1
1
1
3
1
3
1
0
0
3
1
1
1
2
58
20/26 (76.9%)
Severity (n)
Discussion
OA is the most common form of arthritis, and it
is often associated with significant disability and an
impaired quality of life. Clinical and radiographic
surveys have found that the prevalence of OA increases with age from 1% in people <30 years to 10%
in those <40 years to more than 50% in individuals
>60 years of age (19). Although there are no curative
therapies currently available for OA, individualized
treatment programs are available to help relieve pain
and stiffness, and to maintain and/or improve functional status.
In the last few years, various nutritional supplements including chondroitin, glucosamine, avo-
cado/soybean unsaponifiables and diacerein have
emerged as new treatment options for osteoarthritis
(20). In this study, the efficacy of UC-II was studied in
patients identified with moderate to severe OA. The
objective of this study was to determine the effect of
UC-II on disease specific measures and blood measures of OA of the knee compared to G+C. It was hypothesized that UC-II would reduce symptoms of OA
of the knee to a greater extent than G+C.
A meta-analysis of 20 randomized control studies (2570 patients) comparing the effects of glucosamine (glucosamine sulphate, GS or glucosamine HCl,
GH) vs. placebo was done. Of these only eight studies
met the required controlled conditions for adequate
allocation concealment and received a quality score of
4 or higher (rated on the JADAD scale). These studies
failed to show the benefit of glucosamine (GS or GH)
for pain and WOMAC function. When all 20 studies
were included in the meta-analysis, the results favored glucosamine with improvement in pain and
functionality; however, the results were not uniformly
positive and the parameters for WOMAC pain, daily
function and stiffness did not reach statistical significance. Combinations of glucosamine and chondroitin
have been studied in the “GAIT” study. These authors
reported that glucosamine HCl and chondroitin sulphate alone or in combination did not reduce pain
significantly in patients with OA of the knee. However in a subgroup of patients with moderate to severe knee pain the combination of compounds were
found to be effective. Limitations to this study included a high rate of response to placebo (60.1%) and
the fact that 78% of the participants were in the mild
pain subgroup (21).
Previous studies have shown that UC-II is effective in the treatment of RA (8-11), and preliminary
human (12) and animal (13-15) trials have shown it to
be effective in treating OA. In obese-arthritic dogs
given 4 mg or 40 mg per day UC-II for 90 days, significant declines in overall pain, pain during limb
manipulation and lameness after physical exertion
were noted (15). Greater improvement was observed
with the 40 mg dose. No adverse effects or significant
changes in serum chemistry (creatinine, blood urea
nitrogen, alanine aminotransferase, and aspartate
aminotransferase) were noted. Following UC-II
withdrawal for a period of 30 days, all dogs experienced a relapse of overall pain, exercise-associated
lameness and pain upon limb manipulation.
In a recent investigation, efficacy of UC-II was
evaluated in arthritic horses (22). In this study, groups
of horses were orally administered with a daily dose
of placebo, UC-II at 320, 480 or 640 mg, or a combination of glucosamine (5.4 g) and chondroitin (1.8 g) for
150 days. Horses receiving placebo did not show any
improvement in arthritic condition, while those receiving a daily dose of 320, 480 or 640 mg of UC-II
exhibited significant reduction in arthritic pain. Although G+C treated group showed significant reduction in pain compared to baseline values, the efficacy
was less as compared to that observed with UC-II
treatment. In fact, UC-II at 480 or 640 mg/day was
found to be more effective than G+C in treatment of
arthritic pain in horses. Clinical conditions (body
weight, body temperature, respiration rate, and pulse
rate), and liver (bilirubin, GGT, and ALP) and kidney
(BUN and creatinine) functions were not affected by
UC-II treatment, suggesting that UC-II is well toler-
320
ated and does not cause any adverse effects (22).
In a preliminary trial of subjects with OA, taking
a single oral daily dose of 40 mg UC-II on an empty
stomach prior to bedtime for 42 consecutive days, an
average of 26% reduction of pain was noted in four of
five subjects in the study. No side effects were associated with treatment (12). The precise biochemical
mechanism involved in UC-II induced pharmacological anti-arthritic effects in humans, dogs or horses
is not clearly established. Type II collagen is the primary form of collagen contained in cartilage. Type II
collagen extracts contain the amino acids found in the
framework of human cartilage. In addition, these
amino acids are required for the synthesis and repair
of connective tissue throughout the body. These
products reportedly aid in reducing the destruction of
collagen
within
the
body,
may
provide
anti-inflammatory activity, and may improve joint
flexibility (8-12).
The current study indicated that both treatments
reduced the WOMAC scores, which measures the
difficulty in physical function, stiffness and pain in
the knee. However, treatment with UC-II was found
to be more effective in reducing the WOMAC scores
by 33% as compared to 14% in G+C treated groups
after 90 days. Similar results were observed for VAS
scores. Although both the treatments reduced the
VAS score, UC-II was found to be more effective with
40% decrease after 90 days of treatment as compared
to 15.4% in G+C treated groups. The Lequesne’s
functional index was used to determine the effect of
different treatments on pain during daily activities.
Treatment with UC-II reduced Lequesne’s functional
index by 20.1% as compared to 5.9 % in G+C treated
groups. Thus, UC-II supplementation showed improvement in daily activities suggesting an improvement in overall quality of life in the patients
receiving UC-II.
Acknowledgement
This research was supported by InterHealth Research Center, CA.
Conflict of Interest
The authors have declared that no conflict of interest exists.
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Epidemiology of osteoarthritis: Zoetermeer survey. Comparison of radiological osteoarthritis in a Dutch population with
that in 10 other populations. Ann Rheum Dis 1989; 48(4):271-80.
Clegg DO, Reda DJ, Harris CL, et al. Glucosamine, Chondroitin Sulfate, and the Two on Combination for Painful Knee Osteoarthritis. N Engl J Med 2006; 354(8): 795-808.
Gupta RC, Bagchi D, Skaggs P, Burke R, Wegford K, Goad JT,
Canerdy TD, Barnett D and Bagchi M. Therapeutic efficacy of
undenatured type-II collagen (UC-II) in comparison to gluco-
321
samine and chondroitin in arthritic horses. J Vet Pharmacol
Ther 2008; in press.
O 1t96, Amcrican College of Rheumatology
4l
CR.ALTYPE II COLLAGE}"{T'R.EATMENT
XN
EARLY RF{EUE,{ATOID
AR.TI.{R.ITIS
A Double-Blind,Piacebo-controlled,
RandornizedTrial
JOACHIM SIEPER,SONJA KARY, HEI-MUT.SOREhISEN,RIEKE ALTEN,
ULRICH EGGENS,
WER.I.{ERHUGE, FALK HIEPE, ANDREA KUFINE, JOACHIM LISTITq, NOR.BERT
ULBRICH,
JURGEN BRAUN, AFIGELA ZINK. and I'{ICHOLAS avniolq MITCHISON
Objective. To investigate the efficacy of oral type
lI collagen in the treatment of early rheunratoid arthritis (R.A).
Methods.l{inety patients with RA (diseasedura.
tion <3 years) were treated for 12 weeks with orar
ggsspg_tX
collagenat L mg/day(n - 30) or L0
mg/day (n = 30) or with placebo (n = 30), in a
double-blind randomized study.
. Results. There was no signifrcant difference behveen the 3 groups in terms of response to treatment.
However, we observed a highen prevalence of responders in the type II collagen-treated groups: 7 responders
in the tr0-mg type II coltragengroup and 6 in the l-rng
BrouF, versus 4 in the placebo group. Furtherrnore, 3
patients in the 10-rng type trtr collagen group and I
patient in the 1-mg type II collagen group,- but reo
patients in the placebo group, had very gooO response.
a total of 14 patients had to be withdrawn frorn the
study: 2 becauseof side effects(nausea)and 12 because
of lack of efficacy.
Conclusion. Only a rninority of patients re-
. Supported by a grant from the Bundesminisreriumfor
rorschungund Technologie.
JoachimSieper,MD: DeutschesRheumaForschungszen_
ItT' Berlin, and Klinikum BenjaminFrankrin,Free univ-rsity,
Berlin, Germany;Sonja Kary, rrr"O,La."a
Krihne, JoachimListrng, PhD, Norbert Ulbrich, phD, Angela
Zink, phD, Ni.holus
I,litchison, phD: Deutsctr.sii,luma Forschungszenrrum,
*::.""
berlln; Helmut Scirensen,
MD: immanuelHospitai,.Beilin; Rieke
Alten, MD: Schlossparkklinif,
g;riin; Ulrich Eggend,MD, Jrirgen
MD: Klinik'm Benjaminpronfuin,Free University,Berlin;
lf:n,
MD.: RheumaktinikBuch, Beriin; Falk Hiipe, MD:
;,:T3f,fil.q.,
n
u m b o t d tU n i v e r s i t yB
, erlin.
repnnt requeststo JoachimSieper,MD, Depan_
_-_. ol
^.{gdr.ess
Medicine,Rheumarorogy.
Krinikum Benjamin Frankrin.
*,tnl
Htndenburgdamm
30, 12200n.rjin, Germany.
Submittedfor publicarionMay ll , ligS;accepted
in revised
t?.
o r m A u g u s t1 6 , 1 9 9 5 .
spondedto treatrnentwith oral type II collagen.These
resultsjustify further effortsto identify which patients
will havea goodresponse
to suchtherapy.
Oral toierance therapy has long been recog_
nized as being able to induce peripheral immune
toleranceto specificantigen(1,2).Low dosesof orally
administeredantigen favor active suppressionof T
cell-mediatedimmuneresponses,whereashigh doses
caninduceperipheraltolerance.Treatmentwith orally
fed antigenshas proved highiy effectivein various
animalmodeisof humanautoimmunediseases,
including collagen-inducedarthritis (3) and acijuvantafthritis
(4), two models that resemble human rheumatoid
arthritis (RA). Type II coilagenhas beenselectedfor
use in oral tolerance trials in RA because of its
restrictedlocation in cartilage(and the eye) and its
abundance
(5,6),Thereis no convincingevidencethat
collagenitself drives the disease(7,g).
A n t i g e n - s p e c i f i cb y s t a n d e rs u p p r e s s i o nh a s
been describedin severalanimalmodersas a mechanism of oral tolerance,where the antigenused is not
responsiblefor the chronic immune responsein the
target organ (4,9). Thus, in the case of RA, T celis
primed to type II collagenin the gut would release
suppressivecytokines after a secondstimulationby
type II coilagenin the inflamedjoint. From animal
experiments,there is evidence that the peripheral
suppressionis mediatedby Th2 cytokinessuch as
interleukin-4 (IL-4), IL-]0, and transforminggrowth
factor F OGFB;, secretedby T cells that ari specifi_
c a l l y a c t i v a t e di n t h e p e y e r ' sp a t c h e so f t h e g u i 1 i o i .
Thus, oral toierancecan be regardedas one approach
to rectifyingimbalancein T cell cytokineIevels.
Although there has been debatein the past
Ll2
S I E P E RE T A T
a b o u t t h e r o i e o f T c e l l s i n t h e p a t h o g e n e soi sf R A
{ . 1 ,11 2 1 ,t h e r e i s n o w i n c r e a s i n ge v i r l e n c eo f t h e i r
i m p o r t a n c es. e v e r a li n v e s t i g a t o rhs a v e b e e n a b l e t o
cietectT cell cytokinesin RA synoviaimembraneby
moleculag
r e n e t i c( i 3 ) o r i m m u n o h i s t o l o g m
i ce t h o d s
( 1 4 ,t 5 ) . F u f t h e r r n o r et ,h e r e i s e v i d e n c et h a t t h e T h l
cytokine interferon-7 (IFl'|7) predominatesin RA
synorzial
membrane(ij), asit doesin animalrnodeisof
other T cell-mediatedautoimmunediseapessuch as
m u i t i p l es c i e r o s i(s1 6 )a n dd i a b e r e m
s e i l i t u s( 1 7 ) .T h u s ,
the concepthasarisenthat Th I cellssecretingmai'iy
IFI'I7 areresponsible
for the chronicirnmuneresponse
againstan unknownself antigen.while Th2 cytokines
such as IL-4 and II-- 10 would inhibit such a immune
response(18). This provides scope for intervention
strategieswhich aim ai a shift from a Thl to a Th2
pattern. In e;iperimentalallergic encephalornyeliiis
(the animairnodeloi inuitiplescierosis),
in non-obese
diabeticmice (theanimalmoderof diabetesmellitus),
and in coliagen-induced
arthritis (an animal model of
RA), a suppressive
effectof Th2 clones(10),iL-4 (19),
and the II--4 gene(20)has beendemonstrated.
In rhis
context,orai toierancewould work via locai production ("bystandersuppression")
of Th2-typecytokines,
which are beiieved to dovrn-regulatethe damaging
effectof arthritogenicThi cytokines.
Recently,the first clinical trial of oral tiupeII
collagenin the treatmentof RA demonstrated
a trend
toward improvementin the type iI collagen-treated
group comparedwith the placebo group (ZI),The
designof the presentstudy ha,cthreeimportant,lifferencesfrom this earlier study: l) only patientswith
early R.4.(diseaseduration s3 years) were i'ciuded;
2) 2 differentdosesof rypeII collagen(1 mg and l0 rng)
werecomparedwith placebo;and 3) insteadof chicken
type II collagenwe used bovine type II collagen,
which hasa higherhomologyto humantype II coila_
gen. In this study we found a siightlyhigherresponse
rateamongtype II collagen-treated
patientscompared
w i t h t h o s ew h o r e c e i v e dp l a c e t r oe, s p e c i a i l iyn t h e l 0
mg group,althoughthis ciifference
was not sienificanr.
PAT{ENTS AND I\,fETF{ODS
Fatient nec:'uitrnentand characteristics.patients were
recruited at 5 rheumatology ciinics in Berlin.
Incrusion
c r i t e r i a w e r e a s f o l r o w s : r ) i d i a g n o s i so f R A a c c o r d i n g
to
t h e 1 9 8 7c r i r e r i a o f t h e A m e r i c a n c o l l e g e o f R h e u m a t o l , c g y
formerly, the' American Rheumatism Associatio-n)
i19R,.
( 2 2 ) ; 2 ) d i s e a s ed u r a t i o n - 3 y e a r s ; 3 ) n o t r e a t m e n t
with
d i s e a s e - m o r l i f y i n ga n r i r h e u m a t i c d r L r g s ( D M A R D s )
in the
p r e v i o u s 2 w e e k . s ;4 ) c l i n i c a l l y a c t i v E R A w i t h
ai Ieast +
swoilen and tenderjoints; 5) tretrrmentwith a seconcr-rine
d r u g a c c o r d i n gt o n e e d a s s e , s s e bd y t h e p h y s i c i a n ;
6) pred
nisolone dosage -7.5 mg/day rlunng ttre iriat and for
the t.
days before the trial, ancl no intiaarticurar injecrions
o
c o r t i c o s t e r o i d sd u r i n g t h e t r i a r ; 7 ) , J i s e a s eo n s e t b L t r , i , e e n
tht
a g e so f l 6 a n d 6 5 ; 8 ) p a t i e n t ' sw r i t t e n c o n s e n t .
P a t i e n t sw e r e e x c r u d e d t i o m t h e s t u d y i f t h e y h a c
myocardial insufficiency, renai insufficiency (ierum creari.
nine >2.0 mg/dl), disrurbance of liver function (alkaiine
phosphatase >300 unitsr/iiter, serum giutarnic oxaioacetic
transaminase[sGOTJ >50 units/liter, or biiirubin > 1.5
mg,,
d l ) , . m a l i g n a n c y ,o r a c o n s i d e r a b t yr e d u c e d g e n e r a l s t a t e
Ji
h e a l t h a s d e t e r m i n e db y r h e p h y s i c i a n .
._Designand duration of the stuciy. The study was a
controlled, randomized, double-blind, phase II trial that
included 3 groups_c.rf
patients: 2 dift-erent dorug. regimens of
orai bovine type II collagen (l mg and I0 *!; *... testeci
against piacebo.The pianned study size was i0 patient, p.i
study arm. study duration was 12 weeks. The study protocol
was approved by the ethicai committee of the Klinikum
Benjamin Franklin of rhe Freie Universitdt Berlin.
concornitant medication" Al1 patients were treated
with nonsreroidal antiinflammatoiy drugs (NSAIDs)
t h r o u g h o u t t h e t n a l . A n y c h a n g e i n d o s a g ei r p r e p a r a t i o n
was recorded.
clinical and raboratory assessrnents.All patients weie
e x a m i n e da r 0 , 4 , 8 . a n d i 2 w e e k sa f t e r t h e s t a r . ot f t r e a r m e n r .
The following ciinical cliseasevariables were assessed
\23,24): number of painfui joints (29-joint count), number of
j o i n t s ( 2 8 1 o i n tc o u n t ) , p a t i e n t ' s g i o b a l
sw.ollen
aiiessmeni of
pain
.on a iO-pointnumericar rating ..ur., the Funktionsfragebogen Hannover (FFbH; quesiionnaire for measunng
functional disability (zs), patient's globai urr.rr-.ni"o?
d i s e a s ea c t i v i t y o n a l O - p o i n tr a t i n g s . i i e , p h y s i c i a n ' sg l o b a l
assessmeno
t f c h a n g e i n d i s e a s ea c r i v i t y a t t h e e n d
of the
treatment (on a
scale), and erythrocyte sedi_
l-ggint__r_ating
mentation rate (ESR). we decided ro use the FFbH questionnaire for measuring physical function becaui. it
i. tn*
best-validated
i n s t r u m e n tf o r R A p a t i e n t s l i v i n g i n G e r m a n y .
The applied version consists of r2 questioris concerning
activities of daiiy living. patienrs answer on a 3-point
scale:
: y.r, 2 : yesbut with difficulty, :
3_ no or only with help.
!
B a s e do n t h e s er e s p o n s e s a, s c o r e o f 0 ( n o f u n c t i o n ) t o
r\avo
(unimpaired function) is generated.
The Ritchie'arricuiar
index (RAI) (26), duration of morning stiffness,
r.,tp strength
(mean value of 3 measurementswith
a Martin v"igorimeter),
rheumatoid factor (RF), and c-reacrive protein
rinpl levels
were also documented. Radiographs obiained at study entry
w e r e r e v i e w e d r e t r o s p e c t i v e l yb y o n e e ; < a m i n e r .
RF was dererminedby quantitative nephelometric
m e a s u r e m e n ro f l a t e x a g g i u t i n a t i o n( N L a r e x I i F ; B e h r i n g
AC, Marburg, Germany). A levei >40 units *u, ,rgoided
as
p o s i t i v e .C R P w a s d e r e r m i n e r bi y q u a n t i t a t i v e n e p h E l o m e t r i c
m e a s u r e m e n (t N L a t e x C R p r e a g e n t s ;B e h r i n g A G ) . A v a l u e
> 6 m g / m l w a s c o n s i d e r e dp o s i t i v e . H L A c r a s i r y p i n g
u
was
performed by sequence-specifip
c rimed potymrrase chain
reaction(PCR) (27) and consecutives.qrbn.ing-based typi n g a f l e r g r o u p - s p e c i f i cP C R a m p l i f i c a r i o n ( 2 8 ) . "
T o m o n i t o r f o r p o s s i b r es i d c e f f e c r s , t h e f o i r o w i n g
v a r i a b l e sw e r e i n v e s r i g a r e da t e a c h v i s i t : s u b j e c t i v e c o n c l i l i o n , c l i n i c a l s t a t u s ( m e a s u r e m e n to f , w e i g h t L n c i t e m p e r a *
[ u r e , a u s c u l t a t i o no f h e a r t a n d l u n g , a b d o m i n a l p a l p a t i o n
for
tenderncss andlor resistzince,arterial blood pi.oiu.. *-^_
TVFE XI COLLAGEI{ TREATMENT OF EAR.LY RA
Table l.
43
of the 90 rheumatoidarthritispatients,by reatmentgroup
Baseiinecharacteristics
Characteristic*
Age, mean + SD years
No. female/no.male
Diseaseduration,meant SD months
Functionalstatus,no.f
ClassI
ClassII
ClassIII
ClassIV
Elevationof ESR, yes/no
Elevationof CRP,yes/nof
RF positive/negative*
RA-associated
HLA subtype,yes/not
Prednisolone
treatmenl,yes/no$
PreviousDMARD treatment,no.
None
Gold salts
Auranofin
Methotrexate
Hydroxychioroquine
Sulfasaiazine
Placebo
group
(n = 30)
l - m g t y p eI I
collagengroup
(n =30)
l0-mg type ii
collagengroup
(n = 30)
5? -F l?
5?t8
20/10
20.2t 12.0
lq r- 11
7515
1 0 . 3t 7 . 8
)
1
I J
tz
I
t5lt5
16il4
t4il6
16t14
8t22
:
?zl8
l? ? +
)
18
l0
0
2218
20t10
t7113
t9t1l
7t23
It
Q
_1
l6
9
o
t3/17
13fi7
t6/14
16114
12t18
20
2
I
2
22
A
T
I
3
;
J
o ESR : erythrocytesedimentationrate; CRP : C-reactiveprotein;
RA = rheumaroidarthritis;
DMARD = disease-modifying
antirheumaticdrug.
t Accorciingto the revisedcriteria of the American Collegeof Rheumatologl,(ref. 53),
* As definedin Patientsand Methods.
$ Up to 7.5 mg.
surement,and examinationof the skin),completeblood cell
count includingthrombocytes,levelsof SGOT, serumglutamic pyruvic transaminase,alkalinephosphatase,gamma
glutamyl transferase,and creatinine,and urinalysis.
Cniteria for response.According to the definition
proposed by the .ACR (29), improvementin RA in clinical
trials requires a =20vo improvementin both tender joint
count and swollen joint count and an additional >Z\Vo
improvement in at least 3 of the following 5 parameters:
patient global assessmentof diseaseactivity, patient pain
assessment,
patientself-assessed
disabitity,physicianglobal
assessmentof diseaseactivity, and acute-phasereactant
(ESR or CRP). For the FFbH, a chaneeof >11% has been
found to be significantby test-retestcJmparison(25),so this
cutoff was used to designatea >?\Vo improveme.rnt,
and a
changeof >22% to designatea >40Voimprovement.The
percentage change for the variables tender and swollen
joints, ESR, and FFbH refersto the differencebetweenthe
value at the end of study and the value at entry. patient
assessments
of diseaseactivity and of pain were measured
on a l0-point scale.A differenceof I point correspondedto
10%.A modificationwas madewith regardto the physician's
assessment
of diseaseactivity.At the end of the study the
physician assessedthe change in diseaseactivity on a
(1 = much improved,2 : improved,3 = un5-point-scale
changed,4 = deteriorated,5 = much deteriorated).The value
of 2 (improved)correspondedto an improvementof Z}Vo,
and the value 1 (muchimproved)to an improvementof 40%.
Preparationand handling of bovine type II collagen.
Isolationof bovine type II collagenfrom the nasalseptumof
cows was carried out according to describedmethods
(30,31).The isolatedtype II coilagenwas lyophilizedand
storedat -20"C. For the trial, type iI collagenwas dissolved
in 05M aceticacidto final concentrations
of 0.2 mg type II
coilagen/ml05M acetic acid or 2 mg type II coilagen/ml
05M aceticacid. Vials containingeither5 mi of 0.2 mg rype
iI collagenlml0.5Maceticacid (1 mg rype II collagengroup),
5 ml of 2 mg type II collagenlml0.5Macericacid(10mg type
Table 2. Side effects,by treatmentgroup
Table3, Number(%)dropouts,
group
by treatment
Placebo
group
(n = 30)
Pruritus
Exanthema
Gasrointestinalsymptoms
Headache
2
a
:
.+
U
l - m g t y p ei l
coiiagen
group
(p = 30)
2
0
3
a
J
A
0
'-1"1,I*o,ltt
'Ti,if;
Pracebo
"
l O - m gt y p e I I
collagen
group
(n = 30)
1
group
(n : 30)
group
(n = 30)
group
tn : 30)
AII dropouts
Dropoutsdue to disease
6 (20)
5 ,l7)
4 (tj)
4 (j3)
4 (13)
3 (r0)
worsenlng
Dropouts due ro side effects
1 (3)
0 (0)
I (3)
A,l
-t*t
SIEFER.ET A
Tabie 4.
Number
ment group
(%) responders and nonresponders,
by treat-
o f w h o m6 7 ( 7 4 . 4 %w
) e r ew o m e na n d 2 3 ( ? 5 . 6 % )
we,
men. The mean age was 5{ years (range
19-6g
I - m g r y p eI I i0-rngtype iI
Serzenrypatients(78%)had noi been'tr.ut.O
Placebo
prev
collagen
collagen
o
usly with any DMARDs. Only 2 parienrs
grouP
group
group
i
n rt
otttnnmo
,
placebogrouphadreceivedDMARD
(n : 30)
(p = 30)
ti.utmenrbefor
the beginningof the study, in compa.iroo
Improvement
4(ri)
with I
6 (20)
I tlJ)
I m p r o v e m e n t -40Vo*l
patients
in
0 (0)
l-rng type II collagengroup and
I (3)
3 (10)
!h.
U n c h a n g e d o r worsenlng 26(87)
(80)
patientsin rhe r0-mg type II colrag"en
23 (77)
gioup Radic
* According
graphs
from the time of study .nt.y, uiuilubre
to the Arnericancoilegeof Rheumatorogy
for 6
29).
patients'were examinedretrospectivery
".irrJt.J
by one ob
f Includesthosewhoseconditionsimproved -_20Vo.
by
server.Twenty-three
patientsexhibiteoerosionsand
patientshadjoint spacenariowingas
radiorogicsign
of cartiiagedesrruction.Tabre I iummarizes
the pa
I I c o l l a g e ng r o u p ) , o r 5 m l 0 . 5 1 4a c e t i c
a c i d ( p i a c e b og r o u p )
tients'characteristics
at thestartof the trial. The mear
were distibuted once a month to
the_p_atients,-*iro kept
durationof disease,the numberof patienis
them in their home refrigerators...at
with ere
6_.b. E".iV morning
vated ESR.or CRp, the numb*, oi RF_positive
patients swalloweci this so-iution
diiuted i"
gr^ri of water
pa
*,1),rogetherwith a rnuitivit^*i;-p"..p;rarion.ro
tients, and the nurnberof patients iruuing
"
I*
1,m
un
Rd,
lmprove the taste.
associated
FILA classII subtype(DRBt*OiO1,0401
statisticai anaiysis. Ail patients who lvere
enroiled in
04a4,0405,
and0408[33])werenonsignificantry
the study were inciudeci in the
higher
evaruation. Before unbiinclin the 1-mgtype II collagengroup comparecj
ing, the following specifications of trre
with the
siuuv i.oto.or were
determined: the response criteria proposed
by the ACR (29)
l?-*e type II collageng.oup and the placebogroup.
would be usedfbr cssessmenr
The groups were statisticaliysirnilar ln their
oi.mj".y;1"lrr'p^ri"nt who
demo_
fuifiiledthe criteriawourdbe counceaas
graphicand ciinicaicharacteristics.
a responder;and alr
parienrs.
glh:r
incrudingthose who did nof
Side eft'ects
and widrdrawars.side effects were
trial, would be counted as nonresponders.;;;prere trie
The response
mild and were equallydistributedarnong
rates were comparedby Fisher'sr-faired
the groups
exactiest (signifi(Table2). only z patientshad to be *itf,drawn
cance^lever
5%).If the responserate was comparablewith
from
that of methotrexateor gord,the testwouid
the study becauseof a side effect
have a power of
in
lnaus"a
both
at least B0%(29).
cases),and 12 patientswere withdra-wn
due
to
rack
Additionaily, mean changesbetween baseline
of efficacy(Table3). Both rhe i_mg and
and
the end of the rriar were a.u.nu.E for
rhe
l0_mg
the i;lr";;;;
efficacy
coilagen treatment groups had teier
parameters:tend.erjoint count,
*iiho.a*ats
swollenjoint cou"nt,RAI,
E S R, pain a n d d i s e i s ea c ti v i ry ' a s s e s s m ent
d u e t o d i s e a s ew o r s e n i n g( 4 p a t i e n t s
by the pati ents,
and 3 pa_
functionatcapacity, morninj's;ff.;;;;;";;j;;
tients,respectively)
comparedwiih ttreptacebo
Jtrengrn.
$oup
Analysis of covariance(ANbovA) *^,
(5 parients).
v
u LLL,'
lc;
(n
=
3())
>1OO/^4
1d
LA
tit
u..i-i! .o*pu."
theseparameters,
in orderto detecfany differencesbetween
groups. Individual differences(baseiine
value minus our_
come) were adjusredfor their srartingpoint
;".";il;g ro the
modelequationof ANCovA, and these
adjusteddifferences
wer e t hen e v a i u a te dw i th tw o r-ra i re d
,.rr..
T he M ann-w h i tn e yte s t a n c lth e m u l ti"v" ;t;
i . ri en
a ri;;;Ji
ateo,B
test
( 32)w. er eap p l i e d In
. a i l te s ts ,F v a ru e s l e ssthan 0.05
w ere
c ons ider ed
si g n i fi c a n t.
T he s ta ti s ti c aar n a ry s i si n c ru d e da i l 90 pati enrs.
An
analysisof onrv ,hgi." who compreted
the m"f ,"igirr have
bias edr he r e s u rtsw
. e te s re da s i mp rem.tr,oJ oi r?pi a.i ng
m is s ingv alu e s(s u b s ti tu ti nrh
g e Ia s iv a fi A v a l ue)
and a more
c om plexone (e x tra p o l a ti obny re g re s s i o n ).
S
i
nce
i te resutts
wer e t he s ame ,th e ta b re sp ..r.n i
o n l y th e f i rst set of data.
No r eplac em e nwt a s m a d ei n th e c a s e
o f th e FFbH because
t he F F bH wa sa n s w e re do n rytw i c e ,
s o a m issi ngl ast vai ue
c o u l dn o t b e e s t i m a t e d .
R,ESUT,T5
I
I
Fatient characteristlcs at study entry.
Ninety
pat ient s wer e e n ro l l e c li n th e
s tu d y (:O i n .^.f, group),
R'esponders.
Eighteenof the 90 patients had
.
lmprovementaccordingto the ACR criteria.
one of
the piacebogrouppatientswas not treated
with prednisoloneat stuclyentry, but was treatedr.vith
7.5 mg
prednisolone
dairyfrom week 4 untii week r2 during
the trial. This patientwas not countedas
a responder
althoughthe improvement
criteriaas describedabove
werefulfilled.
There was no significantciifi-erence
in response
rates among t!* 3 groups,aithough more
patients
fulfillingthe ACR criteriafor zTvoimprovem.l,
*.r.
found in the 2 type II coilagengroups than
in the
p i a c e b og r o u p : 4 p a t i e n t si n t n e p l a l e b o
group, 6
patientsin the l-mg rype II colla[en g.orJ,
unO 7
patientsin the IO-mgtypeIi coiiagengroup
i-esponded
( T a b l e4 ) . F u r t h e r m o r e3,p a r i e n t i i nt t , .
i b - m g i y p eI I
"typ"
collagengroup and I patient in the 1_rng
Il
collagengroup showecla very good r.*rponi",
either
with an improvementof >40vo by the
ACR critena
TYPE Itr COLLAGEI-{TREATMEhITOF' EARLY RA
( p a t i e n t s4 , 6 , a n d 1 2 ;T a b l e5 ) o r w i t h n o s w o l l e no r
tenderjointsat the end of treatment(patient2), but no
patient in the placebogroup respondedin this way.
The responderpatientsare described
in moredetailin
Table 5. No remissionas definedby the ACR criteria
for complete remission(34) occurred in any of the
groups. In most casesthe improvementstartedafter4
weeks, and usually showed a steady improvement
over the l2-week treatmentperiod(datanot shown).
In comparisonsof the singlevariables,there were no
significantdifferencesbetweenthe groups(Table6).
DISCUSSION
Statistically significant differenceswere not
found in this study comparingtreatmentwith l0 mg
type II collagen/day,I mg type II collagery'day,
and
placebo, in terms of either the ACR criteria for improverflentin RA (29)or the meandifferences
in single
variables.However, we found thatRA patientstreated
with type trl coilagentendedto showbenefit.This was
especiallytrue with the higherdoseof type Ii collagen
(10 mg). In the 10-mg treatmentgroup, 7 patients
showed at leasta Z0%improvement,comparedwith 5
patientsin the l-mg group and4 in the placebogroup.
The rate of withdrawalsdue to diseasedeterioration
was greatest in the placebo group. It is especially
worth mentioningthat 4 patientsin the type II coilagentreated group, but no patient in the placebo group,
showed a very good response:Z patientsin the 10-mg
collagen group and 1 in the l-mg collagengroup had
rmprovementof at least 40V0,and anotherpatient in
the 10-mggroup had no tenderor swollenjoints at the
end of the study. This is similar ro the finding of 4
remissionsin the type II collagen-treatedgroup studied by Trentham et al (21).
Our results appear to be in accordancewith
those of the previous publishedstudy on treatment
with oral type II collagenin RA (21),despitea different
study design. We did not find a significantdifference
for any of the single variables,and the differences
describedin Trentham'sstudy were aiso unimpressive. The salientfeatureof both studiesseemsto be
the good response,with remissionor near-remission,
in a minority of patients.lt does not seemto make
much differencewhether chickenor bovjne type II
collagenis usedor whetherearlyor lateRA is treated,
althoughno dataon the durationof disease
amongthe
patients with remissionwere given in Trentham.s
report. Our findingsindicatethat the higherdosage
of
llp. II collagen,i.e., l0 mglday,may be superiorto
the lower dosage,.Ftrowevei,
the differencesbetween
45
groups were too smallto allow clearconclusions.lt
shouldbe noted that the patientsin the 1-mgtype II
collagengroup had slightlymore severediseaseat
studyentry,asjudgedfrom the clinicaldata(Tablel).
In most cases,there was a continuousimprovement
o v e r t h e 1 2w e e k sa m o n gt h e r e s p o n d e r sT.h i s c o u l d
be taken as evidencethat a longertreatmentperiod
might be more favorable.
In termsof futurestudies,it is notablethat we
found no severe side effects among the collagentreatedpatients,and thatthosethat werefound did not
differ amongthe groups.Furtherrnore,the number of
dropouts was smallerin the type II collagengroups
than in the placebogroup,makingit unlikelythat this
treatmentworsens the disease,althoughthis is a
theoreticalpossibilitysinceabsorptionof immunogens
in the gut is possible(35-37).
If only a minority of patientsrespondto treatment, it becomesdifficultto demonstratea significant
effect. Our findingsposethe questjonof why only a
small numberof patientsrespond,and whetherthese
potentialresponderscouidbe identifiedprior to treatment. No dift"erences
in RF positivity,HLA classII
antigens,or. other variableswere detectedbetween
respondersand nonresponders,
so it is doubtful that
thesefactorsplay a majorrole.
It may be that some patientshandle type II
coliagenin the gut differently.A numberof possibiiities are worth considering
in this context.The amount
of oral antigenand the natureof the fragmentsgenerated seem to be crucial for the induction of oral
toierance.Digestionof the antigenin the gut is essential for the generationof oral tolerance(37,38), and
interferencewith gastrointestinal
proteolysisby neutralizing gastricpH might inhibit the induction of oral
tolerance(35).The digestionof sucha complexmolecule as type II collagenmight differ considerably
betweenindividuals,andit is not yet clearwhere and
how type II collagenis digested
in the gastrointestinal
tract. This problem might be circumventedin the
future if immunodominanttype II collagenpeptides
can be identifiedand fed or inhaled(39), insteadof
administering
the wholeprotein.
Furthermore,it hasbeensuggested
that induction of oral tolerancemightbe depencient
on an intact
mucosa,sincea damagedmucosaallowsthe passage
of immunogenicmacromolecules
(35,37).This could
preventoral tolerancein RA, sinceneariyail patients
are treated with NSAIDs. which have a known
mucosa-damaging
effect(40,,4i).
Anotherpossibiiityis
that a changeof the bacterialgut flora,for exampieby
drugs (42,43)or diet (44), could also influencethe
46
SIEFER.ET A
Tebte 5.
clinical and laboratory parameters
of effcacy and patienr characteristics
in responders*
Patient
no,/sex
Tender Swollen
joints joints
l0 mg type II
collagengroup
Parientl/F
Basefine
Studyencl
l5
9
Difference
Diference in %
Patient 2/F
Baseline
Studyend
Difference
Differencein Zo
Patient3/F
Baseline
Study end
Difi'erence
Differencein %
Fatient4/F
Baseline
Studyend
Difi'erence
Differencein %
Patienr5/F
Baseiine
Studyend
Difference
Dift'erencein Zo
Padent5/F
Baseline
Studyend
Difference
Differencein %
Patient7/F
Baseline
Srudyend
Difference
Dift"erencein Vo
.
l mg rype II
'- . coilagengroup
l,atrentglF
Baseline
Studyend
Difference
_ Differencein o/o
Parient9/Jvf
Baseline
Study end
1
8
2
4
6.0
40.0
4.0
33.3
l'f
L I
1
o
0
ESR.
o
4
3
8
5.0
i0.4
2
1
8
2
L
17
1
I
3.0
30.0
6
)
4
2
12.0
85.7
94.1
16
5
It.0
6B.B
8
tL' 7t
6
11.0
39.3
5.0
50.0
15
9
6.0
40.0
r
0
6
4.0'
4 0 .0
rc
5
lb
ta
] L
4.0
t(n
l0
A
32
2
8.3
<20
IU
25.A
oo./
t
I
A 1
66.7
fi.3
16.7
>20
6
t
4
4
3
2
2 . 0 _ 2 . 0 + 2.0
3 3 .l _20.0t 20.0
100.0
tm.0
o
<20
83
74
',|
50.0
66.7
4.0
40.0
16.7
9.0
10,8
o
o
8
+
4.0
40.0
4I
1
T
6
7
a
'l
>)/l
0101
0401
1
B
2
6.0
60.0
4.0
40.0
-
16
t
a
n
0
fl.O
100.0
Difference
Differerrcein Zo
2.0
66.7
7.0
5g.3
1
-
1
L
f
-
3
0i 0 t
0l0l
3
I
<20
2A
I5
25
)
2
3.0
30.0
6
2
4.0
40.0
50.0
70.8
20.8
>20
2
o
I
8,0
88.9
N-one
No erosions
None
No erosions
\rone
IrIo erosions
hlone
t1
Erosionsin
hands
None
20
30.0
-t n+
*4.0t
No erosions
R7(
3.0
3 0.0
o
None
20
34.0
82.9
7.0
46.7
No erosions
40
6.0
60,0
1 n
l/{1-\/
40
0401
Difference
_ Diference in To
PatienrI0/F
Baseline
Studyend
Difference
^ Differencein %
Patientl1lN4
Baseline
Studyend
No erosions
75.0
1
z
MTX
(25days)
20
75.0
9i.8
20.8
>20
6
Erosionsin
(5 months)
<20
4
I
3.0
30.0
20
2
0
8
10
o
8
r0 .0
20.0
0
5 0 .0 lm.0
0
36
, t a
40
0
1
0
4
2
t
2
I
-2.0i
8.0
3.0
80.0 -20.0t
30.0
0401
o l "
>40
I
'
95.8
5
2
i.u
30.0
I
,
20
5
2
3.0
30.0
z
"
a = - . |
feet
3.0
30.0
5
r
roo.o
5
2
4.0
40.0
4
')-l
-)
r6.0
Iz
.5
2.0
20.0
3.0 -3.0+ 3.0
37.5 - 12.si 30.0
2 .0
20.0
9.0
64.3
3
0
0
5.0
33.3
r\
3
I
5
1
t7.0
12.0
1c0.0 1 0 0 . 0
l0
Pain
Previous
DlvLARD
(washout
phase)
HLA
Disease
Physician
ciassU duration, Radiologic
as.sessrnent
RF DRBI months
findings
Disease
activiry FFbH
/
2?
t7
5
7
5
79.2
9t.7
5.0
22.7
0
0
) n
20.0
t2.5
>1n
No erosions
1
_
0101
12
ALI"R
(10.5monrhs)
No erosions
None
No erosions
SSZ
(lB days)
20
u.0l
20
TYPE II COLLAGEN TREATh4ENT OF' EARLY RA
47
Table 5. (Cont'd)
Patient
no./sex
Patient 12lF
Baseline
Study end
Difference
Difference in 7a
Patient 13/F
Baseline
Study end
Difference
Dfference in 7o
Placebo goup
Patient t4lF
Baseline
Study end
Tender Swollen
Disease
joints
joints ESR Pain activity FFbH
28
2
26.0
q?q
16
30
0 1 0
r6 .0 2 0 .0
100.0 ffi.7
6
2
4.0
40.0
2l
6
15.0
71.4
t2
36
)
2 1 8
10.0 18.0 2.0
8 3 .3 5 0 .0 20.0
l3
0
t2
6
4
1
3.0
30.0
)
a
1
11
7
^
/
3,0
30,0
3
41.7
50.0
Difference
Dift'erencein Zo
Patient l6lF
Base[ne
Study end
11 . 0
64.7
6.0
60.0
Difference
Difference in Va
Patient 17lF
Baseline
Study end
Diference
Difference in Vo
3.0
50.0
L0
75.0
8.0 3.0
18.2 30.0
3.0
30.0
8
6
2.0
25.0
12
6
6.0
50.0
22
5
6
3
16.0 7.0
72J 20.0
)
7,0
20.0
6
3
A
a
t
3
2
0
3
>?o
4
4
r
0
62.5
79,2
16.7
8.3
<20
17
6
4
40
3 .0
30.0
13.0
100.0
6 .0 4 .0 3 .0
50.0 36.4 30.0
1
12.5
>)n
9t.7
r00
Difference
Differencein 7o
Patient 15F
Baseline
Study end
8
6
7.0 7.0
23.3 ?0.0
0
0
8,3
<20
44
36
6
3
79.2
91.7
)
a
Disease
duration, Radiologic
months
findines
Previous
DMARD
(washout
phase)
87.5
r00.0
3
0
0
Physician
assessmentRF
HLA
ciassiI
DRBI
-
0
I
_
1
0
1
0401
7
No erosions
l0
No erosions
None
+U
0401
+
0 1 0 1 , 0 4 0 1 26
t2.5
No x-ray
avaiiable
None
No x-ray
avaiiable
None
No x-ray
availabie
SSZ
(7 months)
No erosions
None
>?n
87.s
95.8
8.3
<20
2
-
0401
l0
20
* Tender and swollenjoint
valuesare from a z8-joint count. Pain and diseaseactivity were rated on a lGpoint scale.physrcianassessmerrwas
s:poin, .:ale. El_R.= eryrhro€)'tesjdimqrtation iare {mn/hour); Ffbd = funktionsiraepb6i"-i'i""""i,lii
q*rtionrBire (.esulrs
-,t::.-:l:
cxprcssed
as percenrage
patienrs
of full funcrionalcapacity;differeoce
in % asdefinedin
andUettrois);-i.F
- = *iu*uioio fa"to.; Ofr anO =
discase-modifyingantilheumaricdrug; MT)l = meihotrexate;AUR = auranofiq;SSZ = sulfasalazini.'
t HLA subtypenot associatcd
with ilA.
* Negative difference indicatesdeterioration.
effect of oral tolerance.It has been shown that oralry
administered lipopolysaccharide,a major component
of bacterial ceil walls, enhancesthe induction of oral
tolerance (45), and the gut flora of untreated RA
patients is significantlydifferentfrom that of non-RA
controls (a6). Finaily, treatmentof anirnalswith IFNT
abrogatesoral tolerance (47). This could be taken as
evidencethat a strongThl responsecounteractsthe
asSumedThz responseinduced by oral tolerance.
various influencescan induceor increasea concomitant Thl response,€.g., viral and bacterialinfections
or a hormonal imbalance,with glucocorticoidsfavoring a Th2 responseand dehydroepiandrosterone
sul-
fate and its derivativedehydroepiandrosterone
favoring a Th1 response(48).
An additionalfactor as tc why only a minority
of patientsrespondedcould be our patientselection.
we choseto treatonly RA patientswith earlyarthritis
(duration<3 years).SinceRA normallyhasa chronic
coursewith eventualdestructionof the joint, a treatment that is startedearly in the diseasehas a better
chanceof cure and/orof preventingirreversiblejoint
damage.Furthermore,anirnal experimentsindicate
that diseaseis best preventedif antigenfeedingstarts
before inductionof diseaseor early in disease.However, the main disadvantage
of concentratingon early
4B
SIEPER ET AL
Table 6.
D i s e a s ev a r i a b l e s i n t h e 2 r y p e I I c o i l a g e n g r o u p s
and the pracebo group*
Variable, group
Tenderjoints (28-joinrcounD
i'.gi;
"*."
s*ott.n-ioint.-fi!-ioin,
[0 mq co]lapen
iq,^ll-*
:^j_"_-74o
Placebo
count;
I mgcoltagen
ror"-gi.rr-ig"r
e1i 1,i""',il,1i?i'
Difference from entry at weeki
fvlean -F SD
vaiue at entry
ii,ii!,i
1o'r= )'v
t6-3 :
5.9
l1'3 = 5'0
12'6! 5'0
L2'5
= 5'0
Placebo
1mgcouasen
6
23
= 4.8
1'4i 4'0 1'7= a.!
24 - 4.1
g.l
4)
+ 1 \
1.9. 3.9
z.z-:.t
,.1;;'.e
.i: * 11|
:i.gi1ti
(10-poin!raringscliej
,,ilt?:;i:i1,,'.",
-,
s, + r c
ii;;i
(1o-pointraringscale)
1^ms
corasen
tr.l:ii
;:0=;'6
'"i'1#t.?:x*:".,
disab'iry
(FF,bH)E
Placebo
I mgcoiraseD
couagen
-,lomg
fhysicianassessrDent
of chansein
. diserseactivity (s-point s;ale)1]
.
,
,.
Aoo[ronal variables
Ritchiea icular iDdex
Placebo
l^mg couagen
=1,:ri::[i#:",
,..,
crip str€ngrhlkpa), right hand
Placebo
I mg co ageo
i':
.::1I'7'2 ! :^
62'3
1.0a 5.1
3.2= 4.9
3.3t 12.6
3.5r r3.5
i.i = t.s
l!:3;?l.l
2.i= 7.0
il i ?Sj
J . 0! 2 . 0
0.9 : 1.9
1.g.11
1.0t r.e
0.6a ?.1
3i=ii 3.iii:; S;i=l;i ili;.;
_3.3
i.j
:6.j;i j.i
-3:3
i 1.3
-3.ii ?.3
S.iI i.f
5.7! )2.6
5.0 a 13.1
l-4 ! 14.8
'i|i;j
ie+.<
i,iiisi 3ii3i ;]=ii
r'4 = 0'4
4.7 t 11.0
49 3 I 53.1,
35.0 1 106.2
48.5!
66,6
.rr.u = 57.0
ru't ! )4 /
23.9 t 88,2
49.0 ! 64.3
,is.-r = .19.5
8.4 = t6 0
Ir.9 r t7.3
8-7: 17.6
b.6 = l6.j
21.8t l6.t
16.8r lz-d
22.8= r8.0
2o.l= 16.3
33:ii3iJ,tliiil:
36'7 = 20'7
0.8a 4.7
5.i = s.:
t.l t 1-9
lli:i|.e
^.jetr:il,i:;:". iiftfii,
Morningosriffniss
{minures)
rr =
76,0 ! 16,8
1 5 . 41 6 . 8
.AdjustecJ
differene"+
,,1:!.i ,,A;Zj
,.i:i.i
iiif
l
2'3! 4'8
4'2r 7-3
z'-i= l.,t
a.0; ;.i
1l.3i3?;1
i l: I
_;;
26.5
:t
21.5
.
il:j
."11"fi'"i$lX'"ij,",,o*"*"
placriviry
l ^ m gc o , t a g e n
12
5.2 r 11.0
:::n:iZ::*?iiii
1._r: i0.l
11.;=,1
iJ:i=x!_i3=lil _ri=fi Li:ii,i ;j;l3j
"ji.,T,,-#ifi;u,,.,.**
i .g.irrrg*
10ms co asen
23 7 = 16'4
37'2t 22-r
_0.1r l6.J
ts.zt ti.i
;;.0 ; i;.;
8.2 ! 17.7
:9.: = rg.:
r:.a. r+.:
i6.0: i6.0
i;.i ; i;j
t7.z! tB.3
wereincluded
in .ne
theanarvsrs;
rn
analysis;
n = 30
n
,tj*,mnY:"F1lj':0,^!'i:Lrô_,is"1l:pn"rs)
30 iin eachgoup. AcR = Americancollege
seeTabte5 t i
o?nniri".."
of
l']:^ll:::at-t:
i NegarivediflerenceinaicaiesO.iJiiJr-",ion.
"ir,ii
mlnusweek12,as dcfinedin paricnrsand Merhods.
I :is-:]l1e
orrurrru,ncrion3r
i ;i:ilr"rlT;.1""il;*rili?. ,.l,,illi *:,"^ll:.:Y":i:d * percertaee
capaci!y;
dropours
notincruded.
dereriorared.
ri ih.-iie "'"lr"f.X'!lJ,iffilg";l'"5""i1iil:
-2.much
ono
l'#iii;Tfl"#:;*jfnii:,i.::l;ffi. r 0.,..,o.",.0.
lim;lTllijl1"',,T,iijri'"Tn'il**",,5j:";s;;il;;;;;;;:i!'li'"lii:"ono,uon.
judsed
*ere
rou",'u"r,
r,,ip.L',l"l,erzuHlil:
i
RA in a study like rhis lies in the possibiiify
thar
non-RA patientsmay be inadvertently
inciuded.Al_
,^h:leh all parienrsd the study met
at least 4 of the
ACR criteria for RA, in the absence
of €rosions
patientswith other,RA-like,diseasesare
likely to be
included,despirethe reporredg5Vo specifi;i,"
of rhe
criteria in eariy RA (22). Diseasesthat
can resemble
earlyRA, suchasprimarySjogren'ssyndrome,
do not
TYPE II COLLAGEh{ TREATMENT OF' EARLY RA
normally lead to erosionsof cartilage/bone.
However,
the conceptof oral tolerancein a diseaselike RA relies
heavilyon the effectof bystandersuppression
induced
by type II collagenin thejoint.
Normally, type II collagenis sequestrated
at a
priviieged site, where it is not recognizedby the
immunesystem.Only in a cartilage-destroying
disease
Iike RA, with formationof new blood vesselsand
invasionof T cells and macrophages,
can it be assumed that T cells migratingfrom the gut recognize
and are stimulatedby collagenin thejoint. This u,ould
meanthat a conditionthat is confinedto inflammation
in the synovium,withoutcartilagedamage,would not
benefit from treatment with oral type II collagen.
Individualswith such conditionswould probablybe
included in any group of patientsbelieved to have
early RA. In our study, erosionswere not a feature
that differentiatedrespondersfrom nonresponders,
but radiologiccriteriamay be lessinformativeearryin
disease.
An irnportant means of identifying which RA
patientsare likely to respondto oravnasartreatment
with type II collagencould be their own level of T cell
reactivity to the protein prior to the start of treatment.
For this purposeit would help to identifyone or more
immunodominantpeptides,becausesuch peptides
have proved useful in assessingT cell reactivity in
cther diseases(49,50).By way of comparison,mice in
which arthritis is induced by foreign rype II collagen
are highly variablein the extent to which they develop
reactivity to their own collagen(51).It would also be
helpful if the successfulinduction of orai tolerance
could be measuredin patients or controls by the
releaseof cytokinesof peripheralblood Iymphocytes
after in vitro stimulationwith type II collagen.Fortyeight to 52 hours after stimulationwith myelin basic
pretein, TGFpsecreting reguiatorycells can be detectedin Peyer'spatches(52).
In conclusion,we found a tendencytoward a
higher responserate in the type II collagen-treated
patientswith RA, especiailyin the 1O-mgtreatmenr
group, which is encouraging,and, in..our
opinion,
justifies further investigations
of oral tolerancein the
treatment of this disease.Oral toleranceis an extremely interestingapproachtoward immunosuppres_
sion becauseit combinesnontoxicitywith antigen
selectivity.Above all, we needto know how to
iden_
tify which RA patienrsare likely to respond
to oral
tolerancetherapy.
49
ACKNOWT,EDGMEI{TS
W e aregratefulto J. K otw asfbr di spensi ng
the st udy
treatments,to R . Fi tzner for performi ngthe rhe um at oid
factor and c-reactiveproteintests,and to R. Blasczyk for
performingrhe HLA typing.
R.EFERENCES
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sl93, lgg4
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m e m b r a n e sC, l i n R h e u m a t ol l4 : 2 3 6 .1 9 9 5
16. Khoury SJ, Hancock WW, Weiner HL: Oral toierance ro
myelin basic proteinand naturalrecoveryfrom experimental
autoimmuneencephalomyelitis
are associitedwith io*nr*nrlation to inflammatorycytokineand differentialupregularion"of
50
SIEPER.ET AL
t r a n s f o . m i n gg r o w t h f ' a c t o rb e r a , i n t e r r e u k i n4 , a n c r
prostaglandie
n x p r e s s i oi n r h e b r a i n .J E x p M e d r 7 6 : r i 5 5 * j i 6 4 ,
I 992
1 7 . S h e h a d e hi r { N , r , a R o s a F , L a f f e r r y K J : A l t e r e d
cytokine
a c r i v i t y i n a d j u v a n ti n h i h i b i t o no f a u t o i m m u n ed i a b e t e s .
J
A u t o i m m u n6 : 1 9l - 1 0 0 .I 9 9 3
I B . L i b l a u R S , s i n g e rS i l { . M c D e v i r tH o : T h r r n d
rh2 cD4- T
c e l l si n r h ep a t h o g e n e soif. os r g a ns p e c i f i ca u t o i m m u ndei s e a s e s .
I m m u n o lT o c i a yl 6 : l a _ 3 g ,1 9 9 5
19. RapoportfulJ,Jaranriilo
A, Zipris D, LazarusAH, SerrezeDV,
L e i r e rE H , c y o p i c kp , D a n s k aJ S , D e r o v i c hr L : I n r e r r e u k i n
4
reversesT ceil proiiierativeunresponsiveness
and.preventsthe
o n s e to t ' d i a b e r ei ns n o n o t , e sdei a b e r i cm i c e .J E x p i z t e c r
17B:97_
qQ I QO?
n i e r C : A r t h r i t o g e n i c i t yo f m i n o r c a r t i l a g e c o l l a g e n s
(types IX
and XI) in mice. Arthriris Rheum li: l_g, 1990
3 2 , o ' B r i e n p c : p r o c e d u r e sf o r c o m p a r i n g
s a m p r e sw i t h m u r t i p r e
e n d p o i n t s .B i o m e r r i c s. 1 01: 0 7 9 _ 1 0 g 7t.9 g 4
'
r
h
o
m
s
o
n
l3' oliier w.
w: popuiation genetics of rheumatoid
a r t h r i t i s .R h e u m D i s C l i n N o r t h . q . mt g : 7 + t _ 7 5 9 ,
tggz
3 4 . P i n a l s R S , M a s i A T , L a r s e n R , A . ,a n d t h e
Subcommittee for
C r i t e r i a o f R e m i s s i o n i n R h e u m a t o i c rA r t h r i t i s
of the American
R h e u m a t i s m _ A s s o c i a t i o nD i a g n o s t i c a n d t h e r a p e u r i c
criteria
Committee: preriminary crireria for crinicai
,..,nir.ion rn rheu_
m a r o i d a r r h n r i s .A r r h n r i s R h e u m i 4 : l 3 0 g _ 1 3 1 5 .
lgBl
l5' Bloch-KBrochD, srearns
M, warkerwa,'r"i.rii.Lt uptut. or
macromolecules.
VI.
tJptake of protein antigen in vivo
in
normai rais and in rats infected with Nippostrorigyrus
2 0 ' B e s s i sN , B o i s s i eM
r - c , c a p u t D , F r a d . e r iD
brasilienz i, F o u r n i e cr : i L - 4
s i s o r s u b j e c t e dt o m i r d s y s t e m i c a n a p h y r a x i s .
or iL-i3 transttcredxenogenicfibrobrastsin rhe treatment
GJstroenterorogy
of
7 7 : 1 0 3 9 - 1 0 4 41, 9 i 7
collageninducedarthritisin mice. crin Rheumator14:26r,
rgg5
36' Cunis cH, Gair DG: Macromorecurar transport
1 1 . T r e n t h a mD E , D y n e s i u s - T r e n t h aRm
by rat gastnc
A , O r a v E J , C o m b i t c hD
i ,
m u c o s a .A m J p h y s i o l 2 5 7 : G l 0 3 j _ C 1 0 4 0 ,1 9 9 2
Lorenzoc, sewelrKL, HafrerDA. weiner HL: E&'ects
of orar
3 7 . I - I a n s o nD , R o y N f , G r e e n G l v I , M i i l e r
administrationof type II coilagen on rheumatoid
s: Inhibition of orally_
arthritis.
i n d u c e d i m m u n e r o l er a n c e i m m i c e b y p r e f e e c i l n g
S c i e n c e2 6 1 t: 7 2 7 _ 1 7 1 1
on endopep_
09
, 93
t i d a . s ei n h i b i r o r . R e g I m m u n o l 5 : 7 6 _ 7 4 , 1 9 9 3
22. ArnettFC, EciworthS
y M , B t o c h D A . M c S h a n eD J , F r i e sJ F ,
38' Michaet JG; The rore of digesrive enzymes
CooperNS, Healey LA, Kaplan SR, Liang MH,Lurirra
in oraily induced
HS,
i m m u n e t o l e r a n c e ,I m m u n o l I n v e s t 1 B : 1 0 4 9 _ 1 0 5 4 ,
MedsgerTAJr, rvritche[DM, NeustadtDH, pinalsRS,
l9B9
schailer
l 9 ' i v { e t z l e rB , w r a i t h D C : I n h i b i r o n o f e x p e r i m e n t o i
JC, sharpJT, wilder RL, Hunciercc: The Amencan
u,rro,-,r,rn.
Rheumae n c e p h a l o m y e r i t i sb y i n h a l a t i o n b u t n o t o r a r
tism Ass'ciation l9g7 revisedcriteriafor the classification
administrarion of
of
r h e e n c e p h a r o _ g e npi ce p t i d e : i n f l u e n c e o f M H C
rheumatoidarthritis.Arthritis Rheum 3l:315_324,
binding affinity.
19gg
Int Immunol 5:i159_t165.lg93
2 3 . F e l s o nD T , A n d e r s o JnJ ,B o e r sM , B o m b a r O i e r
C, ChemoffM,
4 0 . B j a r n a s o nI , W i l l i a m s p , S o A , Z a n e l l i
Fried B, Fur-stD. Goicismith
GD, Levi A J, Gumpei
C, KieszakS, LightfootR, pauius
J M , P e r e r sT J , A n s e i l B : I n r e s t i n a i p e r m e a b i i i t y
H, Tugrvellp, WeinblattM, WidmarkR, Wiiliims HJ,
ano inflammaWoife F:
tion in rheumatoid arthritis: effects or
The ,a.merican
non-rt".oiaa anticoiiegeof Rheumatoragyp..ri*rnurv core set of
inflammarorydrugs. Lancer 2:lI7l_1173, IgB4
diseaseactivity measures
for rheumatoiO-artfrritis
ciinicaltrials.
41. Bjarnason I, Zanelli G, Smirh T,
Frouse p, Wiiliams p,
Arthriri.sRheum 36:729_740.
1993
S m e t h u r s rp , D e l a c e y G , C u m p e l M J , L e v i
2 4 . P r e v o oM L L , v a n ' t H o f M A , K u p e r H H , v a n
AJ: Nonsteroidal
L e e u w e nN I A ,
anriinflammatory drug-induced intestinal
van de Putte LBA, van Rie[ pLC: Modified
inflamrnation in hudiseaseecrivirrr
m
a
n
s
.
G
a
s
t
r
o
e
n
t
e
r
o
l
o
g
y
9 3 : 4 9 0 _ a 8 9 ,1 9 8 7
scoresthat inclucietwenty-eight=ioint
counts:d;;;l;;;;;;;;
42. Bradiey SM, KingB, Troughron pR,
validationin a.prospective
Gooi HC, Bird HA, wrighr
rongitucrinar
stucryof paiientswith
v : c l o s t r i d i u m p e r t r i n g e n sa n c la n r i b o d y
rheumatoidarthritis.ArthritisRh.r* :A:++ig, tggS
,.rponr., io it, untigrn
in arthriris patients on and oft'non-steroidal
25. R.aspe
anti-inflammarory
H-H, Kindelp, vesterlingK, KohlmannT: Die Entwick_
drugs. Br J Rheumatol12:940-941, lgg3
lung der Funktionskapazitiit
und ,r", schmerzintensirrit
von gl
43. Kanerud L, scheynius A, Nord
cP-Patienten
cE, Haftstrcim I: Eft-ectof
unter einer Behancilung
mit AzurfidineRA oder
s u l p h a s a l a z i n eo n g a s t r o i n t e s t i n a rn r i c r o i l o . o
Aurothioglucose.
unJ on mucosal
Z Rheumatol46:71_75,l9g7
heac shock protein expression in patients
2 6 . R i t c h i eD M , B o y l eJ A , M c i n n e sJ l i i , J a s a n i
with rheumatoid
M K , D a l a k o sT G ,
a r r h r i r i s .B r J R h e u m a r o l3 3 : i 0 3 9 _ 1 0 4 8 ,
Grievesonp, Buchananww: crinicarstuclieswith
I994
an articurar
4 4 , P e i t o n e n R , K j e l d s e n - K r a g hJ , H a u g e n
index for the asses.sment
lvl, Tuomine n J, Toiva_
joint
of
tencierness
in foii"ntu *itt-,
n e n P , F o r r e O , E e r o l a E : C h a n g e so f f a e c a l
r|teumaroidarthritis.Q J Med 37:393106, l96g
ilora in rheumatoiO
anhritis during
and one-year vegetarian ciiet. Br J
? 7 ' c l e r u p o ; Z e t t e r q u i sHt : H L A - D R t y p i n gb y p c R
_
f
a
s
r
i
n
g
amprification
R h e u m a r o l 3 3 : 6 3 8 _ 6 4,1 l g g 4
with sequence-specific
primersfpCn_SSplin 2 irours:an alter_
4 5 . K h o u r y S J , L i d e r O , A l - S a b b a g hA , W e i n e r
n a t i v e t o s e r o r o g i c aDrR t y p i n g i n c r i n i c a rp r a c t i c e
H L : S u p p r e s s i o no f
incruding
e , \ p e r r m e n t aar u t o i m m u n e e n c e p h a i o m y e r i t i s
donor-recipien
by oial aciminismta r c h i n gi n ' c a l a v e r i ct r a n s p r o n t o i , oTni.s s u e
,tration of mverin basic prorein. III. sinergistic .n'"., of ripoAnrigens39:225_35,1992
p
o
l
y
s
a
c
c
h
a
r
i
d
e
.
C e l l I r n m u n o l l 3 l : 3 0 2 _ 3f O , t g q O
1 8 . B l a s c z y kR , v a n L e s s e nA , S c h w e i l aN ,
H u h n D , S a l a m aA : A
4 6 . E e r o l a E , M o t t c i n e nT , H a n n o n e n p ,
n o v e lH L A D R r 3 a i l e r e( D R B r " r 3r 4 )i d e n t i f i e d
L u u k l < a i n e nR , K a n t o l a I ,
bysingre-strancl
vuori K. Tuominen J, Toivanen p: Intestinal
conformationporlzmorphism
ffora in earry
anarysisand confirmedIy iiirect
r
h
e
u
m
a
t
o
i
d
a r t h r i r i s .B r J R h e u m a t o l 3 3 : 1 0 3 0 _ 1 0 3 g 1. 9 9 4
s e q u e n c i n gH.u m I m m u n o 4l 3 : 3 0 3 _ l l ? ,1 9 9 _ 5
4 7 . Z h a n g Z , M i c h a e lJ C : O r a i l y i n d u c i b l e
2 9 . F e l s o nD T , A n c l e r s oJnJ , B o e r s M , B o m b a r d i e r
immune ."rpon.iu.n.r.
C , F u r s rD ,
is.abrogateb
d y I F N T t r e a r m e n t .J l r n m u n o l 1 4 4 : 4 1 6 3 { 1 6 5 ,
Cold.smithC, K,ar,z
l9t\)
Lfvi, LightfborR Jr, paulu.sH,'Kieszak
StrandV,
4
8
'
C
l
e
r
i
c
i
M , s h e a r e r c M : T h e T h r - T h 2 h y p o t h e s i so f H I V
Tugwell P, WeinblattM, Williams HJ, Wolfe
infecF,
S:
t i . o n :n e w i n s i g h t s .I m m u n o l T o c l a y l 5 : 5 7 5 _ _ 5 8 l ,
A m e r i c a nC o r l e g eo f R h c u m a r o r . g p
lgg4
yreriminary'definitio
on
f
4 9 ' N i s h i m u r a M , K e r m o d e , { c , c r e r i c i f u r ,s h e a r e r ' c M ,
in
Berzot-sky
rheumaroid
arthrifs.
arrhritis Rheum 3B:72i_
:T!rg::T*nr
J
A
,
U c h i y a m aT , W i l < t o rS Z , p a t e
/ i)
t9q\
30. fticard-Blum S, Tiollier J, Carrone
R, Herbage D: Further
b i o c h e m i c a la n c l p h y . s i , c h e m i c acr h a r a c t e r i z a t i o n
of minor trisulfidc-bonded (type IX) collagen,
cxtriicred tiom tbetal calf
c a r t i l a g e .J C e l l l 3 i o c h e m2 7 : I i 7 _ t 5 8 ,
l9g5
3 1 . B o i s s i e r M - C , C h i o c c h i aC , R o n z i e r e
M_C, Herbage D, Four-
E, Maioney B,'Manns a,
B l a t t n e r w , J a c o b s o ns : D e m o n s t r a t i o no f h u m a n i ' l y m p i r o L r o p i c , r i r u st y p e I ( H T L V - l ) - s p e c i f i c T c e l l
r e : i p o n s e sf *
u..o_
n e g a r l v 0 a n c r p n r y m e r a . s ec h a i n r e r c t i o n n e g a t i v e
persons to
H T L V - t . J i n t - e c tD i s 1 7 0 : i 3 4 _ i 3 g ,l ( ) ( ) 4
5 0 . R o w l a n d - J o n e sS , S u t r t x J , A r i y o s h i
K, Dong T, Cotch F,
McAdam S, Whirby D, Sabally S, Gallimore
A, Con-ah T.
of Typell Collagen
ffects of OralAdministrati6n
Arthritis
on Rheumatoid
DavidE. Tlentham,tRoselynnA' Dynesius-Trentham,
E. John Orav, DanielCombitchi,CarlosLorenzo,
KathrvnLea Sewell,DavidA' Hafler,HowardL. Weiner
umatoidarthdtisis an inflammatorysynovialdiseaselhoughtto involveT cells reactjng
sn antigenwithin theioinL Type llcollagen is ihe majorprotoinin artr lar estilage and
a pote;tial autoafltgen in this diseasg Oral tolerizationto autosniigenssuppresses
dis€ase,includingtwo modelsot ineu_
aLJtoimmune
modelsof T cel!-medialed
arthritis. In this randomized,double-blindt al involvlng 60 patientswith severe,
fieumatoidarthdtis,a decreasein the nurnberof swollenioinb andt€nderjoinls
d in subjectsfed chick€ntype ll collagenfor 3 monthshrt not in thosethat Eceived
bo. Fourpatientsin th€ collagengrouphadcompleteemissionof thedisease.No
efects were evident,Th€se daia demonstrateclinicaleffcacy ol an oral tolerization
roach foarheumaioidarthritis.
afthdlir (RA) is a conunofl
illnessi.r which the synovialnemof multiple joints becohesinnahed,
damage to cartilage and bone. AIrhe pathogenetic nechantums ufl-
ying thc diseareare unknoe,n,rheumaanhricEis associated
with huban lymantigen (HL.A)-DR4 and considto b€ an autoimnune disord€! in
I aciirated T celb p:nicipate (l).
ll collagcn b a candidate aurosnlig€n
thi! diseascbecauseit is the mosl sbunstlllctural protein of caftilagc, and
preses animal models of the auroimune
iiseasesrnultiplesclcrosis,lveiiis, dd diaberes(6-ll). In a dorble-blind Pilot tial
involving 30 parieflt! with mulriple sclerosis, oral adminis$ation of bovine hyilin
anriser$ decr.ased the nunber of T cells
thar reacled $tth nyelin basic prorein
(MBP), wirh no measuEbletoxiciry (12).
Although favorable trcnds oftu!'ed in the
myelin sroup, clinical eficacy could not be
der€mlned b€causc of the small samplc
Oml adminiJtration of nEtive tl"e I
rton of aninal! with rhc nariv€ collagen emcliora€s r$,o animsl rnodcls of
rhcumatoid anhliris indlccd by r'tpe I
teir crcatessrthriti! morpholo$callyrerheumatoidarthrttie(2,3). Pa" collagen (lJ) or completelrcund'g adjuvant (14) . Therc experirncntal fiadings prot! ith rh. disca3c h3vc ihmune re'
vided rhc rationale for a pilot, open-label
co natiyc tj'pe I couacen(4), but
and safctystudy in 10 pa'
dose-escalatton
in
collagcnreactivitypalticipates
primary pElhog€nesisof rheumatoid tienB with recalciFlnt rhcumaloid anh.itb. Subjecrs 1,ere raken og then imrunoor rcficcs tiss'rc degadarion is
suppre'sive and diseasc-modi4'lng drucs
consisring of methotrcxatc, 6-mercaptopuin
Cunenr nertm€nieare inadequate
.t they only panlally concrolestablished dne, a,arhioprine, or aunnofin and fed 0. 1
umatoid arthtitir. They also have side mg ofsolubilizcdq?e II collagendaily for I
thar limir useearly in rhe disease monrh ,nd rhcn swirchedro 0.5 ng lor rhe
and intcrferewith proloncedad- n€xt 2 monfis (15). This dosewas exnap(5). Ar idealthenpy would olared 6om experinenrs in tlE lat adjuvant
drhrirb model where feeding 3 to 30 ps of
i.{a.nmarion in the joint by a
coLla8etr
atrenuareddb€as. (14) and the Et
+peci6c mechanismand would lack
autoimmun€encephalonyeliexperimenlal
roleriation,
a
nerhod
of
, Olal
antigenapecific tolerance, sup- tis (LAE) nodel where feedins 500 to 1m0
(6, 10).Sixof
pg of MBP wassuppressive
Te.|\am, R.A, DFesilnTcnlham, D. combirthe 10 parien$ experienceda substmrial
C. LoreEo,K. L s*dl Dlvisonol Bheumalol
Depanmenl
otMediclne,
a€lhrsd€ HospllEl,lhe clinical esponse, deined by a >50% im"
A DaM Res€4ch hs lure lhe Hatoard- provementin both swol)enand renderjoint
Lrbsrar!ry, Hafrard Medi€l Sdb@|.Bos
meuures
counrswith rwo addirionaldisease
>50%
stihess,
by
improvins
Imomins
Omv, oepanmenlor Mediclne,edqhd and
I5-m walk rime, grip strencth,Vestcrdeh
s Hospiral,
Hadad sclr@rol PlbricH€.rh,
eryrhrocytesedimenlarionmte (ESR), or
Fta er $d ll L wain€r, Conrerlor Nelroloqic
physicianor patientslobalasesmentsland
oitsio. ol Naurology,
ol Medoepadm€nl
lasri.s for ar leasr 2 months afier rhe
Brighao and Wom6ns Hospilal Natoa'd Med
Sch@l BosronMA 02115.
neatment period (i6). A comptete re'
.ehission (14 wirh
sponse,that is, disease
niom on6spondenceshouldbe addressed
sclENCE. VOL.16l ,
9l
2 4S E P T E M B lE9R
discontinlaiion of nontteroidal anti'irF
flamatory drus 0\JSAID) , ecured in one
pacient p.eviously on merhonexate and
continued for 26 months- There were no
advese efece. Baed on the resultsoi rhis
phue t stldy, a ptaebo<ontrolled, phase
li rrial wasunderiakenro deteminc vhether cliniol eficaq could be demonsrmted'
For th; phae II trial, 60 parienrs wirh
*vere, active rheumaoid arthriris and who
met elisibiliry criteria (18) save infonned
consenr (19) md were entered into the
study. They werewithdn*ar Fom irnrnunodrugsif they had been iaking
suppress've
them (20) and endonized (2J) to eithe! a
treadnent identi@l to that used in the
phae I tiial (15) or an indistincuthable
placebo (?2) to be raken onlly for a consecudve 90-day peliod. Both patients and
-investigatoB,
except rhore resporuible for
medicarion (rJ), wele masked a ro rrearheni, Assessmentseerc perfomed by the
sameinvesttgator(D.E.T.) at thc initiaoon
of trearmentand at 1, 2, and I months,
generally a! the same time of day (24) .
A! rll€ conclusion ofthe study, 59 of rhe
60 Dalienr!wereconsid€redevaluable(25)I
28 iad recetvedcollagen and 31 placebo.
On entry, dcnographic, clhical, and labo.atury paErnete$ werc similar in both
eroupsCrablc1) (26). Reladvero enw,
(P < 0.05) improvclhcrc wa36icni.6canr
Tabl. 1 Pati€ characlsrisllcsat 6n1ry.Th€re
b6l'.rcengrcups(P> 0.10)
wsrenodifferences
exacllesl or lha
detecledby €lhBrFisher's
dlrwcoxonrank-sun
tesl(ageanddiseas€
Chs€cterlstic
Age ly€a.s t so)
(yearsi SD)
Collagen
ln = 28)
Placebo
(, = 31)
5 0 . 3r 1 1 , 9 5 5 , 1i 1 2 , 9
68
71
9 . 8 ! 6 . 2 1 0 . 3r E . 1
74 (27)
82 t28l
46 (28)
62l?9)
tactot l%,
resred)l
HLA-DB4+ [%.
t€sted)l
Colagef ll
13
rirer> 2)
25
48
64
58
drawn {%)
rMerho[e€le,emercapropunne,eatnioprm, hysulfasalaineau6.olin, cyclospc
d'oxycn.mquine.
or penicillfrine. S4en Pa.
in cyolophosphande,
ie s Mr€ EceMnq €mbinaidns oi lhese dtugs
(20).fte .emaininopalisnls were .ol on mmdnosutr
pre$lvedrlqs al lhe lm6 oi €nlrybeeuse ol priol
lackolresponse
o. r*i6ily to al leasrh4r ol lhe dtugs.
1727
ffi
ffi
*-W
ffiffffif
ff{ffi
rutffig,ru
mr**uN*td#fli?i;{tfsyl:i:":i
e-rng
;^trecor*ee._i;i:o
;ii|i:"I: :"1il?T:"!":"#l;.
Joinrsswo sn (iuhbt
.rcrntstenderto p.esslreor
on passv€motron
Parntut
Gfoup
aretury
Coltagen
I 1 , 8a 0 . 9
1 2 , 0! a . 8
1 5 . 8t 1 , 3
15.6JO8
Cotlag€n
r"i"ili,iiiii'"s
i"o*
Jo nt-bnderness
or pajn
Grrpskength(mmHo)
hrqht
index
CoIagen
^. Seler€or verysevere
fnys|c/.n
assessment
t%l
rosenr or mrld
Sevse or rery severe
_^Sewe or very srere
2
'2.7 t
2 . 0!
-5,0:
1 3 . 2a 0 , 6
Collagen
1 0 5 . 9
Colrasen
106 j 10
155 a 51
210 J 55
ralent assessmeft/%r
Absenrof mild
Severeq rery severe
'r3.3
1 1.1
1 3 . 2e o . s
1 7 5r 1 . 3
Diiterenceircm entry at
oonlil
0 , 11
-73!
061
-8.9t
6,5*
1,4
0 . 9a
-6.2 *
1,2.
T.0
0.3*
0.6
2.1
0.5"
1,2
6.0
6.2
5.6
5.8
130 a 76
6,3 1
-83 I
-9.3
l6
35
48
Collagen
Collagen
Coilagen
:!
1.q,
2,t
0. '.,
z5.j
istr
7.A
8.4
a5E
lO.1
5 1 . 2: 1 o o
168
1 108
231
46'
31'
15
23
6?
33
26
21
31
48
1.8
r.i*
0.81.8
1.2"
21
CoJlagen
ie
1.s
195 : 100
36'
25'
39'
19
10
la
46
36
6
42
52
35
33
26
21
31
48
a€
2.9
5.0
27
27
12
62
2.8
5.6
32
29
39
19
13
68
it k po$ible thar the diseue
b€ €xacerbatedor an allergy to the
aniken could develop, rhis was nor
ved in olr studr, in aninals (6,1I,
l4), in mulriple sclelosisparienrsgiven
myelin for as long asI yeas (12), o! id
ci6 patientsreated wirh rerinal S-anri(J2). All padents in rhe phaseIl rrial
op€n-labelrrial had collasendisconrinafrer 3 monchs. Four parienrs in rhe
srudywho improvedwhile on coilasen
nced a relapseabour 3 monrhsafrer
ion .,f therapy followed by beneft
reinid.rion of couagen. In aninals,
:ti!e efecrs oforal rolennce app€arro
for 2 to J months afler reminarion of
feeding (6). Recrudescenc€
of disafter discontimrationoI oral roleragen
ako occured in mul'iple 6cle.osis(12)
u\:eiris (J2) patienrs. It thcreforeapthar addirional adminisaacionis reto roinlain rhe clinical ere*s of
a. Fob€ns,M. L sp.rn, H.L W€iner,Proa ,t
tissueslnd reiease.ytokines rhar inacrivare
auioassre$ivecells. In animals, fceding 11. Add. Sci U.SA.39 42111992)
Z, J. Zhane L DavidsonG E6enbanh,H. L
latgedosesof antigen favorsT cell anersy,
W€lnar,P@. ,,vanecad Sci US'4. 33, 10252
(1-.91).
whereasmutiple small doses favoo ihe
12. N. L W€ln6ral.t Sd€nd5g,1321 (1-093)
i.dlcrion ofresularoryT cells (JJ). In rhe
13. C.Na9re.Ande6o.,
L.A Bob$ M, E.Fobinson,
EAE model, hedlns low doses of MBP
G.W SlsklndG.J. Tho.becke.
P@ &all A.ad
acrivatesMBP-lpecinc regulatory cells in
sd: u.s.a. s3 7443(r936)iH. s. c. rhonpson
and N. A Slainer Clin. Eap lnnunal.64 5A1
gut lrnphoid tissue (10). These celis are
(lgeq.
predominan'lyCD8+ and suppressEAE b1
]a Z J.Zhano,C.SY. L6o,O Ljder,N.L. Waif6r,J
ftaftckins to the cenrral neflous system
lMal
145,2aBgI199Ol.
and releasinganriin0ammatory cyrokines, 15 N.liveryp6ll collagen,
kolar€dl.m sr€naler
ilage ol chcks6nd6r€d grhyilc by adminislGsuch as TCF-p and inrerleukin-4, when
lionotg.ahinop@paon
rlle {2),waslsed to rroEl
ihey encounter MBP presentedby MHC
lhe hrclliv€ subi€crsin the pMse I pirotsludy.
nolecules in inflamed brain rissue- This
Slbsequ€nr p3t anb h the pilot td6rand in lh€
doube-blifdsrldy r€ceiredry!€ ll @llsg€nps
proces, teme d antigen-dnven bysrande!
ilied trom nonlatfr/riticch cken s|6ml canilage
suppre$ion (10), implies ihar an orally
bylhe ldenlicallechniqu€
(?)and obrajned
lom
administeredprorein can do{n{esllate or
G€nzyme(8oslo.,NIA).Pf€paGlions
E.e ana.
frzed lor pudlyby sr.ndard bi@hemica Delhods
canapecincauroimuoe diseaseas )ong as
(Z 35).rdresr6d loradhiroqenicryandro.cily
ir is a coruri.ueflrof the ta.gerri$ue and is
/n Brs (2) w n lndl.ss of barch.rdbarclr
oq!iv.capafleofinducing legularoryT cells. 1r is
lency Co agenwassroredin a lrcphlllzedslar€
(2) al -2C"C wilh desiccanl. The prolei. was
not oblisatory io! rhe prorein ro have rhe
soubllzedin 0,1 M ac6ic acld lor -12 hoursal
diseaF-incirlngepitopes. Examplesof bya'C Elerillzed
by membranelillBIio, a.d al.
srander5upp.e$ion include inhibirioo of
quoEd hb ndividlal 1.0-mldoles i sledro
the basisofsrudies of olal rolelance proieolipidlrot€in (PlP)-induced EAE by
lubes Tlb€sslticlenllof -2 wsk ol treatDent
wsE dellv6r€d
on ioolo pafenE &d mdlnlalnad
, tPo ihhunolosic mechanNm! orally adniniltered MBP (J4), delay of
u.der relrlgeElion lntil use. For oral admhistaexplain the clinical rcsponseto coldiaberes
in rhe non-obesediabelicmouseby
lion,lhol.o.ELalquolwas6dded
ro,1lo 6o!nces
observedh rhn srudy.F;edtnsrypeII
(118ro 177 m|) ol .ord oranoojuic6 and he
oral iffulin (ll), and abrocarionof adjumixllredrunklmm€d6l€y OEn93 iuiceprovdin R4 casesmay borh anergize vBnt arthlids by oral coLlacen(14). In all
€d 6n addi onal6c d v.hi. e ro inhibllpreclplta.
t}?e II collagen auroreactivccells
tfuee modeh, autoimaunity to rh€ tole.alon of collag€.and hask€d lne lale olacetlc
gcneratemajor histocornpatibilirycohgen do$ not appear to iniriate dkease.
.cid. Arl dding occudodin lhe homi.g on .n
(MHC) classI- or clasgll-restricted
€nply slon€ch al l€ast20 frih b€roe b.eaKaslor
Accodtnsly, our data do nor deremine
|nEa$onol olhd luids.Smoklngwasnarp€mlr.
ccllsthar sequcsr€r
wirhin joinr wherher t)"c II collagen is rhe primary
t6ddurrnghls l.re0a,
autoantigenLn rheumaroidarrhritls.
i€ M, E. WAnbbn er at, N Enet.J,t.d.312 AlA
(1985)iK, L SewElret at.,Alhtitis BheM.,1^
Alrhough tnirial clinical eficacy of oraL
4 3. Oulcomerneasuresin cotagen,veF
collagcn
quesrions
har
bccn
shown,
conpacebo-lrealedpsli€n16.
vallss are oeF
17, F, S, Ph.6, A. T, Masi F, A. LlFen, /4n riis
ceming optimun dosing and long+crm
of 28 collsgenand 31 placabopaahdud,2a r3o8 (i98i),
control ofdiseaserenain. Nonctheless,thir
18 Tn€,[email protected]€ments
d6roihi.6d 6119
bility:
studydcmonsnabsrhe thcraDeuriceficacv
{r) Ah€dcan FheumatisnAssoclallori (ARA)cn.
l6da
tor
cla6slc
or dollnlteLewatold enhlb
of oral rolerance for a hurirari otoirnmu"e
(16)i0l)on!6rorlhe drseasear.9E 16or orde'
dts€ase
and pronde! rhe foundadonfor rhe
(llr)age
Erleasrlsysarc {iv)AFAtuncrionarctass
C o l a - P a . C o t t a - P t a . developmentof oral coilagen
I or lr (28) (v) s ens o/ sldprome ol Eynwirh
as an eastly
gen
gen
cebo
cebo
unrcsFonriv€
lo ar laag oneimmlnosuDor€ssvo
administelcdnontoxic rlearmenr for rheu.
(Table1)
(vi)sd€re
0
57
43
0
0
58
42
0
71
35
141
39
1a
39
REFEREIICESAND NOTES
t3
19
s rnc€aseor 30% or hore i@6 lhe
valu6lor the joinl.sretingtnder and the i.tnr,
hss or painlndex(t6). comoadson
b€rreen
show€dsignitienttymoreder€r016lonin he
elreatBdpatie.h (P < o.Cl by the Fish€rs
|esl). TNa.colicwilhouranr-i.ltammsrory
!s!aryacekm nophenwilhcodene prop
i of pe.r@cne prescribed at any [he by
r 'Nestigalorin a. anemdto rerainfia.ino
ln Ihs tal. Comp.riso.Oeueen Orolpi
6ignrlcanlygr€aternumbeGor p;.eb+
parenlsrEquning
nar.dric5(P < 004 by tho
s 6rad resu
toeleftined by anq can
sm assocrar
tu crit€ra,o' runcrionat
ctass
| rc I h tariontrofi adhdrc . mtdtt resrfcred:
Ldedr/resrjclediand tv incaoacrv causino
bed or{hee chatexslenceTrendro;ihorove:
I n r h e c o r a a e n q ' o u p n o r r i g n l ' c a . r (too=boy
1 K, L S€welland0. E.Tr€nlha6,Lancet3A\,2a3
(1993).
2. 0 E.Trenlhafi,A.S. loMes, A. H, Ksng,J Ap.
Md1. ft6 A5? (971),
3 J S Coln€nay M J. Oalman,A 0 oayan A
M.din B. Moseda6 Narr'.2A3,666Fgso),E S.
Cahcan d ar, L8b, /rveJr. 54 26 0 905),
4. N A Andiopoulose/at ,4nhila Fheun.19,613
(1976)iD E. rrenlham R A Dynesus R. E.
Focklir, J. R Davjd, N. E.al. J. i.led 2gE, 327
H Carsle. p
{1973)iATa*o$ki L Ktareskoq,
Herbeds,w J. K@pman Anh is Fheun 32.
1 0 3 70 S 3 9 )
5. P.M. Brooksk..et341,236 (1993).
6 P. J.l-iiggnsa.d N t. Weinet,J lmhal 140,
7 0 Bila.a.d c. c \),thta.te,cs hfitnot.1.t2,
364(1s33)iC.C Vihil3cr€,r. E Gienapo,C G.
a.asz A. aB J. tdnunat 141 21ssOsq.t).
3. F B Nusa€nbbner at J. lnnh.l 1u.16sg
I o L d e r , MB S a n r o s , C
S y L e E . PJ H g q n s ,
H L Welneribid 142.743 (rS39)
10. A l,Ii e..O. Lider,HL. Weino.J. E\p Med 17a.
7s1 (1991)S J. Khouryw W Na.cock.H L.
w e j n e rl b r d1 7 6 , 1 3 5 5 { 1 9 S 2 )M: A
i l e r , OL i d e r
SCIENCE .
VOL, ]6I
'
14 SEPTEMBER
I99]
and
aclivedrs€a$dolined
by al loasl$r€€ oi lie,oiown9: anln6palntuor
r6nd$ tohts, >six $orr6n joi.b, :45 mln or
homingsrllhess or >23 ndlhou EsR,E:clu.
3on cnl€.i€includ6da delra ot srtuctlrajoinl
damaQ€
nolahenabl€lo physical@habilirarlon
il
nrrrmmaliof
subsdedaio. l€at@nl or a seious
prcb6m
conclr6nlm€dica
Somepationls(r=
39)roprosent€d
reieftars
ror trealne.t ot retEc.
loryds6asebt tnelmalo€ srs oulsideBosto.
otheis (, = 10)riad jece v€d e&€nmentar rh6.a.
py tor rheumatoid.nhiisrn the p6L
1S. fte sludywasapprovedby th€ BethlsEe Hos.
plal Cohmin66on Cliniel lnv€sligations
and
@ndLcl6du4deran invesrlgatoFinlriated
lNestigaliof.l NN orua 0ND)pemlr rrom rhe L.S
Fed andDrugldhinGtation.
20. Becalseol lhe Fossibir,/ har parenrs @utd
receivginBiecliverherapyor a ptacebo s!ud!
hedic8lonMs begunimmedLarely
afr&rh€pa,
lEn djscoilinuedimmunos
uppressiv6d ru$ (Ta,
b e l)ipalrentsieceivn9 pa.enteElqodwer6nol
ProlonAedcarryeor effecls
couldlnluence ie olbome. Parientsremained
on rheirNSAD, rednlson€dose {<10 mg/day)
or borh,dunnq lh€ 3,month lrealmonlp€fiod.
NSAIDsLbslilurrof,
rncrea3es
in NSAIDor Dred.
nisofedose,or lnlriation
of any olhoranr'6€u.
mahclrEGFywirh rlre excepron .i 4ar9€5ic
aoenlsand iflraarric!a sreroids represented
pr.rocolvioato.s. ll applicab/e pati€dsaeie
.equesled
lo pracrce.onlEceplbn
1129
21 A bosisGricran
{EJ.o ) rlnd.E led €achos efr
;",":uilT,1.l,:,:[:'ff
T#:]gi:lsi?il- eri;,lrmri,qflr?fl#iiffi%1T;l
l {;i:i?1f"r#;:r,e
22. Ttrepacaboconsslodor Lo.nldosesoi
OI M
to nenbrans,jlrato..
^_ acorc aqd subJec,Ed
cr nree nve qatob (0 c. c.1., a.d K.LS I dF
Gr.€o he @domizatEnand preparedmeirrcaflo. DUrordn.r haveaccessro dnicardars No
24 C@onr oial t.skln.nle wereBed Lo
me€sure
'
"
$$$xiruisnli*1ii:ri*ir#
lii
16 c rvr.Kammer
-a o. e r"irr.ii.l
dneun 2t 4Eg(9a4)
1 7 S M .t l e r s o n
e r ; , . L ; c e . 3 s 7 , 3 st7j
$ slpponedby NtNOranlsMOr FRdl
r"r"d*l?"?#:g
"v*.*rlrl"iic
32. F,Nlssnbla , peEonat
mmhmrcltrcn.
- ;,ji5,"fl"Ji,5"":
,"I
$:i"Ll.,rli
HJ::
ft:f"flr"i.#lrr+il#Hi[:"J*:T
ror sareryhoniroring. Llboratory 3ateN *6e*.
menrwEspedohad immedlal€lv
berc,;raiddm.
alon sndat2,4,6,ed 12weel<sthoGafter
rr.
assessn.n cohpris.da 6dpt6tF bt@dcour
o n€@nrd Md ptdreel count, v.r and r€n:l
34. A, Ar.Sabbash,
A, Miit€r,F, A. Sob€rH, L
I
r,n""*::t:.;s
l,#l"g*{;*
havs a ,inancialinleEs i Autoh;;;;
W€in_
I Jln€ r993iacceprcd?3 algusl 1993
TyrosinePhosphorylatien
of DNABindingpro
by MultipleCytokines
**mr:l,#qu"lNffru'l;:
Michaet
David,
GeratdM. Fetdman.
_-- ,1lgg* C..Larner,
rry ano EU mtibody tt€E ro natN€iVo; rj co :.
ssn {*pF6sed ar _lo9,l by arwmi.inraa *
H.,Hacketr,
D"u"i"ns. ,i. wJo,o,
y#,#.::g:,#t?l-j::,1p"1",*l-l_"becca
Tl.Ti,..J#:l
sharon
lltr'""f;
lxfl
t\4.
sweitzer,
Eminuer'rlFliiit"-ii'iii,T"i;ti:";,ir;t
.o. Eebro unbthdhg, d6cl3io.3 Mro
mEd€conc6h.
#,lffti#-*;:i;#F!fr
j,Tijt""ff$
i.li [sf::if"t[]
?rijr;
nFijiJar,'"y:.i':";:;:ui"1%3i
(rseF.sr
;;
i
;;';i;"
#;;""#i
lffil"?I":Je,?:,lli[7:iJl
la,:€-1gei:iacrorr
,nrhepromoteis
mfiftffi{fli{#iiw!ilr"ds*H
of ealy,esponse
;j:;:".#ff".9*:T:^lresenr iffi .'ti""ii!ji
s6n$ s;;.:
il'HlT:lyi:.'^".,:f,::.e""e ii ;#'J;'5,H;giJl::.T.*S
gl,;,$":#;qr"#1"'*ffi#;tr$#
g.qe"h rinil'"]"gil;
|ii':ng
?:'EJTilt'.*0,"to-'.'!:,T:"Totvr_T"9
u
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ryth.tlqr.rgyh.s
DNi
bindrng
dt"h.lii.i,;*sil;,iJ
;j'g:,i:,19*fl:,,1"S:sF).adrv_ated
(GRFJ
rocated
Jn,r.,"p.."i"i li ti"
l?1o-1111f"n,o"
iiiii
;#:fiffii:fl,
o,.Gl.l.c.sF
con'inad
ai,o"rn"_pr,o"pno,yLi"!,i
;31?:?"",:":T'",::l*rll.::
"iutri,ffi*gffffi
-ai-ru$;#;ffi#Jil?,iiiilhg
li""&+#;ir.trnlfi+"$jr'ift
H.,'lf
r
iidfi
[ili].+t"ft:l.e11rypLr:mir
ttran
tnterrerons
can
&?::fi:
;ffi$"i#i:i
Nmhar or
"hote-cel exrracrsprepared tioa of lcM,y, whereasextmc* pre
[:T,?irj"!litr.I':::Hl'1T;::l
"j; CSF con.ainedCAA U,nain" ..^.
migiared
wirh a mobilirydi6eren;
ii:'iii':yti"iLH:.,::.:?',i.j"fl
i: rh8!
rh6ror rheFcRrrtr'c. ral .o-oi;
Eon modocltes Feated wirh IL_3 or
amniryime!nostobutin c Fc receprors;e
rhe fct$-y complex is a
yr-Kuryroshe_pho6phorytated
proternrhar
m a componentofrhe ISCF_l rransdiDflon
comp{e{, which causestp,J_c_stjmujated
exprc$roa oi earty resporoe
sens (Z+).
6ecauserhe peripher.l btoodmonocvte
ts a
cnncar tarser celt for IFN_c, IFN_1,
ond
ohcr clrokrnes, expenmenrsweredone
ro
oeremne whelher any crtokires
other
than ihe inrerfefonj m,shr induceche tnr_
nation of_FcRf1-,N?hote_celt
*nrs
were
preparedrrom nonocyres incubared
wirh
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and
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popolysaccharide-showed
ao
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254
Clinica.lPracticeof AltemariveMcdicine
Volume2, Number4, Wintcr 2001
OruentntPprn
Use of [JndenaturedTypeII Collagen
in the Treatntentof RheumatoidArthritis
David E. Trentham, IvlD,Andrew D. Halpner, phD,RoselynA. Trentham, MS,Manashi
Bagchi, run, ShiI Kothari, tvrs,Harry G. Preuss,lvo,DebasisBagchiornn
ABSTMCT: Meumatoid arthritis is a debiiitating chronic diseasethat lacks an effective treatment It is the ieading cause of disability in the
United States.Rheumatoid arthritis is an inflarDmatoryresponsebeiievedto invoive T cells reacting to an antigen witbin the joints and articular cartilage. Over-tle-counter pain reiievers and anti-inflammatory medications, such as aspiriq acetaminophen,and ibuprofen, are commonly used for preventive measures,but theseproducts only teat the symptoms, not tbe cause,and may also produce serious side effects.
A growing body of evidence indicates that type tr collagen is a major structual protein responsiblefor tcnsile stength and toughless in the
cartilege and also a potential autoantigen in people who have rheumatoidartbritis. If the activity of T cells that releasejoint-destroying factors could be reduced, outcomes for patiens with rheumatoid arthritis could be improved. One metbod of achieving this is termed oral tolerarlce, a concept that is proving useful in the treaunent of autoimmunediseases.Oral tolerance describesa 6tate of tmune hyporesponsiveness following tbe oral ingestion of a protein. It is, thereforc, a method by wtrich a peripheral immune toleraoce (down regulation) to a
particular antigen may be induced by presenting specific amouutsof that antigen to the gastrointestinalsystem. Several clinical studies have
demonstrated the effectiveness and usefulnessof undenaturedcollagenII in auenuatingthe symptoms of rheumatoid arthritis with no serious adverse effects. Thus its adririnisration may demonstratetherapeuticefhcacy by inducing oral toierancefor tp reabnent of this disease.
lntroduction
kttritis is one of the rnost prevalentcbronichealth
problems in theUnited States,affectingnearly43 million
people.'Althoughit is often thoughtof as a diseasethat
predominantly affects the elderly, it is the number I
causeof disabilityaffectingthoseoverthe ageof 15.In
facl more than half of those affected by artbritis are
under the ageof 65, and almost300,000of thoseaffected are children.'Each yearartbritisis responsible
for 44
millibn outpatientvisits and almost I million hospitalizations,andit is secondonly to heartdiseasein termsof
its effect on dayslost from work.2As might be imagined
from thesestatistics,the toll that arthritis takeson the
,healthcareindustry is substantial,costing the United
States approximately$65 billion eachyear in healthreiated expenses.
Unforrunately,the incidenceof artbritis doesnot appearto be decreasing,
andby theyear2020
the Centersfor DiseaseControl (CDC) predictsthat
almost 60 million Americanswiil sufferfrom someform
of this disease.
While the termarthritis may bring to mind a simple
condition characterized
by painful joints and diffrculty
performing certaintasks,it actuallyencompasses
more
than 100 different diseases.Of thesedifferentforms of
All correspondence
concemingthis aniclc shouldbe addrcssedto: Debasis
Bagchi, rm, CreightonUniversity Scbool of PharmacyScienccsand Allied
Health Professions,2500 California Plaza. Omaha, NE 68178 (e-mail:
[email protected]).
arthdtis, osteoarthritis (OA) and rheumatoid arthritis
GA) arethe most common.
Osteoafthrttis, OA currently affects 20 million
joint diseasein which the
Americans.It is a degenerative
cartilagecoveringtheendsofbones deteriorates,resultThis forrt
ing in pain,stiffoess,andlossof movement.3r
generally
and
develafter
tbe
age
of
40
begins
of artlnitis
pain
yeaxs.s
usually
report
People
opsslowly over urany
conbeginninginjoints on only one side ofthe body, in
trast to RA. While inflammationmay be present,joint
pain in OA is typically not accompaniedby the amount
or severiryof inflammationobservedin thosewith RA.
joints suchasthekneeandhip tend to be
Weight-bearing
joints such as the
moreaffectedtlan non-weight-bearing
elbow or shoulder.The generalfeeling of sicknessthat
calraccompanyotherformsof arthritisdoesnot usually
accompany
O/..
"nsw" disease.
RheumaftidAnhitis, RA is not a
of a diseaseresemblingRA
Oneof the fiist descriptions
can be found in rhe CharakaSamhita,a medical text
from india that datesback as far as 500 BC. Another
ancientreferenceto ttre diseasedatesto 100 BC; the
RomanScriboniusLargusdescribeda polyarthritic condition occurringmainly in elderly women that closely
resembled what we now understand to be RA,
Rtre-umatoid
arthritis currentlyaffects approximately 2
world's populartiillion AmericansandaboutIVoof tJ;re
tion. However,the pathologyandprogressionof RA is
somewhatdifferentfrom that of 04.6' It often develops
VOLUME2, NUMBER4, WINTERZwt 255
suddenly,within weeksor months,andgenerallybegins help to providea matrix in which the collagenas well as
betweenthe ages of 25 and 50. Non-weight-bearing watercancoexist.Thereare a numberof different types
joints suchas the hands,shoulders,
and elbowsareusuof collagen,but typeII accountsfor themajorpart of carally affectedbiiaterally,and a significantamountof redtilage.It is composedof 3 identicalchains(termeda-I
ness,tenderness,
swelling,andinflammationis oftenprechains)that form a triple heiix.r3-t5
This interconnected
sent.RA oftenresultsin a feeling of sicknessandfatigue
networkof collagenandproteogiycans
is crucialin mainandmaybe accompanied
by weightlossaswell asfever.6 tainingjoint flexibiliry andresistanceto srressand fracMorning joint stiffnessiasting for an hour or longer is
ture. In RA, autoantigenicresponses,most likely trigrelativeiy common.Subcutaneous
noduiesthatform over
geredby rype II collagen,ultimarely result in the probonesmay alsobe present.Interestingly,3 timesasmany
gressiveinflammation,pain,and destructioncharacteriswomen as men are afflicted with rheumatoidarttriris.
tic of this disease.''
Somepatientswiil experiencea monocycliccourseof the
Regardiess
of the initial cause,a progressivedegendiseasethat may abatewithin 2 years,while otherswiII
eration of the structureand function of the joint takes
experiencea polycyclic,or progressive,course.Of ail the
place,makingnormai activitiesof life increasinglydiffiforms of arthritis,RA tendsto be one of the mostserious
cult. In the caseof RA, the body is unableto recognize
anddisabling;76Vo
of rhosewho havehadthe disease
for
its collagenas normalandin rum attacksit as if it were
1.2or more yearsbecomecompletelydisabled.Lifespan
a foreign invader.r0'r?
A novel approachto treatment,
hasbeenshownto be shortenedby approximately7 years
termedoral tolerance,in which smallamountsof rypeII
in men and 3 yearsin women,which is equivalentto the
collagenare presentedto the gastrointestinaltact, has
increasedmortality observedin thosewith diabetesand
beenthefocusof significantpositivescientificresearch.,s
Hodgkin'sdisease.
Whatis achievedis a downregulationof the body's abiliry to destroyits own coilagen,resultingin improvement
Etiology
in symptomsand.slowing
theprogression
of the disease.
While RA is classifiedas an autoimmunedisease, However,to fully appreciatethe use of oral tolerancein
the exact causesthat resultin its developmentremaina
thetreatmentof RA, it is importantto understandthetypmys0ery.What is known is that manydifferentandcomical treatmentoptionscurrentlyin use.re
plex factorsare involved. Wtrile somecasesof OA may
be the result of yearsof "wear and tear" on joint strucCurrent Treatment Options
tures,otherforms canbe tracedto an injury, infection,or
The featment options for those with rheumatoid
metaboiicdisorder.6rRA, however,doesnot resultfrom
arthritis are rypically nonsteroidalanti-inflammatory
overuseof a joint or from injury, but rather from an
drugs(NSAIDs), aloneor in combinationwith what are
autoimmuneproblemin which thebodyatracksanddamknown as disease-modifyingantirheumatic drugs
ages its own tissue. The damagethat occurs in RA
@MARDs). As is well known,chronicuse of NSAJDs,
appearsto be propagatedby cytokinessecretedby T cells
especiallyin the elderly, is linked to numerous side
in responseto certain autoantigenicstimuli within the
effects,includinggastrointestinal
bleedingandrenalmaljoint.e''oVarious immunological factors are involved,
function.il Even the newer generation of COX-II
including but not limited to CD4-inducerlymphocytes, inhibitors such as rofecoxib (a furanone derivative),,'
CD4 memorycells,macrophages,
neutrophils,andtumor
celecoxib(a 1,5-diarylsubstituted
pyr azole),nandinfl ixnecrosisfactor.rt'r2
One of the most likely candidatesfor
imab (a monoclonalantibody)are not without their own
tiis autoantigenicstimulusis the collagencomponentof
probiems.23pa
While thesedrugsdo reduceinflammation,
cartilage,specificallytype tr collagen.Someresearchers they do not addressthe underlyingcausesof the arthritis
beteve that an infection may trigger the initial inflamandthereforecannotaltertheprogressionof the disease.
mation in a joint through molecularmimicry or other
Furthermore,rofecoxib and celecobixhave been conmechanisms,which in turn initiates the autoimmune traindicatedfor useby patientssufferingfrom hypersenresponse.Geneticsmay also play a role, as may other
sitivity, asthm4 urticaria,or allergic reactions.All of
factors,includingstress.
them can causebleeding,ulceration,perforationof the
The cartilage in joints allows for flexibiliry and
stomachand intestines,and anaphylactoidreactions.In
motionandprovidesa cushionagainsttheimpactof varaddition,rofecoxibhasrecentlybeenassociated
with a
ious forceson the bone,'3The detailedstructureof cartipossible increasedrisk of heart attack and stroke.z0
Iageis complexbut canbe simplifiedinto 2 majorcomProlongeduseof thesenewerCOX-II inhibitorsin the
ponents:collagenand proteoglycans.
Proteoglycans
are
elderlycanresultin sideeffectssimilarto thoseseenwith
large protein molecuiesattachedto iarge carbohydrate traditionalNSAIDs. DMARDs attemprto addressthe
chainscalledglycosaminogiycans.rs
Theseproteoglycans underlyingpathologyof RA morethoroughlyby slowing
256
CUNICAL PMCTICE OF ALTERNATWEMEDICINE
the progressionof the pathology.Oneof the components
in additionto inflammation,is microvasof this disease,
cular injury coupledwith the formationof new capillaries. Many of the DMARDs attemptto inlibit the fonnation of new capiliariesas well as addressthe underlying
inflam:lation. This category of drugs includes azathio'
prine, corticosteroids, gold, hydroxychloroquine,
and a numberof newermedsulfasalaziae,
methotrexate,
Whiie these
icationssuchasieflunomideand etanercept.
medicationshavebeenshown to offer clinical improvement to thosewith rheumatoidarthritis,they canaisobe
associatedwith signifrcant toxicity and side effects,
lymphoproliferativedisoringtu.iing myelosuppression,
ders, maculardamage,thrompocytopenia,osteoporosis'
hyperglycemia,and hepatotoxicify.Another factor that
shouldbe takeninto accountis cost the monthlycostfor
eranerceptandinfliximab, both of which mustbe injected, can be morethan $1000.Also, accordingto a recent
reportby theFDA, Remicade(infliximab)hasbeenasso'
ciatedwith tuberculosisinfection,nervedamage,andrisk
of cancerlymphomain patients.In severecasesof joint
damage,surgeryis often necessary,but this is alsocostly andinvo]vesa lengthyrecoverytime.4
Methylsulfonylniethane, chondroitin, and glucosamine,widely usedfor treatmentof OA,5 areknown
to help rebuild proteogiycansand reduceinfla:nmation
but are unable to help in the processof inactivating
"kilIer" T cells to amelioraterheumatoidarthritis.
ing joint-destroyingfactors,theoutcomefor patientswith
RA canbeimproved,andonesuchmethodto achievethis
is oral tolerance,The conceptof oral tolerancehas existed since1911,and Eaditionalmedicalliteratureis filied
with papersdescribingthis mechanismandhow it might
diseases,'0
benefitthosewith autoimmune
Recentstudieshaveshownthat small dosesof type
II collagenderivedfrom chickencartilageproduceoral
toleranceand work with the immune systemto prevent
the bodyfrom aftackingits joints'8'r8
Oral tolerancecan be inducedby 2 major mechaald clonalanergy'dependnisms,bystandersuppression
Throughout
that
is presbnted.
of
antigen
ing on the dose
gut-associated
of
are
the smail intestine,there
Patches
lymphoid tissue(GALT). Within the GALT canbe found
that contissuethatconsistsof nodules@eyer'sPatches)
and
B
lymphocytes,
of T
tain organizedassemblages
macrophages,and dendritic celis and are the primary
area within the gastrointestinaltract where immune
This immunetissueis designed
aregenerated.
responses
aswell as to
to protectthehostfrom ingestingpathogens
prevent the host from reacting to ingestedproteins. In
fact, scientistshaveattemPtedto usethe GALT asa route
for administeringvaccinesbut havebeendelerredby systhe generation
Nonetheless,
temic hyporesponsiveness.
is
the primary
GALT
tle
within
of immune responses
proteins
can supingested
orally
mechanismby which
presssystemicimmuniry.
0ral Tolerance
BystanderSuppression
This form of oraltoleranceis achievedby presenting
small amountsof antigen to the GALT, which in turn
generatesa T-cell response.After the antigen (in this
case,type II collageri)is consumed,regulatoryTM and
Th3 cells migratefrom the GALT throughthe iymphatic
systemand then into peripheralcirculation.When they
encounteran antigensimilarto that which wasingested,
they secretecytokines,inciuding transforminggrowth
factor-bet4interleukin-4,andinterleukin-10, that result
in the downregulationof activatedhelpelThl cells. It is
theseactivatedhelperT cellstlat are,in Part,involved in
producingthe inflammationand destructionof collagen
in RA. If this activiry againsthealthy collagencan be
the progressionof the diseasecanbe altered.
decreased,
It shouldbe notedthat the oral antigendoesnot need to
enter the systemiccirculation in order to induce a
astheregulatoryT cellsareinducedas a result
response,
of the interactionbetweenthe antigenandtheGAI 'T'ro
''
Whentheimmunesystemis functioningpropedy,it
in orderto
recognizesand identifiesforeign substances
heip eliminate them from the body.lo'rtOne type of
immunecell thatis particularlyimportantin this process
is the T cell, which can be classifiedin a numberof dif"Helper" T
ferent categories,dependingon function.
cells have the function of releasingfactors that help
iacreaseor decreasethe immune response'Thesehave
beenfurther classifiedinto Th-l andTh-2 subsets.Th-I.
cells amplify proinflammatory responses;Th-2 cells
"Killer" T cells attackanddestroy
limit suchresponses,
antigens,The B cell is also crucial to the functioningof
the immunesystem,as it is responsiblefor the production of antibodies.In a normal individual, the immune
systemdoesnot seekout and destoy healthytissuedue
in part to the fact that T cel1sthat have specificityfor
antigens on normal tissue are either suppressedor
destroyedprior to being releasedinto circulation.In the
caseof rheumatoidarthritis,however,T cells with selfantigensfor type II collagenare not properlydestroyed
or suppressed,
resultingin the damagethat is a characteristic hallmarkof this disease.
By decreasing
the activiry of T cells that arereleas-
Clonal Anergy
Anothermechanismby which an orally administered
protein can induce a down reguiation of an immune
calledclonalanergy.'oThis
responseis via a mechanism
VOLUME2, NUMBER4, WINTER2OOI 257
300,000children.Tenpatientsbetweenthe agesof 8 and
14yearswho hadactiveRA weretreatedorally with rype
II collagenfor 3 months.Eight of the i0 patientshad a
reductionin both swollenand tenderjoints at the end of
3 months.One patientin this study also achievedcompleteremission.It wasconcludedthat oral lreatmentwith
nativetype II collagennay be a safeand effectiveform
of treatmentfor juveniie RA.'6
of native Epe tr coilagensuppleA fourth study2T
mentationin RA reportedsignificant improvementin
subjectswho met Pauluscriteria(morningstiffness,joint
joint swelling,and erythrocytesedimentation
tenderness,
of thosetaking type tr coliarate).After 24 weeks,39Vo
gen versusl9Vo toktngplaceboexperiencedsignificant
improvement.While l9%o may appear to be a iarge
responsein the placebo group, it is not unusual to
observethis type of responseia studiesof arthritis.The
impressivefinding wastle high degreeof improvement
type II collagenas
in the grouptreatedwith undenahued,
comparedto the grouptakingtheplacebo.An interesting
observationin this study was that subjectswith a presenceof serumIgA andIgG antibodiesto collagenat the
beginningof the studyhada significantlybefterresponse
to treatmentthanthoselacking suchantibodies.
srudyperIn a fiftLrdouble-blind,placebo-controlled
fomred in Germany,90 subjectswith early RA were
dividedinto groupsreceivingdaily dosesof 1 mg collagen, 10 mg collagen,or placebo.aAt the end of the
study,3 patiensin the 10mg group,I patientin the I mg
group,and no patientsin the placebogroup had experiencedmarkedimprovement.While theseresultsmay not
appearvery impressive,theauthorsweresurprisedby the
degreeof benefit given the small subsetof patients.In
anotherGermanstudy,t6
daily dosesof I mg or 10 mg of
undenatured
type tr collagenresultedin reducedtype II
collagen antibodytites in patientsshowing a clinical
response.This study also suggestedthat 10 mg was a
more effectivedosethan I mg.'6Thesestudiesprovide
the basisandrationalefor the useofnative type tr collagen as a safe and effective modaiity of treatmentfor
thosesufferingfrom RA.
situationresultsfrom the ingeition of high dosesof an
antigen,which in rurninducesa stateof unresponsiveness
from overactiveThl cells.Theseceilsarenot deletedbut
arerenderedincapableofrespondingto a specificantigen.
theyarenrrnedoff, or "anergized,"andwill no
In essence,
longerrecognizetheantigenas a targetfor destruction.'o
Clinical Studies
Via the mechanismof oral tolerance,type tr collagen hasbeenstudiedfor its ability to benefitthosewith
RA. This makessense,becausetype II collagenis the
most abundantstructuralprotein presentin cartiiage.
Numerousanimalmodelsof arttritis havedemonstated
significant benefit from orally administered,native
(undenatured)fype II collagen. Its administrationhas
almostall experimentallyinducible
beenableto supPress
animals,
forms of RA in
including antigen-induced
arthritis, adjuvant arthritis, Upe tr collagen-induced
arthritis, streptococcalcell-wall arthritis, and siliconeinduced arthritis. These impressiveresults led to the
investigationof nativetype tr collagensupplementation
in humanswith RA. In 1993,an open-labelpiiot tial and
a phasetr trid ia humans were conductedat Harvard
Medical School.8Iathe pilot tid, 10 patientsdiagnosed
anddisease-modwith RA had their immunosuppressive
ifying drugs discontinuedand were given 0.1 mg of
nativetypeII collagendaily for 1 month,followedby 0.5
mg of nativetype tr coilagenfor the next? months.Six
of ttre 10 subjectsexperienced
a significantimprovernent
(defined rc >50Vocomparedwith baseline)in swollen
and tenderjoint counts,as well asmorningstiffrtess,50foot walk time, grip strengthtime, and erythrocytesedimentationrate. One subject who had previously been
teated with methotrexateexperiencedcompleteremission.which continuedfor 26 months.No adverseeffects
a
werenoted.EBecauseof theseobservedirnprovements,
placebo-controlledphase II follow-up trial was performedconsistingof 60 subjectswith severe,activeRA.
Participantswere randomly assignedto groups taking
eithera placeboor a daily doseof 0.1 mg nativetype II
collagenfor I month,then0.5 mg for 2 months.At I,2,
significant
and 3 months,thecollagengroupexperienced
improvement(P < 0.05)in the numberof swollenjoints,
the number of painful and tenderjoints (P = 0.06 at 2
months),and 50-footwalk time. Four patientsin the coliagen group, comparedwith no patientsin the placebo
group,experienced
compieteremissionof the disease.
One of the most notablefindings was the lack of side
effects as a result of the teatment, an importantissue
given the side effectsthat can be presentwith various
DMARDs and NSAIDs. Importantly,a recentindepenof type
dent reporthas also confirmedthe effectiveness
II collagenin juvenile RA,26a diseaseaffectingalmost
The Importance of a Native (Undenatured)
Form of Collagen
To conferoral tolerance,lype II collagen must be
used in its undenatured,3-dimensional,triple-helical
structure.r0'2e
Unfortunately,mostproductson the market
containingtype tr collagendo not contain the undenafured form. In theseproductsit has undergoneharsh
manufacturingprocedures
chemicalor high+emperature
which denafureit, thus renderingit inactive and incapable of eliciting an immune responseonce adminisstudiesexist to support
tered.In fact, no peer-reviewed
258
:UNT:ALzMCTICE oF ALTEruNATryrE
MEDICINE
the use of denaturedfype tr collagenin RA, and one
study has showndenaturedrype tr coilagento have no
In orderto insure
impact on the severityof the disease,2e
rype II coliagenis present,highly senthat undenatured
sitive ELISA assaysmust be performedto confirrn that
the collagenis bioiogicallyactive.
Source of Undenatured Type II Collagen
It is well understoodthat type II collagencan be
obtainedfrom all typesof animais,inciudingmice,rats,
chickens, pigs, and dogs, as well as from humans.
However,anidealcommercialsourcewouldbe to obtain
it in a cost-effectiveway from animalshousedandmaintainedin a germ-freeenvironment.Chickensraisedin a
controlled environmentwith ambienttemperatureand
purified air, free from bacteria,viruses,fungi, and other
microorganisms,are currently the best sourceof comtype tr collagen.
mercialundenatured
Dose
Clinical studiessupportthe use of native,undenatured type II collagen and recommendthat it be taken
with waterat bedtime.Furthermore,studieshaveshown
that small doses(rypically 10 mg or less)derivedfrom
chickencartilagework with the humanimmunesystem
to promotehealthyjoints andimprovemobiiiry andflexibility, as well as attenuatingthe symptomsof RA. The
collagenII on an
ideal situationis to ingestundenatured
the
acid
content
in
the stomachis
stomach
wben
empty
protein
absorption
in
a
human
body may
low. Generally,
take from 4 to 8 hours.
Potential Useof Undenatured Type II
Collagenin Osteoarthritis
Therapeuticinterventionsthat work rapidly for RA,
such as NSAIDs or cortisoneinjections,are alsopalliative for OA. Unfortunately,druis that work rapidiy for
OA do not, in general,provide sufficient reductionof
inflammationor pain reiief on their own in rheumatoid
arthritis.OA therapiesin this categoryincludeNSAIDS,
hydrochlohylan g-f 20,andmostprobablyglucosamine
ride andchondroitinsulfate.Presumably,this dichotomy
reiatesto themuchmoresubstantialdegreeof inflammation presentin RA versusOA.
usuallyassociatOA is a wearandtearphenomenon
with rigorousexered with aging;the diseaseprogresses
cise whenmusclesand bonesare alreadyweakeneddue
to aging(exercise
alsocausesmuscleandbonedamagein
patients
by an
with
RA). It is also ch4racterized
aged
joint
to
wear
inflammatorysynovialresponsethat ieads
gradual
deterioraandtear,3o
As RA will effectivelycause
tion and inflammationof certainjoints due to immune
disorders.OA will causewearandteardueto the normal
agingprocessandanincreasein enzymaticactivity.In the
absenceof significant and disfiguring inflammation
(whichis characteristic
of rheumatoidarthritis),wearand
as OA ratherthan RA
tear activity may be misdiagnosed
andtreatedaccordingly.In somecases,OA is addedasan
additionaldiagnosissimply becausewear and tear and
aging persists and exists normally. The biochemical
witb OA inflammation,suchas varimarkersassociated
ous cytokines(interleukin- and interleukin-10),tumor
necrosisfactor-alpha,and interferonsare alsoassociated
Therefore,therapiesusedto
with RA inflammation.rrro'3r
treat RA inJlammationare also used to treat severeOA
that type II
inflarrmation.Earlierresearchdemonstrates
which
inJlammation,
T-cell-mediated
collagensuppresses
is characterizedby cytokines interleukin-4 and interleukin-lO and is seenin the synoviumsof both OA and
RA patients.Anotherbenefitof rypeII collagenis that it
containssmall amountsof glucosamineand chondroitin,
which aregoodfor joint mobility andflexibiiity. In light
of thesefacts,it may be postulatedthat undenatured
rype
II collagenmay alsoprovidebenefitto a significantpopulationof OA patientsas well asthosewith RA.
Conclusion
Finding an effective cure for RA is a major challenge for health professionals.Over-the-counterpain
relievers,NSAIDS and other anti-inJlammatorydrugs,
and mbnoclonalantibodyand COX-[ inhibitors have
major adverseside effects,including liver disease,gasdysfunctions,and, possi8itis, vomiting,cardiovascular
rofecoxib,and
infliximab,
Futhermore,
tuberculosis.
bly,
regular use.
drugs
for
expensive
are
very
celecoxib
which
involves
is
surgery,
Anotherexpensivealternative
have
trials
clinical
a long recoverytime. Severalhuman
undeand usefulnessof
the effectiveness
demonstrated
natured lype II collagenin significantly reducing the
painful symptomsof RA with no adverseside effects'
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other rhcumaticconditions:who is affectednow, will be affected later?
l.
Atthtitis Care Res.1995;8:203-21
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Volume 108, Number 1
Page 159, Abstract No. 769
March 2009
PAIN REDUCTION MEASURED BY GROUND FORCE PLATE IN ARTHRITIC DOGS
TREATED WITH TYPE-II COLLAGEN.
R.C. Gupta1, M. Barnes1, J. Minniear1, J. Lindley1, J.T.Goad1, T.D. Canerdy2, M.Bagchi2, and D.
Bagchi2. 1Toxicology, Murray State University, Hopkinsville/Murray, KY; 2InterHealth
Research Center, Benicia, CA.
Presently, one in four of 77 million pet dogs in the United States is diagnosed with some form of
arthritis. In dogs, osteoarthritis is more common than rheumatoid arthritis and pain is the number
one complaint. This investigation evaluated therapeutic efficacy and safety of glycosylated
undenatured type II collagen (UC-II) in moderately arthritic dogs that received daily placebo or
40 mg type II collagen (10 mg active UC-II) for a period of 120 days, followed by a 30 day
withdrawal. On a monthly basis, dogs were evaluated for overall pain, pain upon limb
manipulation, and pain after physical exertion. In addition, pain was measured using Ground
Force Plate (peak force and impulse area). Dogs on placebo exhibited no significant change in
arthritic conditions. Following 120 days treatment with UC-II, dogs showed significant decreases
in overall pain (77%) and pain after limb manipulation (83%) and exercise (84%). With Ground
Force Plate, peak vertical force value elevated from 7.467 ± 0.419 to 8.818 ± 0.290 Newtons/kg
body wt, and impulse area elevated from 1.154 ± 0.098 to 1.670 ± 0.278 Newtons Sec/kg body
wt, suggesting increase in g-force and decrease in level of pain. Dogs receiving placebo or UC-II
showed no adverse effects in liver, kidney and heart functions (bilirubin, ALT, creatinine, BUN
and CK), or changes in body weight, heart rate, respiration rate, or temperature. In conclusion,
UC-II significantly reduced arthritic pain and is well tolerated.
The undenatured type II collagen (UC-II®) used in this study was supplied by InterHealth Nutraceuticals, Benicia, CA.
J. vet. Pharmacol. Therap. 32, 577–584, doi: 10.1111/j.1365-2885.2009.01079.x.
Therapeutic efficacy of undenatured type-II collagen (UC-II) in comparison
to glucosamine and chondroitin in arthritic horses1
R. C. GUPTA*
T. D. CANERDY*
P. SKAGGS*
A. STOCKER*
G. ZYRKOWSKI*
R. BURKE*
K. WEGFORD*
J. T. GOAD*
K. ROHDE*
D. BARNETT*
W. D E WEES*
M. BAGCHI &
D. BAGCHI *Murray State University,
Murray ⁄ Hopkinsville, KY; InterHealth
Research Center, Benicia, CA, USA
Gupta, R. C., Canerdy, T. D., Skaggs, P., Stocker, A., Zyrkowski, G., Burke, R.,
Wegford, K., Goad, J. T., Rohde, K., Barnett, D., DeWees, W., Bagchi, M.,
Bagchi, D. Therapeutic efficacy of undenatured type-II collagen (UC-II) in
comparison to glucosamine and chondroitin in arthritic horses. J. vet. Pharmacol. Therap. 32, 577–584.
The present investigation evaluated arthritic pain in horses receiving daily
placebo, undenatured type II collagen (UC-II) at 320, 480, or 640 mg
(providing 80, 120, and 160 mg active UC-II, respectively), and glucosamine
and chondroitin (5.4 and 1.8 g, respectively, bid for the first month, and
thereafter once daily) for 150 days. Horses were evaluated for overall pain, pain
upon limb manipulation, physical examination, and liver and kidney functions.
Evaluation of overall pain was based upon a consistent observation of all
subjects during a walk and a trot in the same pattern on the same surface. Pain
upon limb manipulation was conducted after the walk and trot. It consisted of
placing the affected joint in severe flexion for a period of 60 sec. The limb was
then placed to the ground and the animal trotted off. The response to the flexion
test was then noted with the first couple of strides the animal took. Flexion test
was consistent with determining clinically the degree of osteoarthritis in a joint.
Horses receiving placebo showed no change in arthritic condition, while those
receiving 320 or 480 or 640 mg UC-II exhibited significant reduction in
arthritic pain (P < 0.05). UC-II at 480 or 640 mg dose provided equal effects,
and therefore, 480 mg dose was considered optimal. With this dose, reduction
in overall pain was from 5.7 ± 0.42 (100%) to 0.7 ± 0.42 (12%); and in pain
upon limb manipulation from 2.35 ± 0.37 (100%) to 0.52 ± 0.18 (22%).
Although glucosamine and chondroitin treated group showed significant
(P < 0.05) reduction in pain compared with pretreated values, the efficacy was
less compared with that observed with UC-II. In fact, UC-II at 480 or 640 mg
dose was found to be more effective than glucosamine and chondroitin in
arthritic horses. Clinical condition (body weight, body temperature, respiration
rate, and pulse rate), and liver (bilirubin, GGT, and ALP) and kidney (BUN and
creatinine) functions remained unchanged, suggesting that these supplements
were well tolerated.
(Paper received 25 November 2008; accepted for publication 30 March 2009)
Ramesh C. Gupta, DVM, MVSc, PhD, DABT, FACT, FATS, Professor and Head,
Toxicology Department, Murray State University, Breathitt Veterinary Center, PO.
Box 2000; 715 North Drive, Hopkinsville, KY 42240, USA. E-mail:
[email protected]
1
Presented in part at the Annual Meeting of Eurotox, Amsterdam, September
10–12, 2007; American Academy of Veterinary Nutrition, San Antonio, TX,
June 3–5, 2008; and IXth World Conference on Clinical Pharmacology and
Therapeutics, Quebec City, Canada, July 27–August 1, 2008.
INTRODUCTION
Arthritis is a chronic debilitating disease that commonly inflicts
millions of horses around the world, because of excessive
2009 Blackwell Publishing Ltd
running and exercise, injury, immune disorder, aging, or genetic
predisposition (Ruggeiro, 2002). The two most common types of
arthritis are osteoarthritis and rheumatoid arthritis. In horses,
osteoarthritis occurs with a greater frequency than rheumatoid
577
578 R. C. Gupta et al.
arthritis or any other form of joint disease, like in humans and
dogs. Osteoarthritis is an inflammatory joint disease, which is
characterized by degeneration of the cartilage, hypertrophy of
bone at the margins, and changes in the synovial membrane and
fluid, which eventually leads to pain and stiffness of joints
(Goldring, 2000; Bellamy et al., 2001). This disease can wear
down cartilage in a joint to the point that bone rubs against
bone, resulting in loss of cartilage, and, in severe cases, cartilage
fragments can break off and irritate muscles with pain that are
adjacent to the bone. Chronic joint inflammation usually results
in progressive joint destruction, deformity, and loss of function
(van Roon et al., 2001).
Current therapy of arthritis relies upon nonsteroidal antiinflammatory drugs (NSAIDs) alone or in combination with
some other pain killers. Present treatments aim at alleviating
pain, control inflammation, and preserve ability to perform daily
functions. NSAIDs, which are cyclooxygenase (COX) inhibitors,
alleviate pain, but do not eliminate signs and symptoms of active
disease. In general, COX-II inhibitors (such as rofecoxib,
celecoxib, carprofen, and deracoxib) are considered safer than
nonspecific COX inhibitors (such as aspirin and ibuprofen). In
the recent past, chronic use of COX-II inhibitors has been
attributed to various side effects, including gastrointestinal (GI)
ulceration and bleeding, and hepatic, renal and cardiovascular
complications (Richardson, 1991; PDR, 2006; Infante & Lahita,
2000; Matteson, 2000; Schuna & Megeff, 2000; Matheson &
Figgilt, 2001; Lamarque, 2004; Solomon et al., 2004; Muhlfeld
& Floege, 2005). To our knowledge, such side effects have not
been reported in horses.
Presently, nutraceuticals are also used to ease the pain and
discomfort of arthritis in both humans and animals, including
horses (Trumble, 2005; Clegg et al., 2006; Bruyere & Reginster,
2007; Morva, 2007). These products are commonly used in
horses because they are administered orally, well tolerated and
considered safe. Nutraceuticals are defined as functional foods,
natural products, or parts of food that provide medicinal,
therapeutic, or health benefits, including the prevention or
treatment of disease. The present investigation utilized three
supplements (UC-II, glucosamine, and chondroitin), and their
brief description is provided here. Glycosylated undenatured
type-II collagen (UC-II) is derived from chicken sternum and
prepared under good manufacturing practices (GMPs), using low
temperature, which preserves its undenatured form and ensures
intact biological activity with active epitopes. Glucosamine,
extracted from crab, lobster, or shrimp shells, is an aminomonosaccharide precursor of the disaccharide unit of glycosaminoglycan, which is the building block of proteoglycans, the
ground substance of cartilage (Paroli et al., 1991). Chondroitin
sulfate, extracted from animal cartilage, such as tracheas and
shark cartilage, is a part of a large protein molecule (proteoglycan) that gives cartilage elasticity.
Currently, glucosamine and chondroitin are the two most
commonly used nutraceuticals in humans as well as in animals
(including dogs, cats, and horses), to alleviate pain associated
with arthritis (Dechant et al., 2005; Trumble, 2005). However,
based on recent randomized controlled trials and meta-analysis,
these supplements have shown only small-to-moderate symptomatic efficacy in human osteoarthritis (Bruyere & Reginster,
2007), although, this finding is still debated (Clegg et al., 2006;
Rozendaal et al., 2008). In our recent studies conducted in
dogs, daily administration of UC-II at 40 mg (providing 10 mg
active UC-II, respectively) daily dose for 120 days markedly
reduced arthritic pain (DeParle et al., 2005; D’Altilio et al.,
2007). Furthermore, our follow up studies also demonstrated
that UC-II (40 mg daily dose) in combination with other
nutraceuticals (glucosamine plus chondroitin) markedly
reduced the signs associated with arthritis in dogs, and thereby,
tremendously improved daily activity, as climbing stairs and
walking exercise. In a number of in vivo and in vitro
investigations, glucosamine and chondroitin have been found
very effective against osteoarthritis in horses (Fenton et al.,
2000, 2002; Dechant et al., 2005; Neil et al., 2005; Trumble,
2005). In brief, these studies suggested that the combination of
glucosamine and chondroitin appears to be more effective in
preventing or treating osteoarthritis in horses than either
product alone.
The present investigation was therefore undertaken with two
specific objectives: (i) to determine if daily administration of
active UC-II, or glucosamine plus chondroitin, can alleviate the
signs and symptoms of arthritis in horses and (ii) to determine if
these supplements are well tolerated and safe to administer for
the long term in arthritic horses.
MATERIALS AND METHODS
Animals
All horses used in this investigation were diagnosed with
osteoarthritis at the level of moderate severity. They were placed
at the equine center of Murray State University. During the
entire course of investigation, these horses were under the
supervision of licensed veterinarians. The protocol of the present
investigation for using arthritic horses and their treatment was
in compliance with the Murray State University Animal Use and
Care Guidelines. All animals were used routinely in their daily
workout schedule (riding classes). They were lodged into the
amount of time for daily workouts and rest periods. All animals
had the same workout protocol and rest time.
Criteria for inclusion into the study
From a large pool of horses located at the Murray State
University Equine Center, candidates were chosen based upon
outward visual signs of lameness. Once the lame candidates were
identified, the animals with evidence of osteoarthritis based upon
physical examination by two licensed veterinarians (Dr. Terry D.
Canerdy and Dr. William DeWees) were included in the study.
Evidence of osteoarthritis includes joint effusion in one or more
joints of the limbs, reduced joint flexibility, crepitation of the joint
on manipulation, and an increase in lameness upon flexion of
the affected joint.
Horse arthritis treatment 579
Supplements
Glycosylated undenatured type-II collagen (UC-II), in the form of
capsules as a dietary supplement, was provided by InterHealth
Nutraceuticals, Inc. (Benicia, CA, USA). Similar to our previous
studies conducted in dogs, in the present investigation, the
undenatured form of glycosylated type-II collagen was used, as
this form of UC-II is found to be significantly more effective than
denatured type-II collagen against arthritis (Nagler-Anderson
et al., 1986; Bagchi et al., 2002). It should be noted that
undenatured type-II collagen can be denatured (hydrolyzed) by
chemical or high-temperature, altering its molecular structure
and integrity, and denatured collagen does not have active
epitopes rendering it inactive. Cosequin equine powder concentrate (glucosamine and chondroitin) was purchased from
Nutramax (Edgewood, MD, USA).
Experimental design and animal treatment
The present investigation was conducted on moderately osteoarthritic horses. In preliminary dose-dependent studies, horses
received UC-II at 80 or 160 mg (providing 20 and 40 mg active
UC-II, respectively) daily dose for a period of 150 days. Based on
this pilot dose-dependent study, the final investigation was
carried out on five groups of horses (n = 5–6) receiving placebo,
UC-II (higher doses), or glucosamine in combination with
chondroitin daily for 150 days. Group-I horses received placebo.
Horses in Group-II, -III, and -IV received UC-II at 320, 480, and
640 mg (providing 80, 120, and 160 mg active UC-II, respectively), accordingly. Group-V horses received glucosamine and
chondroitin (5.4 and 1.8 g ⁄ day, respectively, bid for the first
month, and once daily thereafter). Treatment in all five groups
was given daily (in the form of capsules administered orally in a
handful of grain) for a period of 5 months. While rationale for
selection of doses of UC-II was based on preliminary studies,
doses of glucosamine and chondroitin were based on the product
information provided on the insert along with Cosequin
(Nutramax).
following flexion. Flexion tests are commonly used in the equine
industry in determining the severity of a joint abnormality.
Scale used in pain measurement
The 0–10 global pain assessment was a scale used because it
provided a broad range of scale for pain. This scale was
consistently used throughout the investigation. In brief, 0, no
pain; 5, moderate pain; and 10, severe and constant pain. To our
knowledge, a universal scale does not exist to assess the pain.
For pain upon limb manipulation, results were graded on a
scale of 0–4: 0, no pain, 1, mild pain; 2, moderate pain; 3, severe
pain; and 4, severe and constant pain. The 0–4 scale was taken
from the American Association of Equine Practitioners (AAEP)
scorecard on lameness. They actually have 0–5, but category 5
was dropped because it indicates inability of an animal to move.
None of our subjects fit this category and therefore it was not
used.
Physical examination
Body weights and physical evaluation were also determined on a
monthly basis for 150 days. On a monthly basis horses were
evaluated for body weight, body temperature, and pulse rate.
Biochemical assays
Blood samples were collected by jugular venipuncture using
20-gauge needles and 12-cc syringes. Serum was separated in a
marble top tube (without anticoagulant) and transferred into
plastic snap-top tubes. Serum samples were frozen immediately
and kept at )80 C until analyzed for bilirubin, GGT, ALP,
blood urea nitrogen (BUN) and creatinine, using Beckman
Coulter CX5-PRO Synchron Clinical System (Fullerton, CA,
USA). Bilirubin, GGT, and ALP were used as markers of liver
function, and BUN and creatinine were used as markers of renal
function.
Statistical analysis
Pain assessment
The horses were evaluated for overall pain and pain after limb
manipulation, on a monthly basis for a period of 150 days.
Overall pain evaluation was based upon a consistent observation
of all subjects when the animal was at a walk and a trot. All
subjects were moved in the same pattern on the same surface
consistently. Gross pain measurement was done and recorded
during the horses movement trials.
Pain upon limb manipulation was conducted after the walk
and trot. It consisted of placing the affected joint in severe flexion
for a period of 60 sec. The limb was then placed to the ground
and the animal trotted off. The response to the flexion test was
then noted with the first couple of strides the animal took.
Flexion test was consistent with determining clinically the degree
of osteoarthritis in a joint. With an increase in osteophytes, the
animal has a degree of discomfort on movement of the limb
The data of body weight in Table 1, serum chemistry in Table 2,
and pain measurement in Figs 1 & 2, are presented as means ±
SEM. Statistical significance of differences was determined by
ANOVA coupled with Tukey–Kramer test using the NCSS 2000
Statistical Software for Windows (Kaysville, UT, USA). Groups
were compared using Duncan’s Multiple-Comparison Test.
Differences with P < 0.05 were considered statistically significant.
RESULTS
Horses used in this investigation were diagnosed with osteoarthritis at a moderate severity. They exhibited some of the
common symptoms, such as difficulty during walking, stiffness
after periods of inactivity, swelling ⁄ tenderness in one or more
joints, steady pain in joints, and lameness.
Table 1. Effect of UC-II or Glucosamine plus Chondroitin on body weight (lbs) of horses
Day
0
30
60
90
120
150
Group-I placebo
1161
1172
1151
1131
1053
1080
±
±
±
±
±
±
40
16
48
43
28
39
(100)
(101)
(99)
(98)
(90)
(93)
Group-II 320 mg UC-II
1080
1070
1066
1068
1069
1082
±
±
±
±
±
±
18
23
22
21
17
23
Group-III 480 mg UC-II
(100)
(99)
(99)
(99)
(99)
(100)
1150
1147
1127
1137
1150
1102
±
±
±
±
±
±
59
58
53
56
66
59
Group-IV 640 mg UC-II
(100)
(100)
(98)
(99)
(100)
(96)
1190
1200
1164
1178
1203
1167
±
±
±
±
±
±
48
45
49
42
47
33
Group-V Gluc. + Chon.
(100)
(101)
(98)
(99)
(101)
(98)
1195
1204
1178
1185
1190
1195
±
±
±
±
±
±
30
27
33
34
31
49
(100)
(101)
(99)
(99)
(100)
(100)
Values are means ± SEM (n = 5–7). No significant change in body weight (P > 0.05). Numbers in parentheses are percent changes compared with
values of day 0 (100%).
Table 2. Effects of UC-II or glucosamine plus chondroitin on markers of liver and renal functions in serum of horses
Days
Parameters
Group
BIL (mg ⁄ dL)
I
II
III
IV
V
I
II
III
IV
V
I
II
III
IV
V
I
II
III
IV
V
I
II
III
IV
V
GGT (IU ⁄ L)
ALP (IU ⁄ L)
BUN (mg ⁄ dL)
Creatinine (mg ⁄ dL)
0
1.18
1.33
0.98
0.93
1.87
12.4
17.5
14.2
14.8
12.0
95.2
79.4
84.3
81.5
82.6
16.4
17.7
17.3
18.7
18.5
1.64
1.50
1.42
1.52
1.43
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
30
0.12
0.10
0.13
0.14
0.35
2.01
9.51
1.99
0.79
0.69
9.61
17.91
8.50
3.33
7.65
0.87
1.19
1.50
0.88
0.50
0.08
0.07
0.06
0.06
0.18
1.24
1.60
1.05
0.77
1.84
11.4
15.4
16.2
16.0
11.7
90.2
58.1
73.2
68.5
77.7
13.6
17.9
17.1
14.2
19.3
1.58
1.56
1.43
1.48
1.64
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.12
0.06
0.13
0.12
0.43
1.21
5.42
1.90
0.52
0.70
7.09
22.97
5.77
2.84
3.98
0.50
1.12
0.83
0.87
0.92
0.19
0.07
0.06
0.06
0.19
60
1.22
1.70
1.05
1.05
1.76
11.2
14.8
13.0
13.1
11.5
86.4
81.3
76.7
75.5
62.6
14.0
17.3
16.0
17.3
18.9
1.66
1.51
1.58
1.48
1.39
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.17
0.04
0.09
0.12
0.27
1.68
4.82
1.51
0.17
1.31
7.42
15.53
5.71
3.23
6.10
0.89
1.08
0.86
0.91
0.99
0.08
0.05
0.12
0.05
0.18
90
1.34
1.30
1.15
1.04
1.87
11.2
15.3
11.5
13.0
12.1
94.2
87.8
74.7
72.8
71.4
16.4
15.3
15.8
19.0
16.6
1.46
1.47
1.33
1.30
1.53
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.19
0.09
0.19
0.12
0.37
1.39
8.46
1.06
0.58
0.73
10.18
19.38
9.43
3.97
4.50
1.21
1.19
0.70
1.13
1.74
0.12
0.07
0.07
0.05
0.17
120
1.22
1.30
1.63
1.30
2.07
11.8
15.1
12.7
14.5
13.1
97.8
84.3
85.7
77.8
75.2
15.2
15.1
16.3
18.5
18.0
1.44
1.50
1.45
1.33
1.50
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.16
0.03
0.27
0.16
0.36
1.59
11.32
1.28
0.99
0.37
14.65
22.66
12.46
3.66
5.50
1.43
1.62
1.23
0.43
1.46
0.14
0.07
0.04
0.02
0.15
150
1.34
1.20
1.17
0.93
2.22
13.4
14.5
12.8
16.6
12.1
95.4
84.5
88.3
97.5
66.5
18.0
14.8
18.8
18.8
17.2
1.66
1.35
1.48
1.30
1.60
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.11
0.10
0.14
0.10
0.48
1.21
7.90
1.35
1.50
0.65
10.17
30.80
9.96
4.61
7.70
1.34
2.00
1.14
1.49
1.32
0.15
0.07
0.16
0.08
0.23
Values are means ± SEM (n = 5–7). No significant change in any parameter (P > 0.0.5).
All horses were grossly and physically examined and flexed for
lameness on a monthly basis for a period of 150 days. UC-II at a
320, 480, or 640 mg daily dose (providing 80, 120, or 160 mg
active UC-II, respectively) provided significant reductions in
arthritic pain by 60 days of treatment (Figs 1 & 2). In fact, with
higher daily dose of UC-II (480 or 640 mg), significant reduction
in overall pain was observed as early as after 30 days of
treatment. With UC-II (320 or 480 or 640 mg), horses showed
maximal pain reduction by 150 days of treatment (overall pain
reduction, 79%, 88%, and 91%, respectively; and pain after limb
manipulation, 71%, 78%, and 80%, respectively). After
5 months of UC-II treatment, the horses became very active,
and performed normally in their daily activities.
Horses receiving glucosamine (5.4 g) plus chondroitin
(1.8 g), bid for the first 30 days, and once daily, thereafter
for the next 120 days showed significant decrease in pain after
60 days of treatment (reduction in overall pain, 36%; and
reduction in pain after limb manipulation, 31%). Maximal
pain reduction was noted after 150 days of treatment (overall
pain, 68%; and pain after limb manipulation, 69%). On
comparison, the UC-II (480 or 640 mg daily dose) was found
to be approximately twice as effective as glucosamine plus
chondroitin, based on pain after limb manipulation on
day 90.
None of the horses in any group showed any adverse effects on
body weight (Table 1), hepatic (bilirubin, GGT, and ALP) or
renal (BUN and creatinine) function markers (Table 2), or body
temperature, pulse rate, and respiration rate (data not shown),
suggesting that these supplements are well tolerated by arthritic
horses and safe to administer for a long term.
Fig. 1. On a monthly basis, overall pain in
horses was measured as a general gross
observation and graded on a scale of 0–10: 0,
no pain; 5, moderate pain; and 10, severe and
constant pain. Values are mean ± SEM
(n = 5–7). * = Indicates significant difference
between the values of day 0 and posttreatment (P < 0.05).
Fig. 2. On a monthly basis, pain upon limb
manipulation was evaluated by animal’s pain
during the flexion of all four limbs for a min.
then jogged after each leg was flexed. Results
were graded on a scale of 0–4: 0, no pain;
1, mild pain; 2, moderate pain; 3, severe pain;
and 4, severe and constant pain. Values are
mean ± SEM (n = 5–7). *Significant difference between the values of day 0 and posttreatment (P < 0.05). **Significant difference
between the values of UC-II-treated and
glucosamine plus chondroitin-treated horses
(P < 0.05).
DISCUSSION
The present investigation evaluated therapeutic efficacy, tolerability, and safety of glycosylated undenatured type II collagen
(UC-II) and glucosamine and chondroitin in moderately arthritic
horses, following a long term of their use. The present findings
revealed that the therapy with UC-II at 320 or 480 or 640 mg
daily dose for a period of 5 months provided significant
improvement in ameliorating the overall pain and pain after
limb manipulation in arthritic horses. Although significant antiarthritic effects were noted after 60–90 days, the maximal
physical improvements were observed after 150 days of treatment and the horses were more playful and active (Figs 1 & 2).
This suggests that prolonged treatment with these supplements
leads to better therapeutic results. Based on this study, it appears
that 480 mg daily dose of UC-II provides the best results, as at
further higher dose (640 mg providing 160 mg active UC-II),
UC-II offered therapeutic efficacy no greater than that observed
with 480 mg daily dose.
Like previous studies conducted in two monogastric species,
humans (Nagler-Anderson et al., 1986; Trentham et al., 1993,
2001; Barnett et al., 1996, 1998; Sieper et al., 1996; Trentham,
1998) and dogs (DeParle et al., 2005; D’Altilio et al., 2007), in
the horses, we used the undenatured form of UC-II. This form of
collagen with triple helix structure and active epitopes is found to
be significantly more effective than denatured form against
arthritis (Nagler-Anderson et al., 1986; Bagchi et al., 2002). In
none of the species has UC-II been found to produce any adverse
effects (Bagchi et al., 2002; D’Altilio et al., 2007), which
demonstrated that once UC-II is ingested, stomach acids and
enzymes perform a partial digestion of the collagen matrix,
resulting in chains of soluble collagen molecules of varying
length, containing biologically active epitopes. These structurally
precise natural epitopes in UC-II interact with Peyer’s Patches
and trigger the complex series of immunological events that, in
case of rheumatoid arthritis, down-regulates the body’s out-ofcontrol autoimmune response (Fig. 3) (Trentham et al., 2001;
Bagchi et al., 2002). In the case of osteoarthritis, which is often
characterized by a subclinical immune disorder and a vicious
cycle of inflammatory events, UC-II can promote a significant
reduction in inflammation (Bagchi et al., 2002). UC-II functions
through a process of oral tolerization that takes place in the
small intestine where the food is absorbed. Through a complex
series of immunological events, patches of lymphoid tissue
(Peyer’s Patches) surrounding the small intestine, screen
incoming compounds and serve as a ‘switch’ to turn the body’s
immune response to foreign substances on or off, depending
upon the substance. In dogs and humans, a small amount of
undenatured UC-II (10 mg active UC-II ⁄ day) taken orally has
been shown to turn off the immune response targeted at type-II
collagen in joint cartilage, and no adverse effects have been
noted (Trentham et al., 1993, 2001; Trentham, 1998; DeParle
et al., 2005). This immunization process helps the body to
differentiate between elements that are foreign invaders to the
body and those that are nutrients and are good for the body
(Weiner, 1997; Trentham, 1998). UC-II stops the immune
system from attacking and damaging its own joint cartilage,
thereby improving joint mobility and flexibility (Trentham et al.,
1993; Trentham, 1998; Bagchi et al., 2002). Type-II collagen is
one of the primary connective tissues of the body, providing
flexibility and support to bone joints. As UC-II is found to be as
equally effective in horses, as reported earlier in humans and
dogs, and it is presumed that the mechanisms described for
humans and dogs may also hold true for horses. Although the
precise biochemical mechanism involved in UC-II- induced
pharmacological anti-arthritic effects in humans, dogs or horses,
is not clearly established.
Glucosamine and chondroitin (5.4 and 1.8 g, respectively,
bid for the first 30 days, and once daily for the next 120 days)
significantly reduced arthritic pain by 60 days of treatment
(Figs 2 & 3), but maximal pain reduction was observed after
150 days (68% in overall pain and 69% in pain after limb
manipulation). Recently, a number of in vivo and in vitro studies
support the use of glucosamine and chondroitin in arthritic
horses (Fenton et al., 2000, 2002; Dechant et al., 2005; Neil
et al., 2005; Trumble, 2005). Unlike UC-II, glucosamine relieves
pain by enhancing proteoglycan synthesis, which is impaired in
osteoarthritic cartilage (Hougee et al., 2006). Chondroitin
sulfate aids in keeping cartilage tissue from dehydrating and
assists in cushioning impact stress and reducing joint pain.
Chondroitin sulfate is also believed to block certain enzymes
that result in the breakdown of cartilage. In an in vitro study,
Dechant et al. (2005) demonstrated that glucosamine plus
chondroitin: (i) reduced total glycosaminoglycan degradation,
which is involved in osteoarthritis and (ii) have no detrimental
effects on cartilage metabolism. Furthermore, from a series of
in vitro studies, Fenton et al. (2000, 2002) revealed that
glucosamine can prevent experimentally induced cartilage
degradation, and therefore support the use of this product in
prevention or treatment of cartilage loss in arthritic horses. In a
recent in vivo study, glucosamine and chondroitin ameliorated
arthritic pain in dogs, but comparatively UC-II was significantly
more effective. Similarly, in horses UC-II (480 or 640 mg daily
dose) was found to be more effective compared with glucosamine and chondroitin based upon limb manipulation on
90 days of treatment.
Aging - Stress - injury
Wear and tear
Acceleration of inflammation
Initiation of inflammation
Adhesion molecules
UC-II
UC-II deactivates killer T-cells
Pro-Inflammatory cytokines
Activate Killer T-Cells
Activate B-cells
Antibodies
Binding to antigens
Antibody-Tagged collagen (ATC)
Recruitment of macrophages to ATC
Collagenase secreted by macrophages
Breakdown of collagens by collagenase
Formation of Collagen-Debris
Removal of debris by macrophage
Erosion of joint collagen
Exposure of bones & bone crunching
Pain, Loss of mobility and flexibility
Osteoarthritis
Fig. 3. Mechanism of action of UC-II in
osteoarthritis.
In conclusion, daily administration of UC-II at varying doses
(320 or 460 or 640 mg) significantly reduced the signs and
symptoms of arthritis in horses. Daily administration of
glucosamine plus chondroitin also provided reduction in
arthritic pain, but the efficacy was less than UC-II. All three
supplements were well tolerated and did not produce any
adverse events.
ACKNOWLEDGMENT
This study was supported by InterHealth Nutraceuticals Inc.
(Benicia, CA, USA).
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SHORT COMMUNICATION
Therapeutic efficacy and safety of undenatured type-II collagen (UC-II)
alone or in combination with ())-hydroxycitric acid and chromemate in
arthritic dogs1
A. PEAL*
M. D’ALTILIO*
C. SIMMS*
M. ALVEY*
R. C. GUPTA*
J. T. GOAD*
T. D. CANERDY*
M. BAGCHI &
D. BAGCHI , à
*Toxicology Department, Murray State University, Hopkinsville/Murray, KY, USA; Research and Development, InterHealth Nutraceuticals,
Inc., Benicia, CA, USA; àDepartment of Pharmacy Sciences, Creighton University Medical Center, Omaha, NE, USA
(Paper received 23 December 2006; accepted for publication 16 February 2007)
Ramesh C. Gupta, Breathitt Veterinary Center, Murray State University, Hopkinsville, KY 42240, USA. E-mail:
[email protected]
The present investigation evaluated therapeutic efficacy and
safety of glycosylated undenatured type II collagen (active UC-II)
alone or in combination with ())-hydroxycitric acid (HCA-SX,
SuperCitrimax) and ChromeMate (chromium niacinate, CM).
Twenty five arthritic dogs in five groups (n ¼ 5) received daily
treatment as follows: group I (placebo), group II (10 mg active
UC-II), group III (1800 mg HCA-SX), group IV (1800 mg HCASX + 100 lg CM), and group V (1800 mg HCA-SX + 100 lg
CM + 10 mg active UC-II). The treatment was given daily for
120 days, followed by 30 days withdrawal. The dogs were
evaluated for overall pain, pain upon limb manipulation, and
exercise-associated lameness, on a monthly basis. Blood-serum
samples were assayed for markers of liver [bilirubin and alanine
aminotrasferase (ALT)] and renal [blood urea nitrogen (BUN)
and creatinine] functions. Group I dogs exhibited no significant
change in arthritic conditions. The dogs receiving active UC-II
alone (group II) or in combination with HCA-SX + CM (group V)
for 90 days showed marked reduction in overall pain (46–57%),
pain upon limb manipulation (50–55%), and exercise-associated
lameness (44–46%). In groups II and V, maximum pain
reduction was seen after 120 days treatment (62–70%, 67–
91%, and 69–78%, correspondingly). All dogs experienced a
relapse of pain after a withdrawal period of 30 days. None of the
dogs in any group showed adverse effects or changes in liver or
kidney function markers, or body temperature. Body weights of
all dogs remained significantly unchanged in all the groups.
1
Presented at the 45th Annual Meeting of the Society of Toxicology, San
Diego, CA, 6–9 March 2006.
Ó 2007 The Authors. Journal compilation Ó 2007 Blackwell Publishing Ltd
These data suggest that treatment of arthritic dogs with active
UC-II alone or in combination with HCA-SX and CM ameliorates
the signs of arthritis, and these supplements are well tolerated as
no adverse effects were noted.
Arthritis is a chronic degenerative disease of the joints causing
pain, stiffness, swelling, and lameness (McLaughlin, 2000;
Burns, 2006). Arthritis commonly affects large breed dogs
(Richardson et al., 1997), because of overweight/obesity, lack of
exercise, physical injury, aging, infection, immune disorder, or
genetic predisposition. Dogs suffer more often with osteoarthritis
than with rheumatoid arthritis (Hielm-Bjorkman et al., 2003).
Osteoarthritis is an inflammatory joint disease, which is
characterized by degeneration of the cartilage, hypertrophy of
bone at the margins in the synovial membrane, and eventually
pain and stiffness of joints (Vaughan-Scott & Taylor, 1997).
Present therapy for arthritis in dogs relies upon drugs that
alleviate pain and control inflammation to preserve daily activity.
Chronic use of cyclooxygenase (COX) inhibitors (nonsteroidal
anti-inflammatory drugs, NSAIDs) is linked to numerous side
effects, including gastrointestinal (GI) bleeding, and hepatic and
renal dysfunction (Lobetti & Joubert, 2000; Bergh & Budsberg,
2005). In the recent past, two commonly used FDA-approved
drugs (Rimadyl and Deramaxx), which are NSAIDs and selective
inhibitors of COX-II, have been shown to cause severe side effects
(Moreau et al., 2003; Sessions et al., 2005).
In recent years, InterHealth Nutraceuticals, Inc. (Benicia, CA,
USA) has developed three supplements (active UC-II, SuperCitrimax, and ChromeMate) that are proven to be very effective in
human arthritis and/or obesity patients (Bagchi et al., 2002;
275
276 A. Peal et al.
Soni et al., 2004; Shara et al., 2005). The structural integrity of
undenatured type II collagen in a active UC-II sample was
determined by Transmission Electron Microscope procedure
using an EM JEOL 100 CX (Peabody, MA, USA), while the
amount of undenatured type-II was analyzed by Capture ELISA
kit (Chondrex LLC, Redmond, WA, USA) (Bagchi et al., 2002).
Twenty-five client-owned arthritic dogs weighing between 62
and 96 pounds were used in this investigation. These dogs
exhibited the signs of osteoarthritis (joint stiffness, lameness,
pain, swollen joints, and difficulty in getting up or down and
walking), which was confirmed radiographically. Dogs were
randomly divided into five groups (n ¼ 5) receiving daily
treatment as follows: group I (placebo), group II (10 mg active
UC-II), group III (1800 mg HCA-SX), group IV (1800 mg HCASX + 100 lg CM); and group V (1800 mg HCA-SX + 100 lg
CM + 10 mg active UC-II). Daily treatment was given for
120 days, followed by a 30-day withdrawal.
Overall pain, pain upon limb manipulation, and lameness
after physical exertion was measured on a monthly basis for a
period of 150 days. Grading for pain measurement is described
in figure legends (Figs 1–3), and in our recent publications
(DeParle et al., 2005; D’Altilio et al., 2007).
Data of pain assessment are shown in Figs 1–3. Dogs receiving
placebo showed no improvement in arthritic pain or lameness.
Dogs receiving active UC-II alone showed significant reduction in
overall pain, pain upon limb manipulation, and exercise-associated lameness. Maximum improvement was noted after 120 days
of treatment. HCA-SX alone did not provide significant improvement in pain reduction, but in combination with CM, it provided
significant reductions in arthritic signs, including pain. Active
UC-II in combination with HCA-SX and CM markedly reduced
overall pain (70%), pain upon limb manipulation (67%), and
exercise-associated lameness (69%). Following a 30-day withdrawal, dogs experienced a relapse of pain and lameness. Data of
dogs’ body weight, body temperature, and serum chemistry
related to liver and renal function (bilirubin, ALT, BUN, and
creatinine), did not show any significant changes at 0, 30, 60, 90,
120, and 150 days.
Recently, in a double-blinded pilot study, we found for the first
time that active UC-II (1 or 10 mg/day) given for 90 days
significantly reduced the pain in arthritic dogs (DeParle et al.,
2005). Dogs given 10 mg active UC-II performed overall better
than those given a 1-mg dose. In a follow-up study, dogs
receiving active UC-II (10 mg/day) alone or in combination with
Glucosamine HCl (2000 mg/day) and Chondroitin sulfate
(1600 mg/day) for 120 days showed significant reductions in
pain (D’Altilio et al., 2007). The present data revealed that daily
therapy with active UC-II alone or with HCA-SX + CM for
120 days provided remarkable improvements in the lifestyle of
dogs by reducing arthritic pain. The majority of anti-arthritic
effects appeared to be obtained from active UC-II, which exerts its
effects through a process of oral tolerization (Trentham, 1998;
DeParle et al., 2005; D’Altilio et al., 2007). Dogs receiving these
supplements were more playful and showed significant reductions in the signalments of the arthritic condition, including pain
and lameness (Figs 1–3).
In conclusion, arthritic dogs treated with active UC-II alone
or in combination with HCA-SX and CM showed marked
reductions in arthritic pain and lameness. Overall, the dogs
became very active and playful. The supplements did not
OVERALL PAIN
Placebo
HCA-SX + CM
UC-II
HCA + CM + UC-II
HCA-SX
7
6
Pain level
5
*
4
*
*
*
3
*
*
*
*
*
2
*
1
0
0
30
60
90
Day
120
150
Fig. 1. Effects of active UC-II alone (10 mg/dog/day) or
in combination with HCA-SX (1800 mg/dog/day) +
CM (100 lg/dog/day) on overall pain in arthritic dogs
(n ¼ 5 dogs/group). Daily treatment continued for
120 days, followed by a withdrawal period of 30 days.
Overall pain was graded on a scale of 0–10: 0, no pain;
5, moderate; and 10, severe and constant pain. For
details, see the text and DeParle et al. (2005). *Significantly different when compared with pretreated
values (P < 0.05). Active UC-II, glycosylated undenatured type-II collagen; HCA-SX, ())-hydroxycitric acid;
and CM, ChromeMate.
Arthritis treatment in dogs 277
PAIN FROM LIMB MANIPULATION
Placebo
HCA-SX + CM
UC-II
HCA + CM + UC-II
HCA-SX
3.5
3.0
Pain level
2.5
Fig. 2. Effects of active UC-II alone or in combination
with HCA-SX + CM on pain after limb manipulation.
Pain was evaluated by animal’s vocalization or other
observations of pain during the extension and flexion of
all four limbs for few min. Pain was graded on a scale of
0–4: 0, no pain; 1, mild; 2, moderate; 3, severe; and 4,
severe and constant pain. For details, see the text and
Fig. 1. *Significantly different when compared with
pretreated values (P < 0.05).
2.0
*
1.5
*
*
*
1.0
*
0.5
*
0.0
0
30
60
90
120
150
Day
PAIN AFTER PHYSICAL EXERTION
4
Placebo
HCA-SX + CM
UC-II
HCA + CM + UC-II
HCA-SX
Pain level
3
*
2
*
Fig. 3. Effects of active UC-II alone or in combination
with HCA-SX + CM on pain after physical exertion.
Lameness was measured after physical exercise for
limping, holding limb up, rigidity of limbs, etc. Signs of
pain and lameness were graded on the scale of 0–4: 0,
no pain; 1, mild; 2, moderate; 3, severe; and 4, severe
and constant pain. For details, see the text and Fig. 1.
*Significantly different when compared with pretreated
values (P < 0.05).
*
1
0
0
produce any side effects and were well tolerated. Relapse of
arthritic signs, seen following a 30-day withdrawal, suggests
that continuous therapy is needed. These data suggest that
active UC-II, HCA-SX, and CM are well tolerated and safe to use
with great efficacy in arthritic dogs.
30
*
60
90
* *
*
*
120
150
Day
ACKNOWLEDGMENTS
The authors would like to thank InterHealth Nutraceuticals, Inc.
for financial support and Mrs Debra Britton and Mrs Shirley
Zafra-Stone for technical support.
278 A. Peal et al.
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Toxicology Mechanisms and Methods, 2010, 1–15, Early Online
RESEARCH ARTICLE
Safety and toxicological evaluation of undenatured type II
collagen
Palma Ann Marone1, Francis C. Lau2, Ramesh C. Gupta3, Manashi Bagchi2, and Debasis Bagchi2,4
1
Eurofins/Product Safety Laboratories, Dayton, NJ, USA, 2Department of Research and Development, InterHealth
Research Center, Benicia, CA, USA, 3Toxicology Department, Murray State University, Murray/Hopkinsville, KY, USA, and
4
Department of Pharmacology and Pharmaceutical Sciences, University of Houston College of Pharmacy, Houston, TX, USA
Abstract
Previous research has shown that undenatured type II collagen is effective in the treatment of arthritis. The present
study evaluated the broad-spectrum safety of UC-II by a variety of toxicological assays including acute oral, acute
dermal, primary dermal irritation, and primary eye irritation toxicity. In addition, genotoxicity studies such as Ames
bacterial reverse mutation assay and mouse lymphoma tests, as well as a dose-dependent 90-day sub-chronic
toxicity study were conducted. Safety studies indicated that acute oral LD50 of UC-II was greater than 5000 mg/kg
in female Sprague-Dawley rats. No changes in body weight or adverse effects were observed following necropsy.
Acute dermal LD50 of UC-II was determined to be greater than 2000 mg/kg. Primary skin irritation tests conducted
on New Zealand Albino rabbits classified UC-II as slightly irritating. Primary eye irritation tests conducted on rabbits indicated that UC-II was moderately irritating to the eye. UC-II did not induce mutagenicity in the bacterial
reverse mutation test in five Salmonella typhimurium strains either with or without metabolic activation. Similarly,
UC-II did not induce a mutagenic effect in the gene mutation test in mouse lymphoma cells either with or without
metabolic activation. A dose-dependent 90-day sub-chronic toxicity study revealed no pathologically significant
changes in selected organ weights individually or as percentages of body or brain weights. No significant changes
were observed in hematology and clinical chemistry. Therefore, the results from the current study show a broadspectrum safety profile of UC-II.
Keywords: Undenatured type II collagen; 90-day toxicity study; acute oral toxicity; acute dermal toxicity; p
rimary
dermal toxicity; primary eye irritation; body and selected organ weights; hematology and clinical chemistry;
histopathology
Introduction
Arthritis and its related chronic conditions affect one in every
five Americans, thus representing one of the most prevalent
causes of disability in the US (Helmick et al. 2008). Indeed,
over 46 million US adults suffered from doctor-diagnosed
arthritis in 2008. This number is estimated to rise to 67 million
by 2030, a massive 46% increase, due in part to increases in
obesity and longevity (Helmick et al. 2008). There are more
than 100 different types of arthritis and among them osteoarthritis (OA) is by far the most prevalent form, affecting ∼ 60%
of all arthritis sufferers Lawrence et al. 2008). Rheumatoid
arthritis (RA) is the second most common form of arthritis,
impinging on 1.3 million US adults (Helmick et al. 2008).
Arthritis describes chronic conditions characterized by joint
pain and difficulty in performing certain tasks resulting in
limited activity (Trentham 1984; 1996; Trentham et al. 1993;
2001; Barnett et al. 1996; 1998). Consequently, arthritis
imposes a tremendous socioeconomic burden on the US
public health system and diminishes the quality of life of
millions of people. OA is the second most common chronic
disease leading to Social Security disability payments due to
long-term absence from work (Bitton 1999). It is prevalent in
the aging population and affects roughly 12% of people aged
60 or older (Felson 2009).
OA is defined by the American College of Rheumatology as
a heterogeneous group of conditions characterized by degeneration of articular cartilage and changes in the underlying
bone at the joint margins (Altman et al. 1986). The etiopathogenesis of OA is multifactorial, and includes inflammatory,
metabolic, and mechanical components. A number of risk
Address for Correspondence: Debasis Bagchi, Department of Pharmacology and Pharmaceutical Sciences, University of Houston College of Pharmacy, Houston,
TX, USA. Tel: 707.751.2800. Fax: 707.751.2801. Email: [email protected]
(Received 09 December 2009; revised 19 January 2010; accepted 23 January 2010)
ISSN 1537-6516 print/ISSN 1537-6524 online © 2010 Informa UK Ltd
DOI: 10.3109/15376511003646440
http://www.informahealthcare.com/txm
2 Palma Ann Marone et al.
factors such as genetics, dietary intake, muscle weakness,
obesity, and trauma may initiate various pathogenic pathways
leading to OA (Felson et al. 2000). In spite of considerable
medical advances in recent years, there is little effective treatment for OA. Common non-surgical treatments of OA include
cyclooxygenase-2 (COX-2) inhibitors and non-steroidal antiinflammation drugs (NSAIDs) targeting pain and inflammation. Unfortunately, many of these agents show limited
efficacy and are associated with serious side-effects and high
toxicities (Sarzi-Puttini et al. 2005). These side-effects include
renal and upper gastrointestinal adverse events, increased risk
for cardiovascular events, and elevated blood pressure (SarziPuttini et al. 2005; Berenbaum 2008). In addition, the recent
negative press and the withdrawal of certain COX-2 selective
NSAIDs from the market have prompted many OA-sufferers
to seek alternative therapies. There is a growing recognition
of the important role of nutraceuticals in the maintenance
of bone and joint health (Goggs et al. 2005). Among these
nutraceuticals, a natural collagen extract known as UC-II has
gained considerable attention recently for its demonstrated
efficacy in the treatment of OA (Crowley et al. 2009).
UC-II is a undenatured type II collagen derived from
chicken sternum cartilage. Animal studies (Deparle et al.
2005; D’Altilio et al. 2007; Peal et al. 2007; Bagchi et al. 2008a;
2009; Gupta et al. 2009a; b) and human trials (Bagchi et al.
2008b; Crowley et al. 2009) have demonstrated UC-II to be
effective and safe in treating OA. A quantitative evaluation of
the therapeutic efficacy of UC-II for 120 days was assessed in
osteoarthritic dogs using a Ground Force Plate (GFP) procedure which objectively measures the peak force and impulse
area (Gupta et al. 2009b). Dogs on placebo exhibited no significant change in arthritic conditions. UC-II supplemented
dogs exhibited a significant improvement, as indicated
by GFP analysis. The peak force was increased by 18% and
impulse area was elevated by 44%, suggesting an increase in
g-force and a decrease in level of pain.
The beneficial effects of UC-II on OA was also observed
in horses (Gupta et al. 2009a). Osteoarthritic horses were
supplemented with placebo, UC-II (320, 480, or 640 mg)
or a combination of 5400 mg of glucosamine plus 1800 mg
of chondroitin for 150 days. Horses receiving 320, 480, or
640 mg of UC-II exhibited significant reduction in arthritic
pain. UC-II at a dose of 480 or 640 mg provided equal effects,
and,therefore, 480 mg was considered optimal. With this
dose, there was an 88% decrease in overall pain and a 78%
decrease in pain upon limb manipulation. UC-II was found to
be more effective in reducing arthritic pain than glucosamine
plus chondroitin (Gupta et al. 2009a).
A recent human clinical trial further demonstrated the
safety and efficacy of UC-II in the treatment of OA (Crowley
et al. 2009). A randomized, double-blind clinical study was
conducted in North America on 52 people with OA of the
knee. A daily dose of 40 mg of UC-II was more than twice as
effective as 1500 mg of glucosamine plus 1200 mg of chondroitin in promoting joint health after 90 days. UC-II significantly decreased joint pain, discomfort, and immobility
compared to baseline, and outperformed the glucosamine
plus chondroitin combination using three standard OA
assessments: Western Ontario and McMaster Osteoarthritis
Index (WOMAC), Visual Analog Scale (VAS), and Lequesne
Functional Index.
The objective of the present study was to determine the
safety profile of UC-II and hence acute oral toxicity, acute
dermal toxicity, primary dermal irritation, primary eye irritation, mutagenicity, and 90-day sub-chronic toxicity studies
were conducted by in vivo and in vitro procedures.
Materials and methods
Study compound
UC-II is a unique, patented natural collagen concentrate
containing 25% undenatured type II collagen. UC-II (UC-250,
off white powder) was obtained from InterHealth Research
Center (Benicia, CA) and used in all the studies reported
here.
Animals and treatment
Safety tests were conducted at Eurofins/Product Safety
Laboratories (Dayton, NJ) in compliance with the Good
Laboratory Practices (GLP) as defined in 21CFR58 by the US
Food and Drug Administration (FDA, 1987) and in accordance with the Organization for Economic Cooperation and
Development (OECD) guidelines for testing of chemicals
(OECD 1998). The mutagenicity studies were performed at
Bioservice Scientific Laboratories (Planegg, Germany) in
compliance with GLP as defined in the Chemikaliengesetz
(Chemical Act) of the Federal Republic of Germany (BGB1.
I Nr. 50 S. 2407), and in accordance with the Environmental
Directorate published by OECD in the Series on Principles
of Good Laboratory Practice and Compliance Monitoring
(OECD 1998). Animals were cared for in accordance with
the most recent Guide for the Care and Use of Laboratory
Animals DHEW (NIH). Detailed animal protocols are provided in individual toxicological assessments.
Acute oral toxicity
The acute oral toxicity evaluation (Up and Down Procedure)
was conducted in rats to determine the potential of UC-II to
produce acute oral toxicity from a single dose through the
oral route. Six healthy young adult female, nulliparous, and
non-pregnant albino Sprague Dawley rats (aged 9–10 weeks
old, initial body weight 188–197 g) were obtained from Ace
Animals, Inc. (Boyertown, PA). Female rats were selected
for the test because they are frequently more sensitive to
the toxicity of test compounds than males. The female rats
were singly housed in suspended stainless steel cages with
mesh floors conforming to the size recommendations in
the most recent Guide for the Care and Use of Laboratory
Animals DHEW (NIH). Litter paper was placed beneath the
cage and was changed at least three times per week. The
rats had free access to standard rat chow (Purina Rodent
Chow# 5012) and filtered tap water ad libitum, and were
maintained at controlled temperature (20–24°C) and light
cycle (12 h light/12 h dark). The animals were acclimated to
Safety of undenatured type II collagen 3
laboratory conditions at least 10–14 days prior to initiation
of dosing.
UC-II was administered in sequence to the animals, as
described in Table 1. The decision to proceed with the next
animal was based on the survival of the previous animal
following dosing. Before each dosing, rats were fasted overnight, examined through the fasting period for health, and
weighed (initial). Individual doses were calculated based
on initial body weights at a dose level of 5000 mg/kg. UC-II
was administered as a 14% w/w suspension in distilled water
using a stainless steel ball-tipped gavage needle. Following
administration, each animal was returned to its designated
cage and the feed was replaced 3–4 h after the final dosing. Individual body weights were recorded again on days
7 and 14 (termination) following dosing. The animals were
observed for mortality, signs of gross toxicity, and behavioral
changes during the first several hours post-dosing and at least
once daily thereafter for 14 days after dosing. Observations
included gross evaluation of skin and fur, eyes and mucous
membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity, and behavioral
pattern. Particular attention was directed to observations of
tremors, convulsions, salivation, diarrhea, and coma. All rats
were euthanized by CO2 inhalation at the end of the 14-day
observation period and gross necropsies were performed on
all animals. Tissues and organs of the thoracic and abdominal
cavities were examined.
Acute dermal toxicity
The acute dermal toxicity evaluation was conducted in rats
to determine the potential for UC-II to produce toxicity
from a single topical application. Five healthy young adult
albino Sprague Dawley male rats (aged 10–11 weeks old,
initial body weight 290–307 g) and five young adult female,
nulliparous, and non-pregnant albino Sprague Dawley rats
(aged 10–11 weeks old, initial body weight 200–215 g) were
obtained from Ace Animals, Inc. (Boyertown, PA). The rats
were singly housed in suspended stainless steel cages with
mesh floors. Litter paper was placed beneath the cage and
was changed at least three times per week. The rats had free
access to standard rat chow (Purina Rodent Chow# 5012)
and filtered tap water ad libitum, and were maintained
at controlled temperature (19–23°C) and light cycle (12 h
light/12 h dark). The animals were acclimated to laboratory
conditions for 21 days.
On the day prior to UC-II application, the five male and
five female animals were prepared by clipping (Oster model
#A5 -small) the dorsal area and the trunk. After clipping and
prior to application, the animals were examined for health,
weighed (initial), and the skin checked for any abnormalities. Individual doses were calculated based on the initial
body weights, taking into account the concentration of the
test mixture. Prior to application, UC-II was moistened with
distilled water to achieve a dry paste by preparing a 50% w/w
mixture. UC-II (2000 mg/kg of body weight) was then applied
to a 2 × 3-inch 4-ply gauze pad and placed on a dose area of
∼ 2 × 3 inches (∼ 10% of the body surface). The gauze pad and
entire trunk of each animal were then wrapped with 3-inch
Durapore tape to avoid dislocation of the pad and to minimize
loss of UC-II. The rats were then returned to their designated
cages. The day of application was considered as day 0 of the
study. After 24 h of exposure of UC-II, the pads were removed,
and the test sites were gently cleansed of any residual test
substance. Individual body weights of the animals were
recorded prior to UC-II application (initial) and again on days
7 and 14 (termination). The animals were observed for mortality, signs of gross toxicity, and behavioral changes during
the first several hours after application and at least once daily
thereafter for 14 days. Observations included gross evaluation
of skin and fur, eyes and mucous membranes, respiratory,
circulatory, autonomic and central nervous systems, somatomotor activity, and behavioral pattern. Particular attention
was directed to observations of tremors, convulsions, salivation, diarrhea, and coma. All rats were euthanized via CO2
inhalation on day 14. Gross necropsies were performed on
all animals at terminal sacrifice. Tissues and organs of the
thoracic and abdominal cavities were examined.
Primary dermal irritation
The primary dermal irritation test was conducted in two
young adult male New Zealand albino rabbits and one young
nulliparous non-pregnant female New Zealand albino rabbit to determine the potential for UC-II to cause irritation
after a single topical application. The rabbits were obtained
from Robinson Services, Inc. (Clemmons, NC), and singly
housed in suspended stainless steel cages with mesh floors,
which conform to the size recommendations in the most
recent Guide for the Care and Use of Laboratory Animals
DHEW (NIH). Litter paper was placed beneath the cage
and was changed at least three times per week. The rabbits
were allowed free access to lab chow (Purina Rabbit Chow
# 5326, St. Louis, MO) and filtered tap water ad libitum, and
maintained at controlled temperature (20–22°C) and light
cycle (12 h light/12 h dark). Animals were acclimated to
Table 1. Acute oral toxicity dosing sequence and observations.
Body weight (g)
Dosing sequence
Dose level (mg/kg)
Initial
Day 7
1
175
182
207
2
550
205
224
3
1750
181
200
4
5000
200
220
5
5000
177
198
6
5000
186
200
Day 14
243
253
246
257
244
246
Cage-side observations
(days 0–14)
Active and healthy
Active and healthy
Active and healthy
Active and healthy
Active and healthy
Active and healthy
Necropsy observations
(all tissues)
No gross abnormalities
laboratory conditions for a period of 28 days prior to initiation of dosing.
On the day before application, rabbits were prepared by
clipping (Oster model #A5 -small) the dorsal area and the
trunk. On the day of dosing but prior to application, the rabbits were critically examined for health and the skin checked
for any abnormalities, and three healthy rabbits without preexisting skin irritation were selected for the test. Individual
doses were calculated based on the initial body weights,
taking into account the concentration of the test mixture.
On the day of application (day 0), UC-II was moistened with
distilled water to achieve a dry paste by preparing a 50% w/w
mixture. Five-tenths of a gram of UC-II (1.0 g of test mixture)
was placed on a 1 × 1-inch 4-ply gauze pad and applied to one
6-cm2 intact dose site on each rabbit. The pad and entire trunk
of each rabbit were then wrapped with semi-occlusive 3-inch
Micropore tape to avoid dislocation of the pad. Elizabethan
collars were placed on each rabbit and they were returned
to their designated cages. After 4 h of exposure to UC-II, the
pads and collars were removed and the test sites were gently
cleansed of any residual test substance.
Individual dose sites were scored according to the Draize
scoring system (Table 2) (Draize et al. 1944) at ∼ 1, 24, 48, and
72 h after patch removal. The classification of irritancy was
obtained by adding the average erythema and edema scores
for the 1, 24, 48, and 72-h scoring intervals and dividing by the
number of evaluation intervals (four). The animals were also
observed for signs of gross toxicity and behavioral changes
at least once daily during the test period. Observations
included gross evaluation of skin and fur, eyes and mucous
membranes, respiratory, circulatory, autonomic and central
nervous systems, somatomotor activity, and behavior pattern.
Particular attention was directed to observation of tremors,
convulsions, salivation, diarrhea, and coma.
Primary eye irritation
The primary eye irritation test was conducted in rabbits to
determine the potential for UC-II to produce irritation from a
single installation through the ocular route. Three female, nulliparous and non-pregnant New Zealand albino rabbits were
obtained from Robinson Services, Inc. (Clemmons, NC) and
singly housed in suspended stainless steel cages with mesh
floors, which conform to the size recommendations in the
most recent Guide for the Care and Use of Laboratory Animals
DHEW (NIH). Litter paper was placed beneath the cage and
was changed at least three times per week. The rabbits were
allowed free access to lab chow (Purina Rabbit Chow# 5326, St.
Louis, MO) and filtered tap water ad libitum, and maintained
at controlled temperature (17–24°C) and light cycle (12 h
Table 2. Primary dermal irritation index (PII) and classification.
Primary Dermal Irritation Index (PDII)
Classification
0
Non-irritating
> 0–2.0
Slightly irritating
2.1–5.0
Moderately
irritating
> 5.0
Severely irritating
light/12 h dark). Animals were acclimated to laboratory conditions for a period of 22 days prior to initiation of dosing.
Prior to instillation, both eyes of rabbits were examined
using a fluorescein dye procedure. One drop of 2% ophthalmic
fluorescein sodium was instilled into both eyes of each rabbit.
The eyes were rinsed with physiological saline (0.9% NaCl)
∼ 30 s after installation of the fluorescein. Using an ultraviolet
light source, the eyes were checked for gross abnormalities
according to the ‘Scale for Scoring Ocular Lesions’ (Draize
et al. 1944). Three healthy animals without pre-existing ocular
irritation were selected for the test. One-tenth of a milliliter
(0.06 g) of UC-II was instilled into the conjunctival sac of the
right eye of each rabbit by gently pulling the lower lid away
from the eyeball. The upper and lower lids were then gently held together for ∼ 1 s before releasing to minimize loss
of the test substance. The left (control) eye of each animal
remained untreated and served as a control. The rabbits were
then returned to their designated cages. Ocular irritation was
evaluated macroscopically using a high-intensity white light in
accordance with Draize et al. (1944) at 1, 24, 48, and 72 h, and 4
days post-instillation. The fluorescein eye evaluation was used
at 24 h to verify the absence of corneal damage. Individual
irritation scores were recorded for each animal. In addition
to observations of the cornea, iris, and conjunctivae, any other
lesions were noted. The average score for all rabbits at each
scoring period was calculated to aid in data interpretation.
Time intervals with the highest mean score (Maximum Mean
Total Score; MMTS) for all rabbits were used to classify the test
substance (UC-II) by the system of Kay and Calandra (1962).
The animals were also observed for signs of gross toxicity
and behavioral changes at least once daily during the test
period. Observations included gross evaluation of skin and
fur, eyes and mucous membranes, respiratory, circulatory,
autonomic and central nervous systems, somatomotor activity, and behavior pattern. Particular attention was directed
to observation of tremors, convulsions, salivation, diarrhea,
and coma.
Mutagenicity test: Ames’ bacterial reverse mutation assay
The Salmonella typhimurium reverse mutation test (Maron
and Ames 1983) was conducted to determine the ability of
UC-II to induce reverse mutation. UC-II was evaluated in the
Ames/Salmonella plate incorporation assay to determine its
potential to induce reverse mutation at selected histidine
loci in five tester strains of Salmonella typhimurium viz. TA
1535, TA 97a, TA 98, TA 100, and TA 102 in the presence and
absence of a metabolic activation system (S9) (Ames et al.
1977). Suspensions of bacterial cells were exposed to UC-II
in triplicate cultures at concentrations of 10.1, 31.6, 100, 316,
1000, 2500, and 5000 μg/plate in the presence and absence of
an exogenous metabolic activation system (S9). The suspensions were mixed with an overlay agar and plated immediately onto minimal medium. After 48 h incubation, revertant
colonies were counted using a ProtoCOL counter (Meintrup
DWS Laborgerate, GmbH) and compared to the number of
spontaneous revertant colonies on vehicle (negative) control
plates.
Mutagenicity test: Mouse lymphoma assay
The mutagenic potential of UC-II was evaluated by in vitro
mammalian cell gene mutation assay (Thymidine Kinase
Locus/TK+/−) in mouse (Mus musculus) lymphoma cell line
L5178Y. The assay was performed in both the presence and
absence of an exogenous metabolic activation system at the
gene locus coding for the enzyme thymidine kinase (TK) in
mouse lymphoma cells.
UC-II was investigated at the following concentrations:
Experiment I with and without metabolic activation, 200, 400,
600, 800, 1000, 1200, 1500, and 2000 μg/ml; Experiment II
with metabolic activation, 300, 500, 700, 1100, 1400, 1800,
and 2000 μg/ml; and Experiment II without activation, 4.4,
17.6, 39.6, 70.4, 110, 264, 330, and 440 μg/ml. The selection of
concentrations was based on data from the pre-experiment.
In experiment I, 2000 μg/ml (with and without metabolic
activation) was selected as the highest concentration. In
experiment II, 2000 μg/ml (with metabolic activation) and
440 μg/ml (without metabolic activation) were selected as
the highest concentration. Experiment II without metabolic
activation was performed as a 24 h long-term exposure assay.
Ethylmethanesulfonate (EMS), methylmethanesulfonate
(MMS), and benzo[a]pyrene (B[a]P) were used as positive
controls. Each trial consisted of duplicate cultures of the
negative (vehicle) and positive controls, and single cultures
treated at each of the dosage levels of UC-II described above.
Treatment consisted of 11 ml of the appropriate treatment
medium (with or without exogenous activation), designated
concentrations of UC-II and 1 × 107 cells in a 25cm2 flask, and
incubated at 37°C in 5% CO2/95% humidified air. After 4 h
incubation, the test compound was removed by centrifugation (200 x g, 10 min) and the cells were washed twice with
phosphate buffered saline (PBS). The cells were suspended
in 30 ml complete culture medium and incubated for an
expression and growth period of 72 h. For the long-term
exposure experiment, 1 × 107 cells were suspended in 50 ml
cell culture medium in a 175-cm2 flask. After expression and
growth period, the relative cloning efficiency (RCE; percentage cloning efficiency of the test group in relation to the
negative control) of the cells was determined as previously
described (Clive and Spector 1975; Clive 1983; Clive et al.
1983; Mitchell et al. 1997).
Dose-dependent 90-day sub-chronic toxicity study
A 90-day oral toxicity study was conducted in male and
female rats at Eurofins/Product Safety Laboratories
(Dayton, NJ) to determine the potential of UC-II to produce
toxicity. A no-observed-adverse-effect level (NOAEL) was
also sought for each sex. Eighty healthy rats (40 males and
40 females) were selected for the test and equally distributed into four groups (10 males and 10 females per dose
level) according to Table 3.
Animal selection
After acclimating to the laboratory environment for 7 days,
the rats were examined for general health and weighed. Only
those rats free of clinical signs of disease or injury and having
a body weight range within ± 20% of the mean were selected
for test. The animals weighed in the range of 195–219 g for
males and 148–174 g for females, and were ∼ 7–8 weeks of
age at test initiation. The 40 male and 40 female rats were
randomly distributed, stratified by body weight, among the
dose and control groups on the day prior to study start.
Dose preparations
The test substance was administered as a 0.4% (low dose),
4.0% (intermediate dose), or 10.0% (high dose) weight/
weight dilution in distilled water. On each dosing day and
for each concentration, an appropriate amount of the test
substance was accurately weighed into a 150 mL glass beaker
and distilled water was added until the desired total weight
was obtained. The dose preparations were used at room temperature within ∼ 2 h, and maintained on a magnetic stir plate
during administration.
Dose calculations
Individual doses were calculated based on the most recent
weekly body weights and were adjusted each week to maintain the targeted dose level for all rats. All doses were administered volumetrically after correcting for dilution. Doses
were administered to all groups at a constant dose volume
of 10.0 mL/kg. The control group received the vehicle only
(distilled water) at the same volume as the test animals.
Dose administration
Each animal was dosed by oral intubation to the stomach
using a ball-tipped gavage needle attached to an appropriate
syringe. Dosing was 7 days per week for a period of 92 days for
males and 93 days for females. The first day of administration
was considered Day 1 of the study. Dosing was at approximately the same time each day ± 2 h, with an exception on
the days the hematology and/or clinical chemistry samples
were collected. On the days of blood collection, food was
returned to the fasted animals for a minimum of 2 h prior to
test substance administration.
Ophthalmologic evaluations
Prior to study initiation, the eyes of a group of rats considered
for study were examined by focal illumination and indirect
ophthalmoscopy. Mydriasis was achieved with 1% tropicamide and the eyes were examined in subdued light. Subdued
light was maintained in the animal room for the remainder of
the day. This procedure was repeated on Day 91 for all surviving test animals.
Clinical observations
All animals were observed at least twice daily for viability.
Cage-side observations of all animals were performed daily
during the study or until death occurred. On Day 1 (prior to
first treatment with the test substance) and approximately
weekly thereafter, a detailed clinical observation test was
conducted while handling the animals, generally on days that
the animals were weighed and food consumption measurements taken. Potential signs noted included, but were not
Table 3. Dose levels and assignment of animals.
Group
Number/group
Number/sex
1
20
10
2
20
10
3
20
10
4
20
10
See Materials and methods section for details.
Oral gavage dose (mg/kg/day)
Control (0)
Low dose (40)
Intermediate dose (400)
High dose (1000)
limited to changes in skin, fur, eyes, and mucous membranes,
occurrence of secretions, excretions, and autonomic activity
(e.g. lacrimation, piloerection, pupil size, unusual respiratory
pattern). Likewise, changes in gait, posture, and response to
handling, as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive
circling), or bizarre behavior (e.g. self-mutilation, walking
backwards) were also recorded.
Body weight, organ weight, and body weight gain
Individual body weights were recorded twice during the
acclimation period, on Day 0 (the day of study start) and
approximately weekly thereafter (7 day intervals ± 1). Mean
daily body weight gains were calculated for each sex and dose
level at each interval and for the overall (Days 1–92) testing
interval. Animals were also weighed prior to sacrifice (fasted
body weight) for the calculation of organ-to-body weight
and organ-to-brain weight ratios. The following organs were
weighed wet as soon as possible after dissection to avoid drying: liver, kidneys (combined), adrenals (combined), brain,
heart, thymus, spleen, ovaries (combined) or testes (combined), epididymides, and uterus and fallopian tubes.
Food consumption and food efficiency
Individual food consumption was measured and was
recorded weekly adjusting for spillage. Mean daily food consumption was calculated for each sex/dose level during each
weekly interval and overall (Days 1–92) testing interval. Mean
daily food efficiency was also calculated for each sex/dose
level based on body weight gain and food consumption data.
Animals were allowed ad libitum access to food throughout
the study. Animals were fasted overnight prior to blood collection on Day 90, and prior to terminal sacrifice on Day 92
(males) or Day 93 (females).
Functional observational battery
A Functional Observational Battery (FOB) was performed on
all animals on Day 86 (females) and Day 87 (surviving males).
Each rat was evaluated during handling and while in an open
field for excitability, autonomic function, gait and sensorimotor coordination (open field and manipulative evaluations),
reactivity and sensitivity (elicited behavior), and other abnormal clinical signs including but not limited to convulsions,
tremors, unusual or bizarre behavior, emaciation, dehydration, and general appearance. In addition to the above
observations, forelimb and hind limb grip strength and foot
splay measurements were obtained and recorded. The grip
strength was measured with a digital force gauge (Wagner
Force Five, Model #FDMV). Triplicate measurements of grip
Dose volume (ml/kg/day)
10.0
10.0
10.0
10.0
% UC-II
0
0.4
4.0
10.0
strength and duplicate measurements for foot splay were
taken for each animal and the means for each group were
calculated.
Motor activity
Motor Activity (MA) was evaluated on all surviving animals
on Day 86 (males) and Day 87 (females). This assessment
was done at approximately the same period during the study
as the FOB. Activity was monitored using an automated
Photobeam Activity System® (San Diego Instruments, Inc.).
An approximate equal number of animals from each dose
group were assigned to the MA assessment for each session.
Each animal was placed into a polycarbonate solid bottom
cage, room lights were turned off, and a white noise generator
was used. The evaluation phase began immediately for that
animal. Each animal was evaluated for a single 1-h phase,
with photobeam counts accumulated over six 10-min intervals. Total movements (consisting of fine movements and
active movements) were considered an appropriate measure for the assessment of potential behavioral effects in this
study.
Clinical pathology
All surviving animals were fasted overnight prior to each
blood collection. Blood samples for hematology (except coagulation samples) and clinical chemistry were collected via
the sub-lingual vein under isoflurane anesthesia during the
12th week of exposure for males and females. Approximately
500 μl was collected in a pre-calibrated tube containing
EDTA for hematology assessments. The whole blood samples
were stored under refrigeration and shipped on cold packs.
Approximately 1000 μl was collected into tubes containing
no preservative for clinical chemistry assessments. These
samples were centrifuged in a refrigerated centrifuge and
the serum was transferred to a labelled tube. Serum samples were stored in a −80°C freezer and shipped frozen in
dry ice. All samples were shipped to DuPont Haskell Global
Centers for Health and Environmental Sciences (Newark,
DE). Blood samples used to determine the prothrombin
time and activated partial thromboplastin time (coagulation)
were collected via the inferior vena cava under isoflurane
anesthesia at terminal sacrifice. Approximately 1800 μl were
collected in a pre-calibrated tube containing sodium citrate.
These samples were centrifuged in a refrigerated centrifuge
and the plasma was transferred to a labelled tube. Plasma
samples were stored in a −80°C freezer and shipped frozen
in dry ice to DuPont Haskell Global Centers for Health and
Environmental Sciences. The day before collection of the
samples for the clinical pathology evaluation, the animals
were placed in metabolism cages. These animals were fasted
after 3 pm (at least 15 h) and urine was collected from each
animal. Urine samples were stored under refrigeration and
shipped on cold packs to DuPont Haskell Global Centers for
Health and Environmental Sciences. All blood samples were
evaluated for quality by visual examination. Upon completion of clinical chemistry, remaining serum samples from
two randomly selected animals were pooled at DuPont
Haskell and sent to Charles River Diagnostics (Wilmington,
MA) for serology.
Sacrifice and macroscopic observations
Scheduled sacrifice. At terminal sacrifice, all surviving
males (Day 93) and all females (Day 94) were euthanized by
exsanguination from the abdominal aorta under isoflurane
anesthesia. All animals in the study (including decedents)
were subjected to a full necropsy, which included examination of the external surface of the body, all orifices, and the
thoracic, abdominal, and cranial cavities, and their contents.
Additional tissues were preserved if indicated by signs of toxicity or target organ involvement.
Histopathology. Histological examination was performed
on the preserved organs and tissues of the animals from both
the control and high dose groups (Groups 1 and 4, respectively) as well as from any animal that died during the course
of the study. In addition, gross lesions of potential toxicological significance noted in any test groups at the time of terminal sacrifice were also examined. Due to findings in the
males and females of Group 4 high dose, the nasal turbinates
were evaluated in the intermediate Group 3 animals. The
fixed tissues were trimmed, processed, embedded in paraffin, microtomed, placed on glass microscope slides, stained
with hematoxylin and eosin, and examined by light microscopy. Slide preparation and histopathological assessment
was performed by Histo-Scientific Research Laboratories (Mt.
Jackson, VA).
Statistical analysis
Eurofins/Product Safety Laboratories performed statistical analysis of all data collected during the in-life phase
of the study as well as organ weight data. DuPont Haskell
Laboratory provided analysis of clinical pathology results to
Eurofins/Product Safety Laboratories. The use of the word
‘significant’ or ‘significantly’ indicates a statistically significant
difference between the control and the experimental groups.
Significance was judged at a probability value of p ≤ 0.05. Male
and female rats were evaluated separately.
Statistical methods (in-life and organ weight data)
Group means and standard deviations were calculated for
body weight, daily body weight gain, daily food consumption,
daily food efficiency, organ weight, and organ-to-body/brain
weight ratio, FOB and MA data. Data within groups were
compared using a One-Way of Analysis (ANOVA), followed
by comparison of the treated groups to control by Dunnett’s
Multiple Comparisons test. Data were evaluated for homogeneity of variances and normality by the Bartlett’s test. Data
that were considered significant by Bartlett’s test were further
evaluated with a non-parametric method (Kruskal-Wallis or
Dunn’s test) (INSTAT Biostatistics, Graph Pad Software, San
Diego, CA). Motor activity data (overall total movements)
were further analyzed using a Two-Way Repeated Measures
ANOVA (SigmaStat, Version 2.03).
Statistical methods (clinical pathology)
Means and standard deviations were calculated for clinical pathology quantitative data. Data within groups were
initially analyzed using Levene’s test for variance homogeneity, and the Shapiro-Wilk test for normality. If variances
were considered not significantly different, groups were
compared using a One-Way Analysis of Variance (ANOVA)
followed by Dunnett’s t-test for multiple comparisons. If
the Shapiro-Wilk test was not significant but Levene’s test
was significant, a robust version of Dunnett’s test was used.
Where variances were considered significantly different by
Levene’s test, groups were compared using a non-parametric method (Kruskal-Wallis non-parametric analysis
of variance followed by Dunn’s test). Differences among
groups were judged significant at a probability value of
p ≤ 0.05. Male and female rats were evaluated separately.
Results
Acute oral toxicity
A single oral administration of UC-II was provided to female
Sprague-Dawley rats to assess its acute toxicity following
Up and Down procedure. UC-II, at the limit dose of level of
5000 mg/kg body weight, did not cause any mortality and did
not demonstrate any signs of gross toxicity, adverse pharmacologic effects, or abnormal behavior in the treated female
rats following dosing and during the observation period of
14 days thereafter. All animals survived, gained normal body
weight, and appeared active and healthy during the study.
No gross abnormalities or pathological alterations were
noted for any of the rats when necropsied at the conclusion
of the 14-day observation period (Table 1). Based on these
results and under the conditions of this study, the acute oral
LD50 of UC-II is greater than 5000 mg/kg of body weight in
female rats.
Acute dermal toxicity
Acute dermal toxicity of UC-II was conducted in male and
female Sprague Dawley rats to determine the potential for
UC-II to cause toxicity from a single topical application.
All animals survived, gained normal body weight, and
appeared active and healthy during the study. There were
no signs of dermal irritation, gross toxicity, adverse pharmacologic effects, or abnormal behavior. No gross abnormalities were noted for any of the animals when necropsied
at the conclusion of the 14-day observation period. The
findings are summarized in Table 4. Under the conditions
of this study, the single dose acute dermal LD50 of UC-II is
greater than 2000 mg/kg of body weight in both male and
female-rats.
Primary dermal irritation
Primary dermal irritation was investigated in male and
female New Zealand albino rabbits to evaluate the potential of UC-II to produce irritation after a single topical
application. Following application of UC-II, all animals
appeared active and healthy. Apart from the dermal irritation noted below, there were no signs of gross toxicity,
adverse pharmacologic effects, or abnormal behavior.
One hour after patch removal, very slight erythema was
observed at all three treated sites. The overall incidence
and severity of irritation decreased with time. All animals
were free from dermal irritation within 24 h. A summary of
Draize primary dermal irritation scoring criteria for dermal
reactions and descriptive rating for mean primary dermal
irritation index (PDII) is presented in Table 2. Under the
conditions of this study, the PDII for UC-II was calculated
to be 0.3, thus classifying UC-II to be slightly irritating to
the skin (Table 5).
Primary eye irritation
A primary eye irritation test was conducted in New Zealand
albino rabbits to determine the potential for UC-II to cause
irritation from a single instillation via the ocular route. All
animals appeared active and healthy. There were no signs
of gross toxicity, adverse pharmacologic effects or abnormal behavior. No corneal opacity or iritis was observed
in any treated eye during the study. One hour following
UC-II instillation, all treated eyes exhibited conjunctivitis
(Table 6). Individual eye irritation scores are presented in
Table 6 in accordance with the Draize Scale for scoring
Eye Lesions and the Kay and Calandra Scheme for classifying eye irritants (Draize et al. 1944; Kay and Calandra
1962). The overall severity of irritation decreased with time
(Table 7). All animals were free of ocular irritation within
48 h. Under the conditions of this study, the maximum
mean total score (MMTS) of UC-II powder was determined
to be 37.7 (Table 7), classifying UC-II to be moderately irritating to the eye.
Mutagenicity test: Ames’ bacterial reverse mutation assay
No toxic effects of UC-II were noted in any of the five tester
strains used up to the highest dose group evaluated (with
and without metabolic activation). No biologically relevant
Table 4. Summary of acute dermal toxicity findings.
Body weight (g)
Sex
Initial
Day 7
Male
252
299
Male
248
302
Male
239
322
Male
257
314
Male
251
299
Female
196
204
Female
201
218
Female
210
223
Female
211
216
Female
199
225
increases in revertant colony numbers of any of the five tester
strains were observed following treatment with UC-II at any
concentration level, in neither the presence nor absence
of metabolic activation. Therefore, UC-II did not cause
gene mutations by base pair changes or frameshifts in the
genome of the tester strains used, indicating that UC-II is
non-mutagenic.
Mutagenicity test: Mouse lymphoma assay
In experiment I with metabolic activation, the relative
total growth (RTG) was 108.55% for the highest concentration (2000 μg/ml) evaluated. The highest concentration
evaluated without metabolic activation was 2000 μg/ml with
an RTG of 83.73%. In experiment II with metabolic activation, the RTG was 90.38% for the highest concentration
(2000 μg/ml) evaluated. The highest concentration evaluated without metabolic activation was 440 μg/ml with an
RTG of 10.11%.
No biologically relevant increases of mutants were found
after treatment with UC-II (with or without metabolic activation) in both experiments I and II. No dose-response relationship was observed. Additionally, in experiments I and
II colony sizing showed no clastogenic effects induced by
UC-II. Therefore, under the experimental conditions of this
study, no evidence of mutagenic activity was detected for
UC-II in the L5178Y mouse lymphoma cell line, and UC-II is
concluded to be negative for the induction of mutagenicity
in this assay.
Dose-dependent 90-day sub-chronic toxicity study
Ophthalmoscopic examinations
Both eyes of all animals were examined by focal illumination and indirect ophthalmoscopy prior to study initiation
Table 5. Summary of primary skin irritation scores (average for three
animals).
Time after patch removal
30–60 min
24 h
48 h
72 h
Erythema
1.0
0
0
0
Edema
0
0
0
0
1.0
0
0
0
Total (PDI*)
PDI: Primary dermal irritation = average erythema + average edema.
Day 14
341
352
364
369
349
234
232
240
245
249
Cage-side observations
(days 0–14)
Active and healthy
Active and healthy
Active and healthy
Active and healthy
Active and healthy
Active and healthy
Active and healthy
Active and healthy
Active and healthy
Active and healthy
Necropsy observations
(all tissues)
Table 6. Individual scores for ocular irrigation.
I. Cornea
II. Iris
III. Conjunctivas
A. Opacity B. Area (A × B) × 5 A. Values A × 5 A. Redness B. Chemosis C. Discharge (A + B + C) × 2 Total
16
36
Rabbit
Hours
1
1
3
15
1
5
3
2
3b
3401 Male
1
5
1
5
3
2
2
14
24
24
1a
4
0
0
0
2
1
1
8
8
48
0a
72
0
4
0
0
0
1
0
0
2
2
Days
4
0
4
0
0
0
0
0
0
0
0
16
36
Rabbit
Hours
1
1
3
15
1
5
3
2
3b
3402
1
5
1
5
2
2
2
12
22
24
1a
Female
4
0
0
0
2
1
1
8
8
48
0a
72
0
4
0
0
0
1
0
0
2
2
Days
4
0
4
0
0
0
0
0
0
0
0
16
41
Rabbit
Hours
1
1
4
20
1
5
3
2
3b
3402
2
10
1
5
2
2
2
12
27
24
1a
Female
4
0
0
0
2
1
1
8
8
48
0a
72
0
4
0
0
0
1
0
0
2
2
Days
4
0
4
0
0
0
0
0
0
0
0
a
2% ophthalmic fluorescein sodium was used to evaluate the extent or verify the absence of corneal opacity.
b
Discharge was white in color.
Table 7. Summary of mean scores of severity and reversibility of primary
eye irritation study.
Time post-instillation
Severity of irritation
1h
37.7
24 h
24.3
48 h
8.0
72 h
2.0
4 days
0.0
Maximum Mean Total Score (MMTS) was observed at 1 h post-instillation. See Materials and methods section for details.
and near experimental completion (Day 91). Both eyes of all
surviving animals were ophthalmoscopically normal. There
was no indication that the test substance, as evaluated, was
an ocular toxicant.
Mortality and clinical observations
Two male animals (one in Group 3 and the other Group 4) were
found dead on study days 86 and 69, respectively. The animal in
Group 3 was active and healthy prior to death and died immediately following the Motor Activity assessment. A cause of
mortality could not be definitively determined; however, there
was no evidence to suggest that mortality was attributable to
test substance administration. Necropsy revealed a distended
stomach filled with gas and food and the kidneys appeared
enlarged. These observations had no histological correlate and
there were no other apparent remarkable findings. The agonal
change of congestion was the notable microscopic finding in
the adrenal glands, kidneys, liver, and lung.
The animal in Group 4 died as a suspected result of a
gavaging error. Prior to death this animal exhibited hypoactivity, hypothermia, moist rales, and irregular respiration
accompanied by a red nasal discharge. Macroscopically, the
trachea and esophagus were punctured, the thoracic cavity was filled with a white liquid substance, and the lungs
were dark red in color. Puncture of the esophagus noted at
necropsy was associated microscopically with the presence
of hemorrhage, inflammation, and myofiber degeneration at
the edges of the puncture wound consistent with an antemortem incident. Puncture of the trachea noted at necropsy
was not observed at the time of trimming. Noted microscopic
findings associated with the esophageal puncture were
marked lung atelectasis, moderate fibrinous inflammation of
the lungs involving the pleura, and slight fibrinous inflammation involving the heart (epicardium). Lymphoid depletion
noted in lymph nodes, spleen, and thymus was a secondary
alteration related to stress/cachexia and was not a primary
finding associated with test substance administration.
There were no test substance-related clinical signs in any
test group (see Table 3) that were considered to be of toxicological significance. Transient clinical signs included black
ocular discharge for one Group 1 (control) male on Days
15–34, one Group 2 (40 mg/kg/day) male on Days 22–35, and
one Group 1 female on Day 39. Red ocular discharges for one
Group 2 male on Days 43–45; red stained fur for one Group 2
male on Days 50–81 and 84–92, one Group 4 (1000 mg/kg/day)
male on Days 50–92, two Group 3 (400 mg/kg/day) females on
Days 59–62 and 60–65, respectively, were observed. Red facial
stainings for one Group 1 male on Days 71–77, one Group 2
male on Days 42–50 and 82–83, one Group 4 male on Days
39–50, and one Group 3 female on Days 63–70 were noted.
Hyperactivity for two Group 3 males on Days 36 and 64 and
Day 50, respectively, and one Group 4 male on Day 50 and
one Group 2 female on Day 92 was observed. One Group 1
male was noted with a swollen right hindlimb (Days 22–24,
28), hind end impairment (Days 28–42), and swollen foot pads
(Days 29–63). One Group 1 male had a wound on the ventral
surface of the head on Days 15–27. One Group 1 male had a
wound on the right ear on Days 78–92. One Group 3 male had
a small scab on the right side of the face on Days 8–18, and one
Group 1 female had a small scab on the top of its head on Days
1–19; one Group 4 male exhibited enophthalmos (right eye)
on Days 50–92. One Group 2 male exhibited variable red nasal
discharge, reduced fecal volume, ano-genital staining, soft
feces, moist rales, hunched posture, and piloerection on Days
53–72. The above findings did not show any adverse effects
and did not appear to be test substance-related because they
were found across all test groups, including control animals.
Body weight, organ weights, and body weight gain
Weekly body weights for male and female rats at 40, 400, and
1000 mg/kg/day were comparable with control values. Overall
(Days 1–92) and mean daily body weight gain for male rats at
40, 400, and 1000 mg/kg/day were comparable with control
values (Table 8). Overall (Days 1–92) and mean daily body
weight gain for female rats at 40, 400, and 1000 mg/kg/day
were generally comparable with control values with the
exception that daily body weight gain was decreased during
Week 3 for Group 4 females.
There were no changes in individual organ weights (Table 9)
or individual organ-to-brain weights (Table 10). The organ-tobody weight ratios were unaffected except that the kidney-tobody weight ratios were significantly decreased in Group 3
males (Table 11). This finding was not associated with any other
clinical finding and did not herald any corresponding pathological changes in the high dose animals. Therefore, this change
was deemed incidental and of no toxicological interest.
Food consumption and food efficiency
Overall (Days 1–92) and mean daily food consumption for
male rats at 40 and 400 mg/kg/day were comparable with
control values. Food consumption was decreased for male rats
at 1000 mg/kg/day (Group 4) during Weeks 5, 7–11, 13, and
overall. Overall and mean daily food consumption for female
rats at 40, 400, and 1000 mg/kg/day were generally comparable
with control values with the exception of the following statistically significant findings. Food consumption was decreased in
females during Weeks 1, 2, and overall at 400 mg/kg/day, and
during Weeks 1, 8, and overall at 1000 mg/kg/day.
Overall and mean daily food efficiency for male rats at
40, 400, and 1000 mg/kg/day were comparable with control
Table 8. Summary of average weekly body weight.
Group (male)
Days
1
2
3
1
207.2 ± 6.1
207.5 ± 6.9
206.8 ± 6.1
8
252.2 ± 9.2
251.8 ± 11.7
247.7 ± 9.1
15
278.5 ± 13.6
277.7 ± 13.8
274.2 ± 10.5
22
302.6 ± 17.9
305.5 ± 18.5
299.4 ± 11.2
29
318.3 ± 24.4
322.8 ± 18.7
315.9 ± 12.6
36
332.6 ± 26.5
336.3 ± 20.9
330.0 ± 12.0
43
349.7 ± 25.2
351.7 ± 23.8
345.7 ± 13.0
50
364.2 ± 24.3
367.2 ± 25.2
359.4 ± 15.5
57
373.2 ± 24.3
368.6 ± 37.2
368.9 ± 17.2
64
379.6 ± 25.0
377.0 ± 36.8
376.4 ± 18.3
71
386.3 ± 25.1
386.5 ± 28.1
385.5 ± 17.6
78
396.2 ± 28.3
397.6 ± 28.0
394.0 ± 18.5
85
400.2 ± 27.9
402.4 ± 28.7
399.2 ± 18.6
92
394.9 ± 25.2
398.3 ± 29.9
390.9 ± 19.5†
values. Overall and mean daily food efficiency for female rats
at 40, 400, and 1000 mg/kg/day were generally comparable
with control values with the exception of the following statistically significant findings. Mean daily food efficiency was
decreased during Week 3 for females at 40 mg/kg/day and at
1000 mg/kg/day.
In summary, the oral administration of UC-II led to some
dose-related decreases in food consumption in males and
females; however, body weight, body weight gain, and food
efficiency remained generally unaffected. Reductions in food
consumption were considered test substance related and
may be of some toxicological interest in light of the pathological findings of nasal turbinate eosinophilia at the high
dose (see Clinical Pathology section).
Functional observational battery
In general, the functional behavioral results of the test groups
of male and female rats were considered comparable to the
control groups. Any decreases in quantitative measurements
or increases in incidence of open field measurements were
minimal and not associated with a constellation of findings
that would support a toxicologically significant behavioral
change. In males, these findings included normal (sleeping)
postures in 5/10 Group 2 males and 2/10 Group 3 males.
Enophthalmos for 1/10 Group 4 males, an inactive/alert
activity level for 1/10 Group 2 males, 1/10 Groups 3 males,
and 1/10 Group 4 males were observed. A slow reaction to
right itself for 1/10 Group 1 males; no approach responses
for 1/10 Group 2 males and 2/10 Group 3 males; as well as no
tactile responses for 2/10 Group 1 males, 1/10 Group 2 males,
2/10 Group 3 males, and 1/10 Group 4 males were noted. In
females, these findings included no tactile responses for 1/10
Group 1 females and 1/10 Group 3 females.
Motor activity
The Motor Activity results of the test groups of male and
female rats were considered comparable to the control groups. In general, all groups of animals (including
control) exhibited a similar level of movement over all
Group (female)
4
1
2
3
4
205.7 ± 6.3
160.7 ± 8.1
159.4 ± 7.3
157.7 ± 6.9
161.0 ± 7.0
247.3 ± 10.9
182.6 ± 8.6
175.6 ± 11.4
174.3 ± 7.4
175.4 ± 9.1
271.1 ± 14.6
195.6 ± 7.4
189.8 ± 12.0
189.9 ± 8.7
191.4 ± 9.4
294.3 ± 19.0
215.3 ± 10.5
202.6 ± 14.7
208.8 ± 14.0 202.7 ± 10.8
311.8 ± 18.4
223.3 ± 13.4
212.3 ± 15.9
211.7 ± 16.5 211.3 ± 12.5
326.3 ± 20.5
225.5 ± 12.6
218.0 ± 13.5
215.2 ± 10.9 215.4 ± 10.9
324.8 ± 23.1
234.1 ± 14.4
222.4 ± 16.5
225.2 ± 17.0 224.8 ± 14.5
352.4 ± 23.6
241.7 ± 16.2
229.3 ± 15.6
229.6 ± 16.8 233.0 ± 17.5
358.5 ± 25.2
246.7 ± 18.7
234.5 ± 18.4
233.7 ± 15.3 233.4 ± 13.6
360.1 ± 29.5
249.1 ± 16.0
238.2 ± 17.7
235.9 ± 15.1 237.1 ± 15.8
250.5 ± 13.6
241.2 ± 16.4
239.8 ± 14.6 241.7 ± 14.9
371.0 ± 27.9†
254.5 ± 15.4
245.2 ± 12.9
244.6 ± 18.5 244.8 ± 14.6
380.8 ± 32.0†
256.3 ± 15.2
247.5 ± 15.7
246.7 ± 16.2 246.3 ± 13.7
388.1 ± 29.8†
250.7 ± 12.7
243.0 ± 14.8
240.3 ± 17.1 242.1 ± 15.0
377.7 ± 26.4†
Values are the Mean ± SD (n = 10 except for † n = 9). No significant difference from control was observed. See Materials and methods section for details.
Table 9. Summary of mean organ weight.
Group (male)
Group (female)
Organ
1
2
3
4
1
2
3
4
0.071 ± 0.009
0.067 ± 0.006
0.067 ± 0.011
0.074 ± 0.006
0.072 ± 0.011
0.070 ± 0.004 0.072 ± 0.008
Adrenals
0.066 ± 0.009†
Brain
1.99 ± 0.10
1.98 ± 0.07
1.99 ± 0.07
1.95 ± 0.10
1.85 ± 0.05
1.81 ± 0.06
1.81 ± 0.07
1.85 ± 0.10
Heart
1.31 ± 0.13
1.38 ± 0.11
1.29 ± 0.08
1.29 ± 0.16
0.90 ± 0.09
0.95 ± 0.09
0.89 ± 0.09
0.93 ± 0.09
Kidney
2.90 ± 0.28
2.92 ± 0.19
2.65 ± 0.13
2.79 ± 0.30
1.76 ± 0.11
1.70 ± 0.08
1.72 ± 0.13
1.74 ± 0.18
Liver
10.07 ± 0.87
10.51 ± 0.79
9.85 ± 0.49
9.25 ± 0.83
6.01 ± 0.50
5.99 ± 0.33
5.81 ± 0.34
5.96 ± 0.41
Spleen
0.76 ± 0.10
0.81 ± 0.09
0.75 ± 0.08
0.69 ± 0.08
0.60 ± 0.07
0.62 ± 0.06
0.59 ± 0.08
0.63 ± 0.09
Thymus
0.300 ± 0.054
0.366 ± 0.129
0.313 ± 0.059
0.271 ± 0.062
0.260 ± 0.025
0.243 ± 0.058
0.253 ± 0.038 0.233 ± 0.055
1.516 ± 0.119
1.569 ± 0.123
—
—
—
—
Epididymides
1.490 ± 0.175
1.434 ± 0.222†
Testes
3.87 ± 0.31
3.98 ± 0.23
3.81 ± 0.33
3.80 ± 0.37
—
—
—
—
Ovaries
—
—
—
—
0.139 ± 0.018
0.131 ± 0.017
0.134 ± 0.018 0.147 ± 0.023
Uterus/
—
—
—
—
0.78 ± 0.19
0.65 ± 0.24
0.75 ± 0.50
0.67 ± 0.21
Fallopian tubes
Table 10. Summary of mean organ-to-brain weight ratios.
Group (male)
Group (female)
Organ
1
2
3
4
1
2
3
4
0.036 ± 0.004
0.034 ± 0.004
0.035 ± 0.006
0.040 ± 0.003
0.040 ± 0.006
0.039 ± 0.003
0.039 ± 0.006
Adrenals
0.030 ± 0.011†
Heart
0.66 ± 0.07
0.70 ± 0.06
0.65 ± 0.05
0.66 ± 0.09
0.49 ± 0.05
0.53 ± 0.05
0.49 ± 0.04
0.51 ± 0.05
Kidney
1.45 ± 0.13
1.47 ± 0.09
1.33 ± 0.09
1.43 ± 0.15
0.95 ± 0.07
0.94 ± 0.05
0.95 ± 0.06
0.94 ± 0.07
Liver
5.05 ± 0.40
5.31 ± 0.42
4.95 ± 0.32
4.76 ± 0.50
3.25 ± 0.24
3.31 ± 0.25
3.22 ± 0.18
3.23 ± 0.24
Spleen
0.38 ± 0.06
0.41 ± 0.05
0.38 ± 0.04
0.35 ± 0.05
0.32 ± 0.03
0.35 ± 0.04
0.32 ± 0.04
0.34 ± 0.04
Thymus
0.151 ± 0.026
0.185 ± 0.066
0.157 ± 0.030
0.140 ± 0.034
0.140 ± 0.012
0.134 ± 0.032
0.140 ± 0.021
0.126 ± 0.029
0.760 ± 0.040
0.807 ± 0.080
—
—
—
—
Epididymides
0.747 ± 0.069
0.725 ± 0.120†
Testes
1.94 ± 0.10
2.01 ± 0.14
1.91 ± 0.16
1.95 ± 0.24
—
—
—
—
Ovaries
—
—
0.075 ± 0.009
0.072 ± 0.011
0.074 ± 0.009
0.079 ± 0.011
Uterus/
—
—
0.42 ± 0.10
0.36 ± 0.13
0.42 ± 0.29
0.36 ± 0.10
Fallopian tubes
Table 11. Summary of mean organ-to-body weight ratios.
Group (male)
Group (female)
Organ
1
2
3
4
1
2
3
4
0.188 ± 0.018
0.180 ± 0.014
0.190 ± 0.038
0.315 ± 0.031
0.317 ± 0.064
0.310 ± 0.031
0.316 ± 0.046
Adrenals
0.178 ± 0.029†
Brain
5.34 ± 0.31
5.27 ± 0.45
5.38 ± 0.31
5.49 ± 0.51
7.86 ± 0.49
7.94 ± 0.56
7.99 ± 0.41
8.11 ± 0.38
Heart
3.48 ± 0.22
3.67 ± 0.41
3.49 ± 0.28
3.62 ± 0.28
3.81 ± 0.29
4.16 ± 0.34
3.91 ± 0.30
4.09 ± 0.41
Kidney
7.72 ± 0.46
7.76 ± 0.62
7.81 ± 0.46
7.47 ± 0.61
7.43 ± 0.50
7.58 ± 0.35
7.62 ± 0.56
7.14 ± 0.34*
Liver
26.84 ± 0.94
27.91 ± 1.91
26.55 ± 0.66
25.92 ± 0.77
25.45 ± 1.43
26.21 ± 1.54
25.65 ± 1.21
26.11 ± 1.50
Spleen
2.04 ± 0.26
2.17 ± 0.30
2.02 ± 0.17
1.92 ± 0.14
2.55 ± 0.32
2.73 ± 0.29
2.58 ± 0.28
2.77 ± 0.38
Thymus
0.801 ± 0.134
0.974 ± 0.374
0.844 ± 0.163
0.766 ± 0.194
1.099 ± 0.086
1.064 ± 0.264
1.119 ± 0.167
1.019 ± 0.229
4.089 ± 0.305
4.421 ± 0.444
—
—
—
—
Epididymides 3.992 ± 0.510
3.876 ± 0.777†
Testes
10.35 ± 0.75
10.59 ± 0.83
10.29 ± 1.11
10.66 ± 0.90
—
—
—
—
Ovaries
—
—
—
—
0.589 ± 0.090
0.570 ± 0.061
0.591 ± 0.059
0.641 ± 0.089
Uterus/
—
—
—
—
3.30 ± 0.90
2.89 ± 1.21
3.32 ± 2.29
2.95 ± 0.86
Fallopian tubes
Values are the Mean ± SD (n = 10 except for † n = 9). * Statistically significant different from control value (p < 0.05). See Materials and methods section
for details.
intervals. No statistical differences were noted in any male
or female group compared to their corresponding control
(Table 12).
Clinical pathology
Hematology. Absolute platelet count (PLT) was significantly decreased in males administered 40 mg/kg/day
compared with control animals (86% of control). This
change in mean hematology parameters was not adverse
and not considered related to exposure to the test substance because the pathological changes did not occur in
a dose-related pattern and because they were not accompanied by any other corresponding clinical- or histopathological change.
Table 12. Summary of motor activity assessment.
Group (male)
Group (female)
Interval
1
2
3
4
1
2
3
1
158.6 ± 30.74
152.5 ± 25.96
154.2 ± 19.93
179.9 ± 41.01
163.8 ± 26.94
168.8 ± 29.90
153.6 ± 23.29
2
93.9 ± 19.3
84.5 ± 20.7
93.3 ± 28.1
102.8 ± 21.9
95.2 ± 26.1
98.4 ± 28.4
79.8 ± 12.9
3
63.3 ± 14.3
65.7 ± 22.6
72.9 ± 25.5
80.7 ± 20.3
66.1 ± 17.0
87.0 ± 28.0
61.3 ± 22.5
4
64.8 ± 23.4
67.9 ± 25.3
70.5 ± 22.6
62.1 ± 15.3
67.8 ± 31.6
62.4 ± 25.6
59.9 ± 18.2
5
62.8 ± 14.4
50.2 ± 12.2
60.9 ± 18.9
54.3 ± 27.1
51.6 ± 14.8
62.5 ± 24.5
56.3 ± 12.5
6
63.3 ± 15.2
59.7 ± 27.0
59.0 ± 23.1
48.6 ± 26.6
72.8 ± 24.8
57.1 ± 28.9
44.5 ± 10.1
Values are the Mean ± SD. No significant difference from control was observed. See Materials and methods section for details.
Absolute eosinophil concentration (AEOS) was
s ignificantly increased in males administered 1000 mg/kg/
day (200% of control). Absolute eosinophil concentration
was also significantly increased in high dose females in a
generalized dose-related response. However, values did not
reach the level of statistical significance due to high variability within the group. Two females, in particular, showed high
eosinophil levels, and this contributed to the overall increase
in the group. Given that this finding occurred in more than
one animal within the group and occurred as a generalized
increase in all the males in Group 4, a test-substance related
effect could not be discounted in females.
In addition to the above findings, one high dose female
displayed detectable concentrations of absolute neutrophil
band (ABAN). This finding, while appearing non-adverse,
might be associated with an individual generalized granulocytic increase in response to test substance administration
at the high dose.
Coagulation. There were no treatment-related or statistically significant effects in coagulation parameters.
Clinical biochemistry. There were no adverse changes
in clinical biochemistry parameters in male or female rats
(Table 13). The following statistically significant changes in
mean clinical biochemistry results were not adverse and not
considered related to exposure to the test substance because
they were not dose-related and because they were not accompanied by any other corresponding clinical- or histopathological change. An increase in the aspartate aminotransferase
(AST) concentration in males administered 40 mg/kg/day and
females administered 400 mg/kg/day (113 and 115% of control, respectively) was observed. A decrease in sorbitol dehydrogenase (SDH) in females administered 40 and 400 mg/
kg/day (66 and 75% of control, respectively) occurred. Both
aspartate aminotransferase and sorbitol dehydrogenase are
hepatocytic enzymes, their leakage suggestive of liver injury.
However, only SDH is liver-specific, and is often accompanied
by significant loss of liver mass. Given the absence of dose-dependent changes, uncorrelative with any microscopic alterations as well as the small magnitude of the change, there was
no evidence to suggest that these changes were toxicologically
relevant to test substance administration.
Urinalysis. There were no treatment-related or statistically
significant effects in urinalysis parameters.
Serology. There were no detectable titers against the
pathogens and antigens tested. In conclusion, there were no
4
156.2 ± 14.52
100.4 ± 15.82
78.1 ± 17.4
61.2 ± 19.9
52.4 ± 24.8
57.0 ± 13.4
adverse changes in coagulation, clinical chemistry, or urinalysis parameters in male or female rats administered UC-II. The
statistically significant increase in eosinophil concentration
in high dose males, with increases in high dose females were
considered related to exposure to the test substance because
this dose-related change was accompanied by potentially
adverse histopathological change in the nasal cavity of both
male and female high dose animals.
Sacrifice,
macroscopic
observations,
and
histopathology. There were no UC-II related macroscopic
findings at scheduled sacrifice, and mortality occurring prematurely was deemed unrelated to test substance administration. At termination, test substance-related microscopic
findings were observed involving the nasal turbinates in
males and females at 1000 mg/kg/day UC-II. An increase in
the incidence and intensity of several findings involving the
respiratory epithelium were noted in males and females at
1000 mg/kg/day UC-II as compared to their respective controls. Findings included goblet cell hypertrophy/hyperplasia,
eosinophilic infiltrates, acute inflammation, and the presence
of eosinophilic cytoplasmic droplets. The incidence and intensity of these microscopic findings are presented in Table 14.
The presence of eosinophilic droplets in the nasal turbinates of
mice has been described as a non-adverse, adaptive response.
Similarly, in this instance, their presence was deemed secondary to the other morphologic alterations described above
for the nasal turbinates. There were statistically significant
increases in absolute eosinophil counts for males at 1000 mg/
kg/day and a non-statistically significant increase in mean
absolute eosinophil counts for females at 1000 mg/kg/day.
These hematologic alterations are likely associated with the
eosinophil infiltrates in the nasal turbinates, which may reflect
a test substance-related hypersensitivity reaction at the highest
dosage tested.
Microscopic findings unrelated to the test-substance
administration include: sporadic alterations involving
the esophagus attributable to repeated gavage procedures, such as minimal-to-moderate esophageal changes
included myofiber degeneration as well as fibroplasia,
hemorrhage, inflammation, and pigmented macrophages
(consistent with hemosiderin and resolving hemorrhage)
involving the esophageal wall. In addition, sporadic findings of minimal chronic inflammation and necrosis involving the Harderian glands were attributable to sequelae of
end of study orbital sinus bleeds. The remaining findings
were incidental and most commonly developmental,
Table 13. Mean clinical biochemistry values.
Group (male)
Parameter (Units)
1
2
3
4
1
Aspartate
91 ± 27
96 ± 15
97 ± 19
85 ± 7
103 ± 15*
Aminotransferase
(AST, U/L)
Alanine
44 ± 7
49 ± 5
44 ± 5
50 ± 10
36 ± 4
Aminotransferase
(ALT, U/L)
Sorbitol
9.2 ± 2.5
8.6 ± 3.7
8.2 ± 2.7
9.2 ± 1.7
10.8 ± 2.4
Dehydrogenase
(SDH, U/L)
Alkaline Phosphatase
126 ± 32
137 ± 22
139 ± 34
135 ± 27
103 ± 30
(ALKP, U/L)
Total Bilirubin (BILI,
0.14 ± 0.02
0.14 ± 0.03
0.14 ± 0.003
0.16 ± 0.03
0.18 ± 0.02
mg/dL)
Blood Urea Nitrogen
21 ± 3
21 ± 3
20 ± 1
21 ± 5
20 ± 2
(BUN, mg/dL)
Creatinine
0.29 ± 0.03
0.31 ± 0.03
0.31 ± 0.02
0.31 ± 0.04
0.39 ± 0.04
(CREA, mg/dL)
Cholesterol
79 ± 10
82 ± 10
80 ± 9
80 ± 8
90 ± 18
(CHOL, mg/dL)
Triglycerides
49 ± 9
45 ± 8
45 ± 12
38 ± 8
28 ± 5
(TRIG, mg/dL)
Glucose
159 ± 24
160 ± 36
155 ± 23
160 ± 30
119 ± 15
(GLUC, mg/dL)
Total protein
6.3 ± 0.2
6.3 ± 0.3
6.4 ± 0.2
6.4 ± 0.3
6.5 ± 0.3
(TP, g/dL)
Albumin (ALB, g/dL)
3.2 ± 0.2
3.2 ± 0.1
3.2 ± 0.1
3.3 ± 0.2
3.5 ± 0.2
Globulin (GLOB, g/dL) 3.1 ± 0.2
3.2 ± 0.3
3.1 ± 0.2
3.1 ± 0.2
3.0 ± 0.2
Calcium
9.5 ± 0.5
9.6 ± 0.5
9.7 ± 0.2
9.6 ± 0.3
9.8 ± 0.4
(CALC, mg/dL)
Inorganic Phosphorus 6.3 ± 0.7
6.6 ± 1.0
6.5 ± 0.5
6.5 ± 0.5
6.0 ± 0.9
(IPHS, mg/dL)
Sodium (NA, mmol/L) 144.6 ± 6.0
144.8 ± 3.9
145.0 ± 6.6
145.0 ± 4.1
146.1 ± 5.5
Potassium
6.06 ± 0.60
5.99 ± 0.75
5.86 ± 0.47
6.14 ± 0.32
5.17 ± 0.44
(K, mmol/L)
Chloride
103.5 ± 3.1
103.8 ± 3.2
104.2 ± 4.6
103.2 ± 1.7
105.8 ± 2.6
(CL, mmol/L)
Values are the mean ± SD (n ≥ 8). * Statistically significant different from control values (p < 0.05).
Table 14. Incidence and severity of microscopic nasal turbinate findings.
Group
1
Dose volume (mg/kg/day)
0
Sex
Male
Goblet cell hypertrophy/hyperplasia: respiratory epithelium
1
Grade 1
0
Grade 2
1
Grade 3
0
Eosinophil infiltrates: respiratory epithelium
1
Grade 1
1
Grade 2
0
Acute inflammation: respiratory epithelium
0
Grade 1
0
Grade 2
0
Eosinophil droplets: respiratory epithelium cytoplasmic
0
Grade 1
0
Grade 2
0
See Materials and methods section for details.
Group (female)
2
3
97 ± 11
98 ± 17*
4
85 ± 9
41 ± 6
41 ± 6
37 ± 3
7.1 ± 2.1*
8.1 ± 2.8*
8.6 ± 1.7
103 ± 27
104 ± 23
88 ± 18
0.20 ± 0.04
0.19 ± 0.03
0.18 ± 0.03
21 ± 3
22 ± 4
23 ± 2
0.39 ± 0.06
0.41 ± 0.05
0.39 ± 0.04
84 ± 10
84 ± 13
85 ± 18
31 ± 6
28 ± 6
27 ± 7
120 ± 12
125 ± 14
116 ± 15
6.8 ± 0.5
6.8 ± 0.2
6.8 ± 0.2
3.6 ± 0.1
3.2 ± 0.4
9.9 ± 0.5
3.5 ± 0.2
3.3 ± 0.3
9.9 ± 0.3
3.6 ± 0.2
3.2 ± 0.2
10.0 ± 0.3
5.8 ± 0.7
6.2 ± 0.5
5.5 ± 0.5
145.4 ± 7.7
5.37 ± 0.45
146.7 ± 4.4
5.33 ± 0.47
148.0 ± 7.6
5.32 ± 0.54
105.3 ± 4.4
105.7 ± 3.1
107.1 ± 4.5
3
400
Female
0
0
0
0
0
0
0
0
0
0
0
0
0
Male
0
0
0
0
1
1
0
0
0
0
1
0
1
4
1000
Female
1
0
1
0
1
1
0
0
0
0
1
1
0
Male
9
0
5
4
9
5
4
4
3
1
9
4
5
Female
9
0
8
1
9
2
7
1
1
0
7
4
3
inflammatory, or degenerative changes that can be seen
in the age and strain of rat used in this study. Examples
included, but were not limited to, nephropathy, pulmonary
alveolar histiocytosis, pituitary gland cyst, and ectopic thymus in thyroid gland. UC-II related microscopic findings
were observed involving the respiratory epithelium of the
nasal turbinates in males and females at 1000 mg/kg/day
UC-II. Salient microscopic observations included eosinophil infiltrates, goblet cell hypertrophy and hyperplasia,
and acute inflammation. Therefore, under the conditions of
this study, the anatomic pathology no-observed-adverseeffect level (NOAEL) for UC-II was 400 mg/kg/day following daily oral gavage to male and female Sprague-Dawley
rats for at least 90 days.
Discussion
Given that OA is the most prevalent form of arthritis and that
the number of persons affected with OA will increase significantly in the near future, finding alternative, safer pharmacological therapies for OA is of considerable importance. With
the continued growth of the elderly population in the US,
OA is becoming a major medical and financial concern. In
the last few years, various nutritional supplements including
chondroitin, glucosamine, avocado/soybean unsaponifiables, and diacerein have emerged as new treatment options
for osteoarthritis. Among these nutraceuticals, the efficacy of
UC-II was repeatedly demonstrated in animal (Deparle et al.
2005; D’Altilio et al. 2007; Peal et al. 2007; Bagchi et al. 2008a;
2009; Gupta et al. 2009a; b) and human (Bagchi et al. 2008b;
Crowley et al. 2009) studies without any significant adverse
events.
The current study demonstrated the broad-spectrum
safety of UC-II in animals over the dose levels and routes
of administration tested. Acute oral toxicity did not reveal
any significant changes for all examined tissues. Based on
these results, the oral LD50 of UC-II was concluded to be
> 5000 mg/kg in female rats. Acute dermal toxicity study
conducted with a single 2000 mg/kg dose of UC-II applied
directly to the skin of male and female rats for 24 h revealed
no dermal irritation, adverse pharmacological effects, or
abnormal behavior. Based on these results, the acute dermal LD50 of UC-II was > 2000 mg/kg. The primary dermal
irritation assay using a single 1000 mg dose of UC-II applied
directly to the skin of rabbits for 4 h caused an initial redness
of the skin. The overall incidence and severity of irritation
decreased with time and irritation completely subsided by
24 h. Based on these results, UC-II was classified as slightly
irritating to the skin. There were no other signs of gross toxicity, adverse pharmacologic effects, or abnormal behavior.
Primary eye irritation was tested in rabbits using a single
dose of 60 mg. One hour after UC-II application, treated eyes
exhibited corneal opacity, iritis, and positive conjunctivitis.
The overall incidence and severity of irritation decreased
gradually with time. All animals were free of ocular irritation
within 96 h. Based on these results, UC-II was classified as
moderately irritating to the eye.
Ames’ Bacterial Reverse Mutation Assay using five strains
of Salmonella typhimurium (TA98, TA100, TA1535, TA1537,
and TA102) was used to evaluate the mutagenic potential
of UC-II in the presence and absence of metabolic activation. UC-II was determined to be non-mutagenic. Cell gene
mutation assay in mouse lymphoma cells was conducted to
test the mutagenic potential of UC-II in the L5178Y mouse
lymphoma cell line. UC-II did not induce mutagenic effects
either with or without metabolic activation.
The results from the 90-day sub-chronic toxicity study did
not show any adverse effects in individual body weight or
individual organ weight after 90 days of UC-II administration
in increasing doses. No significant changes in organ-to-body
weight ratios were observed except for the kidney-to-body
weight ratio, which was significantly decreased in Group
3 males. This finding was not associated with any other
clinical findings, and did not indicate any corresponding
pathologic changes in the high dose animals. Therefore, this
change was deemed incidental and of no toxicological interest. Mortality of a single Group 3 male and a single Group
4 male were not associated with test substance administration. Test substance-related microscopic findings were
observed involving the respiratory epithelium of the nasal
turbinates in males and females at 1000 mg/kg/day UC-II.
Salient microscopic observations included eosinophil infiltrates, goblet cell hypertrophy and hyperplasia, and acute
inflammation. Therefore, under the conditions of this study,
the anatomic pathology no-observed-adverse-effect level
(NOAEL) for UC-II was 400 mg/kg/day following daily oral
gavage to male and female Sprague-Dawley rats for at least
90 days.
Overall, results from the current study combined with the
animal (Deparle et al. 2005; D’Altilio et al. 2007; Peal et al.
2007; Bagchi et al. 2008a; 2009; Gupta et al. 2009a; b) and
human (Bagchi et al. 2008b; Crowley et al. 2009) data demonstrate the broad-spectrum safety of UC-II.
Declaration of interest
This study was supported by a research grant from InterHealth
Nutraceuticals Inc. The authors report no conflicts of interest.
The authors alone are responsible for the content and writing
of the paper.
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March 2010
USA & International
UC-II® Product Sampler
September 2011
Trenker Laboratories
Product: Ortho UC-II®
Market: France
NeoCare
Product: Mobiflex®
Market: Belgium
Catalent Pharma
Solutions
Product: JointEze
Market: South Africa
Ryusendo
Product: UC-II
Market: Japan
Ushizu Pharmaceutical Co., Ltd.
Nippo Pharmaceutical Ind., Co.
Product :
Rheumasoft UC-II
Market:
Japan
UC-II
Solid Dose Products
USA
Body Ammo
Nutraceuticals
Product: RheumaGuard
Market: USA & Internet
Vitamin World
Product: UC-IITM
Contains: UC-II only
Market: USA
Sunburst Biorganics
Product: UC-II Undenatured
Type II Collagen
Contains: UC-II, Potassium
Chloride, Microcrystalline
Cellulose, Magnesium Stearate,
Silicon Dioxide.
Market: USA and Internet
Myogenix
Product: Joint and Tissue
Repair
Contains: UC-II, Vitamin C,
Glucosamine sulfate KCl,
MSM and Bromelain
Douglas Labs
Product: RheumaShield
Contains: UC-II, Devil’s
Claw Extract and Bromelain
Sun Naturals
(Arnet Pharmaceuticals)
Product: UC-II
Contains: UC-II only
NOW Foods
Product: Joint-UC-II
Contains: UC-II
Market: USA - Practitioner
Channel
Thank You

Safety and efficacy of undenatured type II collagen in the

Transcription

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