Paolo Mainardi

Paolo Mainardi
Mincep, October 21, 2008
Paolo Mainardi
2
Mincep, October 21, 2008
The problem of refractory epilepsy has not been solved,
despite the increasing number of new AEDs
History of AEDs in the US
Zonisamide
18
Levetiracetam
16
Topiramate
Tiagabine
Lamotrigine
Gabapentin
Felbamate
Valproic acid
Clonazepam
Ethosuximide Carbamazepine
14
N°of AEDs
12
10
8
6
4
Bromides
Oxcarbazepine
Phenytoin
Phenobarbital
Primidone
2
0
1850
1870
1890
1910
1930
1950
1970
1990
2010
2030
years
Novel mechanisms of action are needed to solve drug resistance
W. Löscher, I.E. Leppik . Critical re-evaluation of previous preclinical strategies for the discovery and the development of
new antiepileptic drugs. Epilepsy Research 50 (2002) 17–20
“to solve this problem, ….a better hypothesis of the basic mechanisms of pharmacoresistance
are needed”.
Paolo Mainardi
3
Anticonvulsive effect of Serotonin:
Mincep, October 21, 2008
1956 Bonnycastle: DPH increases brain serotonin
1957 Bonnycastle: AEDs increases brain serotonin
1973 Boggan WO et al. Antiepileptic effect of 5-HTP in animals
1973 Wenger GR et al. Decreasing in brain 5-HTP increases seizure in animals
1975 Maynert E et al. Decreasing in brain 5-HTP increases seizure in humans
1983 Lazarova M. Serotonin mediation of the protective effect of clonidine against
pentylenetetrazol-induced seizures in rats.
Anticonvulsive effect of SSRIs:
1975: Truscot TC et al. Anticonvulsant effect of Fluoxetine in mice. Pharmacol Biochem
Behav 3, 909 1975.
1992: Leander JD. Fluoxetine, a selective serotonin-uptake inhibitor, enhances the
anticonvulsant effects of phenytoin, carbamazepine, and ameltolide. Epilepsia. 1992
May-Jun;33(3):573-6.
1992: Pasini A, Tortorella A, Gale K. Anticonvulsant effect of intragranial fluoxetine.
Brain Res. 1992 Oct 16;593(2):287-90.
1993: Prendiville S, Gale K. Anticonvulsant effect of fluoxetine on focally evoked limbic
motor seizures in rats. Epilepsia. 1993 Mar-Apr;34(2):381-4.
Paolo Mainardi
4
Mincep, October 21, 2008
In 1994 our group started clinical trials by adding SSRIs to
AED therapy in drug-resistant patients.
As SSRIs we used both
• fluoxetine
• citalopram
Our data showed the anticonvulsant effect of SSRIs:
potentiation of the serotoninergic system helps
to control epileptic seizures.
Jobe, 2005: high doses of SSRIs are pro-convulsants, low doses are anti-convulsants
Favale E, …, Mainardi P, …, Albano C. Anticonvulsant effect of fluoxetine in humans. Neurology. 1995
Oct;45(10):1926-7
Favale E,…, Albano C. The anticonvulsant effect of citalopram as an indirect evidence of serotonergic impairment in
human epileptogenesis. Seizure. 2003 Jul;12(5):316-8.
Cupello A, …, Albano C. Decrease of serotonin transporters in blood platelets after epileptic seizures. Neurochem
Res. 2005 Apr;30(4):425-8.
Albano C, et al. Successful treatment of epilepsy with serotonin reuptake inhibitors: proposed mechanism.
Neurochem Res. 2006 Apr;31(4):509-14. Epub 2006 May 13
Cupello A, …., Albano C. Epileptic seizures but not pseudoseizures are associated with decreased density of the
serotonin transporter in blood platelet membranes. Neurochem Res. 2008 Nov;33(11):2263-8. Epub 2008 Apr 22.
Paolo Mainardi
5
Mincep, October 21, 2008
Tryptophan, the only precursor of serotonin
An essential amino acid
A large neutral amino acid (LNAA)
All LNAAs compete to the same carrier to cross BBB
LNAAs: Tyr, Val, Met, Ile, Leu, Phe, Trp
The rate of Trp brain uptake depends on Trp/LNAAs plasmatic ratio
R=Trp/LNAAs
Cughani, by PET, confirmed
that brain serotonin synthesis
depends on brain Trp uptake
6
Low Trp levels in blood of epileptic patients:
•
•
Paolo Mainardi
Mincep, October 21, 2008
Marion JL, Bigot JC, Goas JY . Presse Med 1985;14(12):681–3
Ko FJ, Chiang CH, et al.. Ko Hsiung I Hsueh Ko Hsueh Tsa Chih 1993;9(3):131–42
Our own data:
Lunardi G, Mainardi P, et al. Tryptophan and epilepsy. In: Recent Advances in Tryptophan Research
(Eds G. Allegri Filippini and V. L. Costa) New York and London, Plenum Press, 1995: pp. 101–102
Lunardi G, Mainardi P, et al.. Tryptophan and epilepsy. Adv Exp Med Biol. 1996;398
R=Trp/LNAAs
*
*
*
*
*
1/3 reduction in brain influx rate!
Paolo Mainardi
7
A new idea:
Mincep, October 21, 2008
To increase plasmatic
R=Trp/LNAAs
To increase brain Trp
uptake
To increase brain
serotonin synthesis
To increase seizure control
8
Paolo Mainardi
Mincep, October 21, 2008
In the late 80’s 14 million people were assuming oral tryptophan to:
Clinical studies on oral Trp administration in more severe diseases, i.e.
epilepsy and depression, carried out controversial results.
9
Trp in experimental models
Paolo Mainardi
Mincep, October 21, 2008
• DBA/2J,
• Phe increases seizures,
• Trp decreases seizures
• O'Grady mice,
• 5-HTP decreases seizures
• electrical stimulation of the
dorsal hippocampus of rats.
• systemic injections of 5-HTP
• a great reduction in the
electrographic seizure activity
• GEPR.
• varied doses of 5-HTP ip + fluox. (12.5, 25, 50 + 15 mg/kg)
• the severity of audiogenic seizures was decreased dose-dependently
•
•
•
•
GEPR-9s
15 mg/kg fluox. reduced seizures in 33% of rats
12.5 mg/Kg 5-HTP no anticonvulsant effect
Together decrease seizures in 83% of rats
Conclusion:
I.p. or i.v. Trp administration is effective, while oral administration, is not.
Paolo Mainardi
10
Mincep, October 21, 2008
Free amino acids vs protein intestinal absorption
Free amino acids are not fully absorbed
In the GI system LNAAs compete for the same carrier as in BBB
• Whey proteins do not coagulate under
acidic conditions
• They are considered to be “fast proteins”
• They reach the jejunum rapidly after
ingestion
Peptides arising from protein demolition are freely adsorbed by GI system
Enzymatic proteolysis continue in the blood
Whey proteins produce a higher postprandial level of plasma amino acids
compared to casein (Hall WL,et al. Br J Nutr 2003;89:239-248)
Frenhani PB, Burini RC. Mechanism of action and control in the digestion of proteins and peptides in humans.
Arq Gastroenterol. 1999 Jul-Sep;36(3):139-47
Paolo Mainardi
11
Mincep, October 21, 2008
•
•
•
•
•
Alpha-lactalbumin (ALAC) is a whey protein
naturally occurring in human milk (2 g/l)
high Trp/LNAAs ratio: contains 4.8% trp.
fully adsorbed by gastro-intestinal system as short peptides
increases the Trp/LNAAs ratio up to 48% compared to casein
produced by Davisco Foods Intl as ultrapure protein
Paolo Mainardi
12
Mincep, October 21, 2008
Serplus (Giofarma Srl) is a food supplement
containing 0.75 g/tablet of ultra pure ALAC
ALAC is supplied as pure protein by Davisco Foods
Paolo Mainardi
13
Mincep, October 21, 2008
3
2
2
1
1
0
0
3
6
1
0
12
0
1
months
5
4
4
3
3
2
2
mg Alac/die
seizures
1
0
0
0
15
10
1
3
6
12
1
1
0
1
2
5
6
2
25
3
20
2
15
10
1
5
0
0
3
6
12
mg Alac/die
seizures
2
1
0
2
3
months
pt 6: sex F, age 28, PC II gen
35
30
4
mg Alac/die
seizures
25
3
20
2
15
10
1
mg Alac/die
seizures
5
0
0
1
2
3
SERPLUS TRIAL
pt 8: sex F, age 45, PC II gen
3
1
12
5
0
200
180
160
140
120
100
80
60
40
20
0
0
6
6
12
months
4
mg Alac/die
4
seizures/ month
30
3
SERPLUS TRIAL
12
5
35
months
3
SERPLUS TRIAL
pt 7: sex F, age 34, Postural
seizures
0
1
months
SERPLUS TRIAL
2
pt 5: sex F, age 67, PC II gen
3
3
2
2
0
mg Alac/die
5
0
0
5
4
4
months
mg Alac/die
20
2
months
2
1
1
seizures
25
3
12
3
1
0
mg Alac/die
mg Alac/die
4
mg Alac/die
5
2
6
SERPLUS TRIAL
6
seizures/ month
mg Alac/die
pt 4: sex F, age 46, PC II gen
5
1
3
30
months
SERPLUS TRIAL
0
2
35
4
6
12
mg Alac/die
seizures
mg Alac/die
2
2
seizures/ month
1
seizures
3
seizures/ month
0
mg Alac/die
5
seizures/ month
3
100
90
80
70
60
50
40
30
20
10
0
seizures/ month
4
pt 3: sex F, age 51, PC II gen
pt 9: sex M, age 53, Mioclonic
5
3
4
2
3
2
2
1
1
1
0
0
0
1
2
3
months
6
12
seizures/ month
5
4
SERPLUS TRIAL
mg Alac/die
5
4
mg Alac/die
5
pt 2: sex M, age 39, PC II gen
seizures/ month
SERPLUS TRIAL
pt 1: sex F, age 41, PC II gen
seizures/ month
mg Alac/die
SERPLUS TRIAL
mg Alac/die
seizures
Paolo Mainardi
14
Mincep, October 21, 2008
5
15
10
1
mg Alac/die
seizures
25
3
20
2
15
10
1
5
0
0
0
3
6
0
12
1
2
100
90
80
70
60
50
40
30
20
10
0
mg Alac/die
4
3
2
1
0
3
6
SERPLUS TRIAL
seizures
2
1
0
0
1
4
10
4
8
3
6
2
4
mg Alac/die
seizures
mg Alac/die
5
3
6
2
1
0
0
0
3
6
12
1
2
12
pt 15: sex F, age 56, PC II gen
35
5
30
4
mg Alac/die
seizures
25
3
20
2
15
10
1
mg Alac/die
seizures
5
0
0
0
1
2
3
SERPLUS TRIAL
pt 17: sex M, age 35, PC + GM
100
90
80
70
60
50
40
30
20
10
0
0
6
6
12
months
2
2
3
SERPLUS TRIAL
12
3
1
months
2
SERPLUS TRIAL
12
seizures/ month
mg Alac/die
pt 16: sex M, age 40, PC
5
2
pt 14: sex M, age 50, PC II gen
3
12
seizures
months
4
mg Alac/die
mg Alac/die
0
1
months
SERPLUS TRIAL
1
1
0
200
180
160
140
120
100
80
60
40
20
0
months
0
0
12
5
mg Alac/die
5
2
2
2
1
mg Alac/die
pt 13: sex M, age 51, PC
seizures/ month
SERPLUS TRIAL
1
6
3
3
months
months
0
3
seizures
3
months
6
12
mg Alac/die
seizures PC
mg Alac/die
2
mg Alac/die
0
seizures/ month
1
4
5
seizures/ month
0
4
seizures/ month
20
2
mg Alac/die
3
5
30
4
seizures/ month
25
35
pt 12: sex M, age 59, PC
seizures/ month
30
SERPLUS TRIAL
pt 17: sex M, age 35, PC + GM
5
12
4
10
8
3
6
2
4
1
2
0
0
0
1
2
months
3
6
12
seizures/ month
35
4
pt 11: sex F, age 50, PC
mg Alac/die
5
mg Alac/die
SERPLUS TRIAL
pt 10: sex M, age 60, PC II gen
seizures/ month
SERPLUS TRIAL
mg Alac/die
seizures GM
Paolo Mainardi
ALAC on experimental models
Audiogenic mice: GEPR-9s
15
Mincep, October 21, 2008
5 days chronic administration
Single oral administration
vehicle
250
500
750
1000
6
3
9
Seizure score
Seizure score
9
vehicle
250
500
750
1000
6
3
0
0
0
1
2
3
4
6
12
24
0
48
1
2
3
4
6
12
24
48
Time (hours) after last ALAC administration
Time (hours) after last ALAC administration
12 days chronic administration
Seizure score
9
vehicle
250
500
750
1000
6
3
N=5 animals per dose and time group
0
0
1
2
3
4
6
12
24
Time (hours) after last ALAC administration
48
Results obtained by De Sarro, Confidential data
Paolo Mainardi
ALAC on experimental models
16
ALAC in DBA/2 mice
Mincep, October 21, 2008
N=10 animals per dose and time group
% OF CLONUS
100
1 day
80
5 days
60
40
12 days
20
0
0
250
500
750
ALAC dose (mg/kg)
8
Duration(sec)
MES test:
5 days repeated ALAC administrations
MES test
1000
7
6
5
4
0
2
4
6
8
g/Kg ALAC for 5 days
Results obtained by De Sarro, Confidential data
ALAC on experimental models
ALAC on seizures induced by Pilocarpine
17
Chronic seizures
Acute seizures
ALAC 15 days
Animal group
Dose (mg/kg)
50
40
30
20
10
0
200
400
600
ALAC dose (mg/Kg)
CBZ
n EEG
n EEG
70
60
0
70
60
50
40
30
20
10
0
0
10
20
30
40
CBZ dose (mg/Kg)
Paolo Mainardi
Mincep, October 21, 2008
Pilocarpine (mg/kg)
250
270
300
Control
10/10
10/10
10/10
CBZ 20
10/10
10/10
10/10
CBZ 40
10/10
10/10
10/10
CBZ 80
10/10
10/10
10/10
ALAC 125
4/10
6/10
9/10
ALAC 250
0/10
3/10
9/10
ALAC 500
0/10
8/10
10/10
50
Results obtained by De Sarro, Confidential data
ALAC on experimental models
18
Paolo Mainardi
Mincep, October 21, 2008
Chronic administration of ALAC increases
Trp/LNAAs plasmatic ratio.
Our own data
R trp/LNAAs
Fuertè et al.
R trp/LNAAs
n= 7
0,30
0,30
0,25
0,25
0,15
Rtrp/LNAAs
R
0,20
0,10
0,05
0,20
ALAC
CAS
0,15
0,10
0,00
0
5
10
days of ALAC (250 mg/Kg) chronic
administration
15
0,05
0,00
0
5
days 190 g/kg die
10
(Fuerte S
Our initial hypothesis has been confirmed: an increase in
Trp/LNAAs ratio permits to improve the seizure control.
et a l)
Paolo Mainardi
19
Mincep, October 21, 2008
Is the anticonvulsant effect due to
Trp/LNAAs increase only?
• Only 1% Trp is uptaked in the brain
• Cortisol increases the liver catabolism of Trp
• Cortisol levels are elevated in epileptic and depressed pts
• ALAC can decrease cortisol levels
Markus CR et al. The bovine protein alpha-lactalbumin increases the plasma ratio of tryptophan to the other large neutral amino
acids, and in vulnerable subjects aises brain serotonin activity, reduces cortisol concentration, and improves mood understress.
Am J Clin Nutr. 2000 Jun;71(6):1536-44.
• Trp (i.v.) decreases other LNAAs, increasing protein synthesis
Paolo Mainardi
20
Diet and epilepsy: Ketogenic diet
Mincep, October 21, 2008
Is the antiepileptic effect of the ketogenic diet due to ketones?
Mainardi P, Albano C. Med Hypotheses. 2008;70(3):536-9.
APR: Acute phase reaction
Stig Bengmark , Acute and ‘‘chronic’’ phase reaction - a mother of disease.
Clinical Nutrition (2004) 23, 1256–1266
Brain synthesis:
• Leptin
• NPY “endogenous anticonvulsant”
• Norepinephrine
NE
All of them are reported to be anticonvulsant when injected in the brain
Intestinal stimuli:
• Starvation….... (Biblical times)
• Fats…….. (Ketogenic diet)
• Medium Chain Triglycerids… (MCT diet)
• Tryptophan and or Serotonin…. (SSRIs, AEDs, ALAC)
Paolo Mainardi
21
Mincep, October 21, 2008
Brain synthesis
Norepinephrine, Leptin and NPY are reported to be
anticonvulsant when injected in the brain
NPY “endogenous anticonvulsant”
Leptin
Norepinephrine
NE
•El Bahh B et al. The anti-epileptic actions of neuropeptide Y in the hippocampus are
mediated by Y and not Y receptors. Eur J Neurosci. 2005 Sep;22(6):1417-30.
•Richichi C. et al. Anticonvulsant and antiepileptogenic effects mediated by adeno-associated
virus vector neuropeptide Y expression in the rat hippocampus. Neurosci. 2004 Mar
24;24(12):3051-9.
•Woldbye DP et al. Neuropeptide Y inhibits in vitro epileptiform activity in the entorhinal cortex
of mice. Neurosci Lett. 2002 Nov 22;333(2):127-30.
•Sabrina Diano1,2 and Tamas L. Horvath1,2,3 Anticonvulsant effects of leptin in epilepsy J.
Clin. Invest. 118:26–28 (2008).
•Lin Xu, et al Leptin inhibits 4-aminopyridine– and pentylenetetrazole-induced seizures and
AMPAR-mediated synaptic transmission in rodents. J. Clin. Invest.. 118:272
Intestinal stimuli
Serotonin
Tryptophan
MCTs
Fats
Starvation
Paolo Mainardi
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Mincep, October 21, 2008
1) 5-HTP, i.p. injected, increases serum leptin
Jun YAMADA, et al. Involvement of Leptin in Hypophagia Induced by the
Serotonin Precursor 5-Hydroxytryptophan (5-HTP) in Mice. Biol. Pharm. Bull.
29(3) 557—559 (2006)
2) Leptin increases Insulin secretion
Ceddia RB, et al. Modulation of insulin secretion by leptin, General PharmacologyVolume
32, Issue 2, , February 1999, Pages 233-237.
3) Fats (KD) increases insulin secretion
C. Cruciani-Guglielmacci, et al. Early changes in insulin secretion and
action induced by high-fat diet are related to a decreased sympathetic tone
Am J Physiol Endocrinol Metab 288
4) MCT diet increases insulin secretion
Valls E, et al. Modifications of insulin and growth hormone after medium
chain triglycerides ingestion An Esp Pediatr. 1978 Oct;11(10):675-82
Paolo Mainardi
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Mincep, October 21, 2008
5) ALAC increases insulin secretion
Glucose level
ALAC, orally administered, has the
same effect as Trp (i.p. or I.v. injected)
180
140
120
100
80
60
40
20
0
0
30
60
90
120
150
time after glucose administration (min)
Insulin / Glucose ratio
Insulin levels
300
2,50
2,00
250
200
I/G
Insulin (m U/m l)
glucose (mg/dl)
160
1,50
150
1,00
100
0,50
50
0,00
0
0
0
30
60
90
120
time after glucose administration (min)
150
30
60
90
120
time after glucose administration (min)
150
Paolo Mainardi
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Mincep, October 21, 2008
The role of catecholamine in epilepsy
• Augmentation of norepinephrine increases anticonvulsant effect of KD
Martillotti J, Weinshenker D, Liles LC, Eagles DA. A ketogenic diet and knockout of the norepinephrine transporter both reduce
seizure severity in mice. Epilepsy Res. 2006 Mar;68(3):207-11.
• Pharmacological stimulation of noradrenergic system increases anticonvulsant activity
of drugs. Conversely pharmacological suppression reduces the activity of AEDs.
Fischer W, Müller M. Pharmacological modulation of central monoaminergic systems and influence on the anticonvulsant
effectiveness of standard antiepileptics in maximal electroshock seizure. Biomed Biochim Acta. 1988;47(7):631-45.
• Norepinephrine has both anticonvulsant and antidepressive effects
Ahern TH, Javors MA, Eagles DA, Martillotti J, Mitchell HA, Liles LC, WeinshenkerD. The effects of chronic norepinephrine
transporter inactivation on seizuresusceptibility in mice. Neuropsychopharmacology. 2006 Apr;31(4):730-8.
In agreement with anticonvulsive and antidepressive effect of SSNRIs
Paolo Mainardi
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Mincep, October 21, 2008
Ketogenic diet has been tested in other neurological diseases, as
depression, ALS, MS, Alzheimer, …
Nayanah Siva. Can ketogenic diet slow progression of ALS? The Lancet Neurology, Volume 5, Issue 6, June 2006, Page 476
Samuel T. Henderson, Ingrid Van der Auwera, Stefaan Wera and Fred Van Leuven. A ketogenic diet reduces amyloid beta 40 and 42 in a mouse model of
Alzheimer’s disease. Alzheimer's and Dementia, Volume 2, Issue 3, Supplement 1, July 2006, Pages S637-S638
Patricia Murphy, Sergei Likhodii, Kirk Nylen and W.M. Burnham. The antidepressant properties of the ketogenic diet. Biological Psychiatry, Volume 56,
Issue 12, 15 December 2004, Pages 981-983
Gasior M, Rogawski MA, Hartman AL. Neuroprotective and disease-modifying effects of the ketogenic diet. Behav Pharmacol. 2006 Sep;17(5-6):431-9
Trp/LNAAs plasmatic ratio was found lower in:
epilepsy, depression, MS, ALS,…
Leptin was found lower in: epilepsy, depression, SM,….
Further studies are needed to better understand the
neuroendocrine response of ALAC
Paolo Mainardi
26
Possible future perspective and co-operation:
Mincep, October 21, 2008
• ALAC controlled clinical trials
• Studies on ALAC neuroendocrine response
• …. other proposals are welcome!
Keep in touch !
[email protected]
Thank you for your patience
and your kind attention ☺