Small Molecule Inhibitors in

Rheumatoid arthritis

Venkatesh.S, MD, DM fellow

Dept of Clinical Immunology SGPGIMS Lucknow

India drsvpai@yahoo.com

Introduction

• Rheumatoid arthritis – autoimmune synovitis

• 0.5-1.0% of the population

• Adaptive + Innate immune responses synovial hyperplasia/ pannus formation cartilage and bone destruction

• Search for newer treatment options – DMARDS, biologics, and now small molecule inhibitors

• Signal transduction – kinases in the nexus of pro-inflammatory pathways

Kinase activity and inhibition

Kinases enzymatically transfer a phosphate group from ATP to the target molecule

Inhibition of kinase function by small molecules occurs by one of two basic mechanisms

Kinases and their inhibition

• Tyrosine kinases – Syk, JAK

• MAP kinases – p38, JNK, ERK

• NF-kB kinase 2

Tyrosine kinases: the front runners

Tyrosine kinases involved in RA pathogenesis

Activation of Tyrosine kinases

Receptor tyrosine kinases [RTKs]

Increase in stability of transmembrane RTK dimers Autophosphorylation of intracellular domains, including a conformational change that allows ATP and substrate binding

Active kinase then catalyzes transfer of phosphate from ATP -OH ions of tyrosine residues on the receptor itself or on substrate proteins, creating binding sites or activating substrate proteins, respectively, to promote signal transduction

Activation of Tyrosine kinases

Non-Receptor tyrosine kinases [Non-RTKs] – Src-family kinase [Lck]

[Folded upon itself]

Activation loop

Interacts with target signaling molecules

Lipid moiety attaches here Plasma membrane localisation

Specific interaction with RTKs & NRTKs

Catalytic domain

C-terminal tail domain

[unfolding – revealing the Ty 416 for autophosphorylation]

Expression of select tyrosine kinases in human RA

Small molecule inhibitors of select Tyks

The Spleen tyrosine kinase (Syk)

• An intracellular cytoplasmic tyrosine kinase[non-RTK]

• Important mediator of immuno-receptor signaling in macrophages, neutrophils, mast cells, and B cells

• Present in the synovium of patients with RA

• TNF-α induced activation of Syk in FLS Cytokine and MMP

• In rodent models of collagen-induced arthritis, R788 (fostamatinib disodium), an oral prodrug R406, had potent anti-inflammatory activity

• A 12-week, ascending-dose, randomized, placebo-controlled trial (ClinicalTrials.gov number, NCT00326339) - 189 patients with active RA despite MTX, a significant reduction in arthritis activity and in serum levels of IL-6 and MMP-3

Arthritis Rheum 2008;58:3309-18

457 patients

R788

304

100mg bd

152

150mg od

152

Placebo

153

bd dose

77

od dose

76

# Multi-center, randomized, double-blind, placebo-controlled trial # at 64 sites in six countries (Bulgaria, Colombia, Mexico, Poland, Romania, and the United States) # between May 2008 and June 2009 # designed jointly by the sponsor (Rigel) and the principal investigators

Follow up - at weeks 1, 2, 4, 6, and 8 and monthly

Patients who completed the study or withdrew early were eligible to enter a long-term open-label study in which all patients received R788 according to the dosing regimen to which they had been assigned in the randomized study

Inclusion criteria Exclusion criteria

Active rheumatoid arthritis for at least 6 months, on a stable dose of MTX (between 7.5 and 25 mg per week) for a minimum of 3 months Active RA - TJC ≥6 + SJC ≥6 + at least ½[↑ESR or CRP]

# Active or untreated latent infection, including hepatitis B or C, # Cancer (except for basal-cell or squamous-cell carcinoma of the skin) within the previous 5 years # Uncontrolled hypertension # ALT >1.2x ULN # Hb < 10g/dL # Platelet count < 125,000/ cu.mm # S.Creat > ULN

Previous Rx with biologics - allowed if patient has had a washout period

Concurrent treatment with stable doses of sulfasalazine, chloroquine/ HCQS, NSAIDs, or oral corticosteroids (≤10 mg of prednisone per day or the equivalent) was permitted

Clinical responses over 6 months

67%

57%

35%

43%

19%

32%

10%

14%

28% 31%

21%

7%

3 deaths: septicemia (possibly pneumococcal), cerebral hemorrhage on heparin therapy for pulmonary emboli, and one a sudden death of unknown cause with past h/o MI

27 serious adverse events: including the three deaths, one case of B-cell lymphoma, one case of cervical ca, and one documented MI

Previous h/o non-responsiveness to biologics - 15% patients R788 100mg bd dose 43% achieved ACR 20 at 6 mon Placebo 14%

Conclusions of this R788 phase II study…

• R788 at dose of 100mg bd/ 150mg od superior to placebo

• Significant difference in rates of ACR 20, 50,70 and remission rates [DAS28<2.6]

• Earliest response at 1 week of initiation of Rx

• Patients with previous non-responsiveness to biologics also significantly benefited from R788 compared to placebo

• Adverse events – diarrhea[MC], infections, reversible neutropenia, elevated ALT, hypertension

• Whether R788, alone or in combination with methotrexate, will slow or prevent the development of erosions will be of great interest

The Janus kinase (Jaks)

• Intracellular/ Cytoplasmic Tyks Cytokine receptors

• Cytokine receptors with common γ-chain subunit Jak 1/ Jak 3

• Cytokine receptors for hematopoietic growth factors Jak 2

Jak 1 Jak 2 Jak 3

Site of expression Ubiquitously expressed Immune cells

Critical function Lymphopoeisis Hematopoesis Lymphocyte activation/ proliferation/ functioning

Kinase Inhibitors INCB18424/ INCB28050

CP690,550/ Tasocitinib

Adverse effects of inhibition expected

More serious side effects since ubiquitously expressed

Likely to have lesser side effects

Jak mutations – severe

immunodeficiency

• Study 28050-201, a randomized, double-blind, placebo-controlled, dose-ranging trial • 125 patients with active RA with an inadequate response to any disease modifying anti-rheumatic

drug (DMARD) therapy including biologics • duration of the study was six months with the primary endpoint assessed at three months • patients were randomly assigned to one of three doses (4, 7 or 10 mg QD) of INCB28050 or placebo • Primary Outcome Measures: Safety and tolerability; Efficacy as determined by percent of patients

achieving ACR20 improvement at three months

Objective: To determine the efficacy, safety, and tolerability of 3 different dosages of CP-690,550, a potent, orally active JAK inhibitor, in patients with active rheumatoid arthritis (RA) in whom methotrexate, etanercept, infliximab, or adalimumab caused an inadequate or toxic response Methods: n = 264 were randomized equally to receive placebo, 5 mg of CP-690,550, 15 mg of CP-690,550, or 30 mg of CP-690,550 twice daily for 6 weeks, and were followed up for an additional 6 weeks after treatment The primary efficacy end point was the American College of Rheumatology 20% improvement criteria (ACR20) response rate at 6 weeks

Results: By week 6, the ACR20 response rates were 70.5%, 81.2%, and 76.8% in the 5 mg, 15 mg, and 30mg twice daily groups, respectively, compared with 29.2% in the placebo group (P < 0.001)

Adverse effects - headache and nausea

The infection rate in both the 15mg twice daily group and the 30 mg twice daily group was 30.4% (versus 26.2% in the placebo group).

No opportunistic infections or deaths occurred

Increases in mean LDL-C, and HDL-C and mean serum creatinine level (0.04–0.06 mg/dl) were seen in all CP-690,550 treatment arms

Tasocitinib (CP-690,550), an Orally Available Selective Janus Kinase Inhibitor, Exhibits Sustained Safety and Efficacy in the Treatment of Rheumatoid

Arthritis over 24 Months.

Carol A Connell, Richard Riese, Susan Wood, John Bradley, Samuel H Zwillich. 1Pfizer Inc

• Tasocitinib (CP-690,550) is an oral, small molecule Janus kinase inhibitor

• Phase 2/3, open label study of 1070 patients

• Either 5 or 10 mg tasocitinib twice daily

• Primary endpoints were laboratory safety and adverse event (AE) reports

• Secondary endpoints included ACR20, ACR50 and ACR70 response rates, Disease Activity Score using the erythrocyte sedimentation rate (DAS28-4 [ESR]), and the Health Assessment Questionnaire-Disability Index (HAQ-DI)

• 1070 patients

• Total duration - 1295.7 patient-years

• Median days of treatment 518.5 (ALL, n=1070), 655.0 (M12, n=648), and 796.0 (M24, n=207)

• Adverse effect related discontinuance - 70 patients

• Total of 929 treatment-related AEs (TRAEs) were reported; the most frequently reported classes of events were infections and infestations (197, 18.4%), gastrointestinal disorders (109, 10.2%),

• 188 serious AEs (SAEs) were reported, of which 48 (25.5%) were considered possibly related to treatment; the most frequent SAE was infection (34, 18.1%, 2.62/100 patient-years of tasocitinib treatment)

• Mean DAS28-4(ESR) at baseline was 6.41 (ALL); at month 12, 3.70 (ALL) and at month 24, 3.55 (ALL)

• Tasocitinib is effective in the long-term treatment of RA with a manageable safety profile.

• Efficacy is demonstrated by sustained improvement in ACR response rates, DAS28-4(ESR), and HAQ-DI over a 24-month period.

Other Tyk inhibitors studied in preclinical/animal models

• Imatinib – attenuation of CIA due to suppression of c-Fms activation in synovial macropahges, of PDGFR activation on FLS, and c-Kit activation in mast cells

• C-Fms and its ligand M-CSF – integral to MQ and osteoclast formation

• PDGFR – fibroblast proliferation FLS pannus formation

• C-Kit – mast cell activation [Masitinib – C-kit inhibitor]

• BTK transduces BCR signaling in B cells, FceR1 signaling in mast cells, TLR signaling in monocytes

• VEGFR inhibitors – cardiotoxicity and hypertension

Mitogen-Activated Protein kinases(MAPK)

• 3 interrelated parallel interactive signaling pathways – each mediated by a distinct terminal MAPK family

– p38 MAPK(α,β,γ and δ)

– Extracellular signal regulated kinase(ERK1 and ERK2))

– c-Jun-N-terminal kinase(JNK1, JNK2 and JNK3)

• MAPK pathways – sequential activation of multiple kinases, with the terminal kinases being p38, ERKs, JNKs

• All three are activated in the RA synovium and proposed as therapeutic targets in Rx of RA

The MAPK signaling cascade

p38 (MAPK)

• Inflammation and cytokine production – TNFα, IL-1, IL-6

(A) Endothelial cell function / Angiogenesis

(B) Synovial inflammation

(C) Bone and Cartilage Degeneration

Functions of p38MAPK activation in RA

Enthusiasm for p38 Inhibitors - subsided!!

• Many tested in preclinical studies – did not extend to the Rx of RA • First generation molecules – pan-p38 inhibitors Clinical trials – failed due to liver, brain & skin toxicity

• Identification of p38α as the important isoform in RA – selective p38

inhibitors [SCIO-323 – skin toxicity and AMG-548 – liver toxicity; some which entered the phase II trials proved ineffective]

• Recently – p38α has some anti-inflammatory role also • Components downstream of p38α may yet constitute viable

therapeutic targets MK2[post-transcriptionally promotes expression of pro- inflammatory genes] MK2 deficient mice – protected against CIA

c-Jun-N-terminal kinases(JNKs)

• Matrix regulation, apoptosis, and inflammation

• 3 isoforms – JNK1, JNK2 and JNK3

JNK1 JNK2 JNK3

Site of expression Ubiquitously expressed Brain, heart, testes

RA synovium Phospho-JNK1/JNK2 detected in RA synovium but not in OA

Not thought to be involved in RA

Role in RA Expression of MMP-3 and IL-6 JNK1 regulates diffn of T cells Th1

Inhibitors Pan-JNK inhibitors - SP600125 and AS601245 Long tem suppression of JNK Tumorogenicity JNK1 def mice – intestinal tumors

Poor specificity for JNK – unfavorable pharmacokinetic profile

The Master Regulator

The NF-ĸB pathway

• NF-κB regulates transcription of many genes that encode cytokines, chemokines and cell adhesion molecules

• The NF-κB family of proteins, including RelA (p65), RelB, c-Rel, p50/p105 and p52/p100, normally bind to inhibitor of NF-κB (IκB), which retains NF-κB in the cytoplasm

Role of NF-ĸB in pathogenesis of RA IKK inhibition should work wonders!

NF-ĸB is regulated by IKK complex

The IKK Complex - 2 catalytic components - A regulatory comp: [NEMO]

/ NEMO

/ Dominant / Dominant

P100/ NF-ĸB2

IKKα def-MQs (+) thru TLRs prolonged expression of pro-inflam: cytokines by NFĸB pathway

Selective inhibition of IKKβ – potential target in RA

IKK inhibitors tested in preclinical models

Safety concerns of NF-ĸB inhibition Possible mechanism based adverse effects:

Embryonic development • IKKβ-def mice & mice deficient in other components of the NF-kB

pathway, including p65-/- mice extensive liver apoptosis • Heterozygotes – develop normally [though NF-kB activation is

inhibited by 70–80% in these animals] • IKKα knockouts are also embryonic lethal - skin and limb

abnormalities - massive epidermal hyperproliferation defect in keratinocyte differentiation

Hepatotoxicity - would not lead to mechanism-based hepatoxicity in a developed organ and, in fact, could be hepatoprotective

Lymphopoiesis Infection

Summary of clinical trials

Summary

• Success of small molecule kinase inhibitors in cancer Rx identification of kinase targets in RA

• Many kinases – convincingly implicated and their inhibitors proved efficacious in preclinical models

• Few have made it into clinical development – narrow therapeutic index

• All clinical trials – pharma sponsored

• Promising molecules – Syk inhibitors, JAK inhibitors