ProMetic BioSciences Inc.

ProMetic BioSciences Inc.
Triazine derivatives as protein
A mimetics for the treatment
of autoimmune disease
Presented by: Boulos Zacharie, Ph.D.
February 7, 2008
1
ProMetic Life Sciences Inc.
ProMetic
Life Sciences Inc.
Protein Technologies
ProMetic
BioTherapeutics,
(USA)
ProMetic
BioSciences Ltd
(UK)
BSafE
Innovations Inc.
(Canada)
ProMetic BioSciences Inc.
(Canada)
PBI-1402
anemia
Oncology/Hematology
and
Autoimmune Disease
2
Corporate core technology
PROMETIC = PROTEIN MIMETIC
A core technology based on the
“Mimetic Ligand™” approach
OBJECTIVE
To replace complex (expensive) proteins (antibodies)
with synthetic “drug-like” protein mimetics
CHALLENGE
Replacement of a high MW protein biopolymer
with a lower MW synthetic compound
ADVANTAGE
Increased probability of success with validated
biochemical target and accompanying human data
3
First proof of concept
™
™
Protein A linked to a filtration (Prosorba) column
is an FDA approved product for the treatment of:
¾
ITP (1987)
¾
Rheumatoid arthritis (1999)
Protein A too toxic for in vivo use
4
First proof of concept (Cont’d)
™
Develop a protein A mimetic or synthetic
equivalent as an in vivo replacement for the
Prosorba column
™
Identify compounds which function as
antagonists of the IgG-protein A interaction
™
Optimize the affinity of IgG-protein A
antagonist(s) by means of the structure activity
relationship (SAR) approach
5
First proof of concept (Cont’d)
™
PBI-1308 is the current lead molecule
™
Confirmed in vivo activity in standard
animal models
™
Potential therapeutic indications include
rheumatoid arthritis, systemic lupus
erythematosous (SLE) and psoriasis
™
Three families of patent applications covering
use and composition of matter
6
Protein A
From an X-Ray crystal
structure, the B domain
of protein A.
PHE132
Protein A (MW = 42,000)
is found on the surface
of the bacteria S. aureus
and consists of five
domains or regions,
each can bind to the Fc
portion of antibodies.
TYR133
7
Protein A
An X-Ray crystal
structure of the B
domain of protein A
complexed with human
immunoglobulin (CH2
and CH3 domains of the
Fc tail).
8
1,3,5-Substituted triazines as protein A mimetics
General structure of triazine compounds
R2
X1
N
R1
N
N
N
N
H
Linker
N
H
N
N
X2
X1, X2, R1 and R2 = substituents
™ General procedure developed for the synthesis of high yield,
high purity product
™ ~3,000 analogues of protein A mimetics synthesized
™ > 100 compounds show equipotent in vitro binding activity with
PBI-1308 as the lead compound
™ Some compounds display significant in vivo activity when
administrated by i.v. or oral routes
9
Structure-activity relationship of triazine dimers
Effect of R1 and R2
™
N
SAR studies demonstrate the
importance of the presence of at least
one 1,3-phenylenediamine substituent
Entry
R1
R2
HN
IC50 (nM)
R1
Entry
HN
1
>10
2
N
N
N
N
H
NH2
117
R2
HN
NH2
Cl
SO2Me
HN
6
192
HN
209
NH2
HN
(CH2)2
OH
450
HN
(CH2)2
NH2
749
HN
(CH2)2
OH
>105
NH2
8
NH2
IC50 (nM)
F
3
HN
OH
HN
NH2
7
NH2
N
208
HN
HN
R2
HN
F
NH2 HN
N
5
HN
HN
H
N
R1
HN
5
F
OH
HN
HN
HN
4
204
9
NHSO2CH3
F
IC50 (Protein A) = 334 nM
10
Structure-activity relationship of triazine dimers
NH2
N
Effect of the diamine linker
N
H
Entry
Diamine Linker
1
2
3
4
NH
HN
IC50 (nM)
OH
HN
Entry
N
N
N
N
H
Linker
N
H
Diamine Linker
NH
NH
HN
NH
HN
NH
165
HN
O
HN
1020
HN
HN
7
117
NH
422
HN
NH
519
IC50 (nM)
NH
284
OH
6
N
H
NH
10
5
N
248
424
9
HN
N
HN
321
8
HN
OH
NH2
HN
NH
11
NH
460
HN
617
12
112
N
IC50 (Protein A) = 334 nM
11
Structure-activity relationship of triazine dimers
OH
HN
N
Effect of the X2
X2
Entry
H2N
N
H
H
N
N
N
X2
IC50 (nM) Entry
N
N
H
64
1
HN
3
HN
HN
NH2
HN
77
4
8
PBI-1308
NH2
HN
N
687
NH
196
HN
990
14
OH
77
9
HN
51
162
N
HN
HN
5
78
12
OH
58
HN
NH 2
NH 2
85
13
233
IC50 (nM)
11
NH2
7
H 2N
X2
HN
6
130
N
IC50 (nM) Entry
HN
2
NH2
X2
OH
HN
H
N
N
F
15
N
16
HN
2560
10
N
69
N
OH
143
IC50 (Protein A) = 334 nM
12
Representative example of the synthesis of the
lead compound PBI-1308
NHBoc
N
H
N
Cl
THF/acetone/H2O,
Cl RT, 48h, 94%
OH
NHBoc HN
4-Aminophenethylamine
DIPEA
N
N
N
THF, 70 °C, 48h, 99%
N
H
NH2
N
N
N
H
Cl
N
N
H
N
H
Ethanolamine
5% aq. NaHCO3
N
OH
NHBoc HN
N
Cl
N
Acetone/H2O,
-10 °C, 2h, 99%
NH2
NHBoc
NHBoc
Cyanuric chloride
5% aq. NaHCO3
N
N
OH
NHBoc HN
H
N
Cl
N
5% aq. NaHCO3
THF/acetone/H2O,
RT, 48h, 96%
1) 4-Aminophenethylamine
DIPEA, THF, 70 °C, 48h,
85%
2) HCl 4M in dioxane
10% H2O, 0 °C, 2h,
51%
N
H
N
N
NH2
N
N
H
N
H
N
Cl
NHBoc
OH
HN
N
H
N
N
H
N
N
N
N
N
H
H
N
N
N
NH
PBI-1308
NH2
.5 HCl
H2N
13
Postulated binding site of PBI-1308 onto
Fc portion of IgG
1
2
3
4
5
SDS-PAGE of human total IgG bound and
purified by PBI-1308 linked on a solid support
1:
2:
3:
4:
5:
Prestained SDS-PAGE standard broad range
Human total IgG
Flow through fraction
Washed fraction
Eluted fraction
14
More soluble analogues of PBI-1308
NH2
OH
HN
N
N
H
H
N
N
N
N
N
H
N
N
H
N
NH2
HN
PBI-1308
NH2
™
Alteration of functional groups improves the solubility of PBI-1308
General structure:
OH
NH2
HN
N
N
H
OH
N
N
OH
HN
N
Linker
OH
N
N
N
OH
H
15
Effect of the diamine linker
OH
OH
HN
N
H 2N
Entry
N
H
Diamine Linker
OH
N
N
N
N
H
Linker
IC50 (nM, PBS) Entry
HN
117
1
OH
HN
N
H
N
N
OH
N
H
Diamine Linker IC50 (nM, PBS)
HN
167
4
NH
NH
NH
173
2
HN
3
HN
NH
HN
5
NH
554
451
IC50 (protein A) = 189 nM (PBS)
16
Effect of the diamine linker
OH
OH
HN
N
H
Entry
N
N
N
N
H
OH
HN
N
N
H2N
OH
Linker
N
H
Diamine Linker
N
N
H
F
IC50 (nM, PBS)
HN
158
1
NH
HN
294
2
NH
NH
500
3
HN
IC50 (protein A) = 189 nM (PBS)
17
Effect of R2
OH
OH
HN
N
H2N
R2
Entry
HN
N
H
1
253
HN
2
444
F
HN
N
N
F
3
5
HO
6
H2N
7
HO
8
348
HN
4
1080
F
HN
N
OH
OH
R2
R2
IC50 (nM) Entry
OH
N
N
N
138
NH2
803
NH2
80
NH2
HN
H
N
N
N
N
H
R2
IC50 (nM) Entry
NH2
H
N
9
11
NH2
HN
N
N
37
2440
1370
NH2
N
10
IC50 (nM)
12
N
O
830
58
NH2
IC50 (protein A) = 334 nM
18
Effect of R1 and R2 (linker 4-aminoaniline)
R1
N
H 2N
R
X1
Entry
HN
HN
NH22 HN
HN
NH
N
N
H
N
R22
X
HN
3
HN
HN
OH
HN
OH
N
R2
X
R11
Entry
X
R22
7
OH
HN
248
>105
8
HN
HN
OH
9
HN
501
OH
HN
HN
OH
HN
430
OH
10
11
12
HN
OH
HN
OH
HO
OH
N
OH
HN
OH
HN
499
298
N
O
834
N
O
398
868
13
F
6
303
HN
HN
OH
F
Cl
OH
OMe
5
414
HN
NH2
HN
IC50 (nM)
HN
OH
4
OH
NH22
NH
107
OH
H
N
N
N
N
H
IC50 (nM)
1
2
H
N
N
O
F
292
14
N
N
N
N
791
IC50 (protein A) = 334 nM
19
Effect of R1 and R2
(linker 4-aminophenethylamine)
OH
HN
H
N
N
N
N
H 2N
N
N
HN
R2
IC50 (nM) Entry
R2
R1
N
N
H
H
Entry
R1
N
R1
NH2 HN
R2
HN
51
1
4
IC50 (nM)
F
OH
209
HN
NH2
HN
NH2
HN
2
NH2
OH
HN
78
5
HN
HN
293
F
1330
HN
OH
3
IC50 (protein A) = 334 nM
20
In Vivo Results – Effect of oral and intravenous
administration of PBI-1308: delayed-type
hypersensitivity model
OH
HN
N
0.45
H2N
N
H
H
N
N
N
NH2
N
HN
PBI-1308
0.40
Inflammation (mm)
N
N
H
H
N
N
NH2
0.35
0.30
0.25
0.20
0.15
Control PO Methotrexate
PBI-1308 (mg/kg)
150
per os
50
25
5
0.5
21
In Vivo Results – Effect of intravenous administration
of PBI-4117 and PBI-4122: delayed-type
hypersensitivity model
OH
HO
HO
N
H2N
N
H
H
N
N
N
N
H
H
N
N
N
O
N
PBI-4117
OH
N
H2N
HN
N
H
H
N
N
OH
OH
N
OH
HN
PBI-4122
OH
H
N
N
N
N
H
N
O
NH2
N
100
N
90
N
80
Inlammation (mm )
OH
HN
NH
H2N
H
N
N
N
H
N
H
N
N
N
N
H
N
F
N
70
PBI-3668
60
50
N
OH
40
OH
HN
30
N
20
H2N
10
N
H
H
N
N
N
N
H
H
N
N
N
N
OH
OH
N
0
PBI-4121
Control
Methotrexate
PBI-4117
PBI-4122
N
™ Similar results were obtained with PBI-4121 and PBI-3668
22
In Vivo Results – Effect of topical application of
PBI-1308: delayed-type hypersensitivity model
OH
HN
N
H2N
0.50
N
H
H
N
N
N
N
N
H
H
N
N
NH2
N
HN
PBI-1308
Inflammation (mm)
NH2
0.40
0.30
0.20
0.10
0
Control
Hydrocortisone (0.5%)
PBI-1308 (0.5%)
23
In Vivo Results – Effect of intravenous and oral
administration of PBI-1308: adjuvant-induced arthritis
model
OH
HN
N
H2N
N
H
Control
MTX
PBI-1308 IV
4.30
H
N
N
N
N
N
H
H
N
N
NH2
N
HN
PBI-1308
NH2
Inflammation (mm)
PBI-1308 PO
3.80
3.30
2.80
2.30
1
2
3
4
5
6
7
8
10
12
13
14
15
16
18
19
20
21
22
DAYS
Inflammation significantly inferior to Control (P<0.05)
MTX: Day 14, 15, 16, 18 and 20
PBI-1308 IV: Day 13, 14, 15, 16, 18 and 20
PBI-1308 PO: Day 13, 14,15,16,19, 20, 21 and 22
24
In Vivo Results – Inhibition of inflammation by
intravenous administration of PBI-1308: adjuvantinduced arthritis model
OH
HN
N
H2N
N
H
H
N
N
N
N
N
H
H
N
N
NH2
N
HN
PBI-1308
5
NH2
CTL
4
mm
MTX
3
PBI-1308
2
1
0
D1
D2
PBI-1308 is significantly less inflammed than control on day 1 and 2.
PBI-1308 is significantly less inflammed than MTX on day 1.
MTX is significantly less inflammed than control on day 2.
25
In Vivo Results – Inhibition of inflammation by
intravenous administration of PBI-1308: adjuvantinduced arthritis model
OH
HN
N
H 2N
DAY 1
15000
5000
Control
Control
H
N
N
N
N
H
NH2
N
HN
PBI-1308
400
10000
0
N
PGE2
pg/mg of tissue
[Rat TNF] (pg/g tissue)
TNFα
N
H
H
N
N
NH 2
300
200
100
0
MTX
PBI
PBI-1308
Methotrexate
PBI-1308
Control
Control
MTX
PBI-1308
Methotrexate
PBI-1308
DAY 2
PGE2
4000
500
pg/mg of tissue
[Rat TNF] (pg/g tissue)
TNFα
3000
2000
1000
0
*
Control
Control
400
300
200
100
0
Methotrexate
PBI-1308
Control
PBI-1308
Control Methotrexate
MTX
PBI-1308
MTX
PBI-1308
* Significantly different from control p < 0.05 ** Significantly different from control p < 0.03
**
26
In Vivo Results – Inhibition of inflammation by
intravenous administration of PBI-1308: LPS-induced
air-pouch model
OH
HN
N
H2N
N
H
H
N
N
N
N
N
H
H
N
N
NH2
N
HN
PBI-1308
NH2
3000
120
TNF-α (pg/ml)
PGE2 (pg/ml)
100
80
60
40
20
0
*
*
Control
Indomethacin
PBI-1308
* Significantly different from control p < 0.01
2000
1000
*
0
Control
Indomethacin
PBI-1308
* Significantly different from control p < 0.001
27
Autoimmune Disease – Achievements
1. Developed a general procedure that is:
R1
a) High yield
R4
N
4-6 steps
N
b) Reproducible
Linker
N
N
N
N
R2
R3
R1, R2, R3 and R4 = substituents
c) Amenable to scale-up; multi-gram scale
synthesis of high purity lead compound PBI-1308
without the use of column chromatography
d) Yields a diverse range of analogues;
approximately 3,000 synthesized to date
28
Autoimmune Disease – Achievements (Cont’d)
2. By means of the drug discovery process:
a) Identified compounds with significant in vitro
activity as mimetics of bacterial protein A, e.g.
some equipotent with protein A
b) Discovered compounds which display significant
in vivo activity when administered by i.v. or oral
routes
3. In June 2007, an agreement was reached between
ProMetic and Laboratorios Dermatológicos Darier
(Mexico) for development of PBI-1308 to treat
dermatological disorders.
29