IMMUNOMODULATORY PROPERTIES OF THALIDOMIDE

1
IMMUNOMODULATORY PROPERTIES OF THALIDOMIDE

• Steve Teo PhD DABT
• Celgene Corporation
• Warren, NJ
• Steo_at_celgene.com
• September 23 2003

2
HISTORY AND ACTIVITY (Stephens and Brynner 2001)
3

• 1953 thalidomide synthesized by Ciba
• Small molecule glutamic acid derivative
• Barbiturate analog without toxicity
• Used as safe sedative and anti-emetic in Europe
• Late 50s - 60s fetal deformities seen in humans
• Early 60s removed from European market
• Never approved in the US (AE concerns)
• 1965 thalidomide reported effective for Erythema
  Nodosum Leprosum (ENL) in leprosy
• 1970 drug of choice for ENL (WHO)
• 1960s present experimental use for various
  diseases

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(No Transcript)
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ENL Treatment(Teo et al., 2002)
Before
6 weeks 200 mg
1 week 200 mg
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Selected Activities and Side-Effects

• Inhibition of TNF?
• Stimulation of IL-10
• Stimulate T-cell function
• Inhibition of angiogenesis

• Teratogenicity
• Sedation
• Constipation
• Peripheral neuropathy

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Current Thalidomide Disease Applications

• HIV
• Aphthous Ulcers
• Cachexia
• Protozoal Diarrhea
• Kaposis Sarcoma

• GI
• Crohns Disease
• Ulcerative Colitis

• NEUROLOGY
• Dementia
• Adrenoleukodystrophy
• Multiple Sclerosis

• CANCER
• Multiple Myeloma (Phase 3)
• Myelodysplastic syn
• Glioblastoma
• Breast
• Prostate
• Cachexia
• Liver
• Melanoma
• Leukemias
• Renal Cell
• Head/Neck
• Lung

• ENDOCRINOLOGY
• Type II Diabetes

• OPHTHALMOLOGY
• Macular Degeneration
• Diabetic Retinopathy

• DERMATOLOGY
• Behçets Disease
• Discoid Lupus

• CARDIOLOGY
• Congestive Heart Failure

• INFECTIOUS DISEASE
• TB Meningitis
• ENL/Leprosy APPROVED USE

• RHEUMATOLOGY
• Rheumatoid Arthritis
• Sarcoidosis
• Ankylosing Spondylitis
• Scleroderma

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THALIDOMIDE ANALOGUES(Corral et al., 1996
1999a 1999b)
9

• Immunomodulatory analogues (IMiDs) CC-5013
  (REVIMID?), CC-4047 (ACTIMID?)
• 2000 20,000X more potent in inhibiting TNF-?
  from LPS-stimulated monocytes
• Fewer side-effects in humans
• Similar immunomodulatory profile with increased
  potency
• Currently in Phase 2 (CC-4047, prostate cancer)
  and 3 (CC-5013, multiple myeloma and metastatic
  melanoma)

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Thalidomide
CC-5013 (REVIMID?)
CC-4047 (ACTIMID?)
11

• ORAL PHARMACOKINETICS
• (Teo et al., 2004)

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Noncompartmental Pharmacokinetic Parameters for 3
Thalidomide Treatments in Healthy Subjects
Parameter
Treatment A
Treatment B
Treatment C
1 X 50 mg
4 X 50 mg
8 X 50 mg
Cmax (?g/ml)
0.62 ? 0.32
1.76 ? 0.52
2.82 ? 0.80
Cmax (?g/ml) - normalized
1.00 ? 0.52
2.84 ? 0.96
4.55 ? 1.29
Tmax (hr)
2.9 ? 1.9
3.5 ? 2.0
4.3 ? 1.6
AUC0-t (?ghr/ml)
3.44 ? 0.66
15.6 ? 3.8
31.4 ? 9.7
AUC0-? (?ghr/ml)
4.90 ? 0.77
18.9 ? 3.3
36.4 ? 9.6
AUC0-? (?ghr/ml) normalized
1.00 ? 0.16
3.86 ? 0.67
7.43 ? 1.96
Percent AUC extrapolated
17
14
30 0.138 ? 0.040
? (1/hr)
0.132 ? 0.032
0.104 ? 0.030
Terminal t1/2 (hr)
5.5 ? 2.0
5.5 ? 1.4
7.3 ? 2.6
V/F (L)
81.1 ? 26.2
87.8 ? 20.2
121.9 ? 47.5
Cl/F (L/hr)
10.4 ? 1.8
10.9 ? 1.9
11.7 ? 2.8
   
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Dose Proportionality
10
A
1
Plasma concentration
800 mg
0.1
50 mg
200 mg
0.01
0
10
20
30
40
Time (hr)
10
6
B
C
max
(
m
C
5
3
max
g/ml)
AUC
0
0
0
100
200
300
400
500
Dose
(mg)
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Noncompartmental Pharmacokinetic Parameters Under
Fed and Fasted Conditions after Single Oral
Administration to 13 Healthy Volunteers

 
 
 
Fasted
Fed
Treatment A 4 X 50 mg capsules
Treatment B 4 X 50 mg capsules
Difference between means
Mean Ratio ()
 
 
 
 
1.99 ? 0.41
2.17 ? 0.51
8.54
109
4.62 ? 2.47
6.08 ? 2.33
31.4
131
23.00 ? 5.11
22.10 ? 4.29
-4.83
95.2
24.70 ? 5.13
23.50 ? 3.73
-5.47
94.5
0.126 ? 0.023
0.141 ? 0.025
 
 
5.80 ? 1.72
5.09 ? 1.03
 
 
262 ?103
259 ? 75
 
 
10.40 ? 2.34
10.90 ? 1.96
 
 
 
 
 
 
0.66 ? 0.21
0.75 ? 0.24
 
 
3.11 ? 0.23
3.07 ? 0.25
 
 
3.19 ? 0.22
3.15 ? 0.18
 
 
 
 
 
 
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Food effect
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SUMMARY OF IN VITRO EFFECTS OF THALIDOMIDE ON
HUMAN LEUKOCYTE RESPONSES
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• Cytokines are soluble glycoproteins released by
  cells of the immune system which act
  non-enzymatically through specific receptors to
  regulate immune responses. Some cytokines
• Interleukins
• Interferon
• Tumor necrosis factor
• Colony-stimulating factor

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Thalidomide and IMiDs have different effects on
different cytokines in monocytes and T cells
Monocytes TNF-?, IL-1?, IL-6, IL-12,
GM-CSF IL-10 T cells TNF-?, IL-2, IFN-?,
proliferation, CD40L, GM-CSF
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INHIBITION OF CYTOKINES (Moreira et al., 1993
Corral and Kaplan 1999a)
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Role of TNF-? in Immune Response

• TNF-? is a cytokine produced by monocytes,
  macrophages, lymphocytes and NK cells.
• Plays important role in host immune and
  inflammatory response to viral, parasitic, fungal
  and bacterial infections.
• TNF-? also responsible for pathogenesis of
  infectious and autoimmune diseases.
• Increased TNF-? can cause or aggravate septic
  shock, cancer cachexia, joint inflammation in
  rheumatoid arthritis, IBD, Crohns disease, ENL
  in leprosy.
• Reduction in TNF-? levels is an effective
  treatment for these conditions.
• Thalidomide has been shown to be effective in
  treating some of these diseases.
• Thalidomides anti-inflammatory action is due to
  its inhibition of TNF-? production by degrading
  the mRNA encoding its synthesis in monocytes.

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Effect of IMiD on LPS Activated Human PBMC
450.0
400.0
350.0
300.0
250.0
of Control
200.0
150.0
100.0
50.0
0.0
0.012
0.036
0.12
1.2
12
IMiD1 uM
TNF
IL-8
IL-1b
IL-6
IL-10
IL-12
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Dose Response Curves For TNF? Inhibitionin LPS
Activated Human PBMCs
100
90
80
IMiDs
70
60
Thalidomide
Inhibition of TNFa
50
40
30
20
10
0
0.001
0.01
0.1
1
10
100
1000
?M
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TNF? Inhibition in LPS-Stimulated Human PBMCs
100
90
80
CC-5013
70
60
Inhibition of TNFa
50
40
Thalidomide
30
20
10
0
1000 µM
0.0001
0.001
0.01
0.1
1.0
10.0
100.0
Thalidomide IC50 194 µM
CC-5013 IC50 90 nM
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Thalidomide and IMiDs Inhibit TNF-a Production in
Mouse LPS Shock Model
Inhibition Serum TNF-a
0.01
0.1
1
10
100
0.01
1
10
0.1
100
Dose (mg/kg i.p.)
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Effect of Thalidomide and CC-4047 on CD3 and
LPS-Induced TNF Levels
(T cell)
(monocyte)
CC-4047
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Effect of Thalidomide and CC-4047 on CD3 and
LPS-Induced IL-12 Levels
(T cell)
(monocyte)
CC-4047
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Differential Immunomodulation/Secretion of GM-CSF
by IMiDs
hPBMC
Monocytes and T cells
250
250
T cells (OKT3)
T cells (OKT3)
Monocytes (LPS)
Monocytes (LPS)
200
200
150
150
GM-CSF ()
GM-CSF ()
100
100
50
50
0
0
0
0.001
0.01
0.1
1
0
0.001
0.01
0.1
1
IMiD (?M)
IMiD (?M)
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Effect of Thalidomide and CC-4047 on CD3 and LPS
Induced IL-10 Levels
T cell
(T cell)
(monocyte)
CC-4047
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CO-STIMULATION OF PRIMARY HUMAN T CELL
PROLIFERATION (Corral et al., 1999b)
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Stimulation of T Cell Cytokines by Thalidomide
and CC-5013
IFNg
IL-2
Cytokine levels (pg/mL)
Concentration of compounds (?M)
Thalidomide
CC-5013
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Differential Co-stimulation T cells by IMiDs
Proliferationof T cell subsets (3H-thymidine
incorporation)
CD4/CD8 ratiosin unsorted T cells
350
3
T cells
IMiD 1
300
CD4
2.5
IMiD 2
250
CD8
2
200
CD4/CD8 ratio
CPM x 1000
1.5
150
1
100
0.5
50
0
0
0
0.01
0.1
1
10
0
0.001
0.01
0.1
1
IMiD 2 concentration (?M)
Drugs concentration (?M)
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MODIFICATION OF SURFACE ADHESION MOLECULES IN
LEUCOCYTES(Geitz et al., 1996)
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• An inflammatory reaction requires the recruitment
  of leucocytes
• and their transmigration into tissues. The
  endothelial cell surface
• antigens ICAM-1,VCAM-1, E- and L-selectin mediate
  the cell
• adhesion cascade leading to an inflammatory
  response.
• Thalidomide
• Up-regulates ICAM-1 (down-regulation seen with
  IMiD1 analogue) more recent data however showed
  that ICAM-1 is down-regulated
• Down-regulates VCAM-1, E- and L-selectin
• Inhibition of TNF-?-induced up-regulation of
  VCAM-1, E- and L-selectin could give rise to the
  anti-vasculitis effects of thalidomide.

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Stages of Leucocyte Binding to Vascular
Endothelium
Capture
Rolling
Tight Binding
Transmigration
Chemotaxis
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Adhesion Molecules Involved in Leucocyte Binding
to Vascular Endothelium
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Effect of Thalidomide on Adhesion Molecules
Expression in HUVEC Cells
300 250 200 150 100 50 0
160 140 120 100 80 60 40 20 0
VCAM-1
E-Selectin
MFI
MFI
Thalidomide (mg/m)
Thalidomide (mg/m)
100 80 60 40 20 0
L-Selectin
MFI
Thalidomide (mg/m)
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EFFECT OF THALIDOMIDE ON TH1 AND TH2
IMMUNITY(McHugh et al., 1995 Oliver et al.,
2000 Schafer et al., 2003 Verbon et al., 2000)
38
Th1 and Th2 Responses

• Protection against
• intracellular bacteria
• Organ-specific
• autoimmune diseases
• Crohns disease
• Allograft rejection
• Unexplained abortions
• H. pylori peptic ulcers

Macrophage activation
IL-2
IL-12
IgG2a
IFN-g
Th1
B
IgG3
TNF-b
Th0
IL-10
Mast cell growth

• Protection against
• metazoan parasites
• Allergy
• Atopic disorders
• Omenns syndrome
• Chronic Graft vs. Host
• disease
• Successful pregnancy
• Progression to AIDS
• in HIV infection

IL-4
IL-5
Th2
Eosinophil growth
IL-4
IgE
B
IgG1
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• Pro Th1
• A single 400 mg dose of thalidomide to healthy
  volunteers increased IFN-? and decreased IL-5
  production by PBMC stimulated ex vivo (Verbon et
  al.)
• In scleroderma patients, thalidomide (dose esc.
  50 to 400 mg/day) elevated plasma IL-12 and
  sIL-2R levels within 4 to 12 weeks (Oliver et
  al.).
• Pro Th2
• Thalidomide decreased IFN-? and increased IL-4
  production by PBMC in vitro (McHugh et al.).

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IMiDs Enhance Both Th1 and Th2 Differentiation
Th1
Th2
41
IMiDs Enhance Cytokine Production from
Differentiated Human Th1 and Th2 Cells
Th1
Th2
Thalidomide CC-4047 CC-5013
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CC-4047 Promotes Th1 and Suppresses Th2 Cytokines
from Naïve Mouse Splenocytes In Vitro
200
150
of anti-CD3 stimulated cultures
IL-4
IL-10
100
IFN-g
IL-2
GM-CSF
50
43
INDUCTION OF NATURAL KILLER CELL ACTIVITY(Davies
et al., 2001)
44
Natural Killer (NK) CellsNatural Killer (NK)
cells are a type of lethal lymphocyte. NK cells
serve as a rapidly acting first-line defense
mechanism Like cytotoxic T cells, they contain
granules filled with potent chemicals. They are
called "natural" killers because they, unlike
cytotoxic T cells, do not need to recognize a
specific antigen before swinging into action.
They target tumor cells and protect against a
wide variety of infectious microbes.
45
Effect of Thalidomide and Analogues on Human
Multiple Myeloma Cells
PBMCs from 2 MM patients were cultured with IL-2
for 72 hr and then with Thal/IMIDs for 72
hr. Lysis of MM cells were measured by a 51Cr
release assay.
46
Effects of Thalidomide on Paraprotein, NK cells,
IL-2 and IFN-? Levels in Relapsed MM Patients
47
EFFECT ON ANGIOGENIC FACTORS VEGF AND bFGF(Gupta
et al., 2001)
48
Effects of IMiDs on Angiogenic Factors in An In
Vitro Multiple Myeloma Model
BMSC bone marrow stromal cell (stimulate growth
factors and maintain bone marrow environment)
HSS HS-sultan (a human multiple myeloma cell
line)
49
ACTIVITIES OF THALIDOMIDE AND IMIDS IN MULTIPLE
MYELOMA(Richardson et al., 2002)
50

• Thalidomide and IMiDs have numerous activities
  and
• multiple sites of action towards multiple
  myeloma
• 1) decrease cytokine activity and production
• 2) enhance/induce host immune system
• 3) induce anti-angiogenesis through
  inhibition of
• growth factors
• 4) produce cell cycle arrest at G1 and
  apoptosis
• 5) decrease binding of multiple myeloma
  cells to
• bone marrow stromal cells
• This combination of actions has produced
  significant
• responses in relapsed multiple myeloma
  patients.
• Thalidomide and IMiDs therefore represent a class
  of
• novel compounds with potent immunomodulatory
• and anti-angiogenic activities.

51
Inhibition of Human Multiple Myeloma Cell
Proliferation Through 3H-Thymidine Incorporation
8
7
Thalidomide
(x104)
6
5
CPM
4
IMiDs
3
2
1
0
0
0.0001
0.001
0.01
0.1
1
10
100
THAL / IMiDs
(?M)
52
Demonstrated Mechanisms of Action of
Thalidomide/IMiDs in Multiple Myeloma
Thal/IMiDs
MM Cells ?
IL-6 ? TNF-a ?
Thal/IMiDs
Thal/IMiDs
Bone Marrow Stromal Cells
ICAM-1
?
Bone Marrow Vessels
IL-2 ? IFN? ?
Thal/IMiDs
VEGF ? bFGF ?
Thal/IMiDs
T Cells ?
MM Cells
NK Cells ?
Adapted from Richardson et al. 2002
53
SUMMARY
54

• Thalidomide and its more potent IMiDs analogues
  have
• significant immunomodulatory and
  anti-inflammatory activities.
• These include
• Inhibiting the cytokines TNF-?, IL-12, GM-CSF,
  IL-1?
• Co-stimulating T cells and increase IL-2 and
  IFN-? levels
• Modulating cell surface adhesion molecules
  (ICAM-1, VCAM-1, E- and L-Selectin)
• Upregulating Th1 (cytotoxic) immunity through
  production of IFN-? and IL-2
• Inducing NK cell activity
• These immunomodulatory activities along with
  others (anti-
• angiogenic, apoptotic, etc) play a major role in
  the responses
• seen in various inflammatory and oncologic
  diseases.

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FURTHER READING

• Corral et al., Selection of novel analogs of
  thalidomide with enhanced tumor necrosis factor
  alpha inhibitory activity. Molecular Medicine. 2,
  506-515, 1996.
• Corral and Kaplan. Immunomodulation by
  thalidomide and thalidomide analogues. Annals of
  Rheumatic Disease. 58, I107-I113, 1999a.
• Corral et al. Differential cytokine modulation
  and T cell activation by 2 distinct classes of
  thalidomide analogues that are potent inhibitors
  of TNF-?. Journal of Immunology. 163, 380-386,
  1999b.
• Davies et al., Thalidomide and immunomodulatory
  derivatives augment natural killer cell
  cytotoxicity in multiple myeloma. Blood, 98,
  210-216, 2001.
• Geitz et al., Thalidomide selectively modulates
  the density of cell surface molecules involved in
  the adhesion cascade. Immunopharmacology. 31,
  213-221, 1996.
• Gupta et al., Adherence of multiple myeloma cells
  to bone marrow stromal cells upregulates vascular
  endothelial growth factor secretion therapeutic
  applications. Leukemia, 15, 1950-1961, 2001.
• McHugh et al., The immunosuppressive drug
  thalidomide induces T helper cell type 2 (Th2)
  and concomitantly inhibits Th1 cytokine
  production in mitogen- and antigen-stimulated
  human peripheral blood mononuclear cell cultures.
  Clinical and Experimental Immunology. 99,
  160-167, 1995.
• Moreira et al., Thalidomide exerts its inhibitory
  action on TNF- by enhancing mRNA degradation.
  Journal of Experimental Medicine. 177, 1675-1680,
  1993.

56

• Oliver et al., Immune stimulation in scleroderma
  patients treated with thalidomide. Clinical
  Immunology. 97, 109-120, 2000.
• Richardson et al., Immunomodulatory drug CC-5013
  overcomes drug resistence and is well tolerated
  in patients with relapsed multiple myeloma.
  Blood. 100, 3063-3067, 2002.
• Schafer et al., Enhancement of cytokine
  production and AP-1 transcriptional activity in T
  cells by thalidomide related immunomodulatory
  drugs. Journal of Pharmacology and Experimental
  Therapeutics. In press 2003.
• Stephens and Brynner. Dark remedy. The impact of
  thalidomide and its revival as a vital medicine.
  Perseus Publishing, Cambridge, MA, 2001.
• Teo et al., Thalidomide in the treatment of
  leprosy. Microbes and Infection, 4, 1193-1202,
  2002.
• Teo et al., Clinical pharmacokinetics of
  thalidomide. Clinical Pharmacokinetics. In press,
  2004.
• Verbon et al., A single oral dose of thalidomide
  enhances the capacity of lymphocytes to secrete
  gamma interferon in healthy humans. Antimicrobial
  Agents and Chemotherapy. 44, 2286-2290, 2000.