Anwar Murtaza

Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin

For complex diseases in which multiple mediators contribute to overall disease pathogenesis by distinct or redundant mechanisms, simultaneous blockade of multiple targets may yield better therapeutic efficacy than inhibition of a single target. However, developing two separate monoclonal antibodies for clinical use as combination therapy is impractical, owing to regulatory hurdles and cost. Multi-specific, antibody-based molecules have been investigated; however, their therapeutic use has been hampered by poor pharmacokinetics, stability and manufacturing feasibility. Here, we describe a generally applicable model of a dual-specific, tetravalent immunoglobulin G (IgG)-like molecule—termed dual-variable-domain immunoglobulin (DVD-Ig)—that can be engineered from any two monoclonal antibodies while preserving activities of the parental antibodies. This molecule can be efficiently produced from mammalian cells and exhibits good physicochemical and pharmacokinetic properties. Preclinical studies of a DVD-Ig protein in an animal disease model demonstrate its potential for therapeutic application in human diseases.

The Bcl-2 Family Antagonist ABT-737 Significantly Inhibits Multiple Animal Models of Autoimmunity

The Bcl-2 family of proteins plays a critical role in controlling immune responses by regulating the expansion and contraction of activated lymphocyte clones by apoptosis. ABT-737, which was originally developed for oncology, is a potent inhibitor of Bcl-2, Bcl-xL, and Bcl-w protein function. There is evidence that Bcl-2–associated dysregulation of lymphocyte apoptosis may contribute to the pathogenesis of autoimmunity and lead to the development of autoimmune diseases. In this study, we report that ABT-737 treatment resulted in potent inhibition of lymphocyte proliferation as measured by in vitro mitogenic or ex vivo Ag-specific stimulation. More importantly, ABT-737 significantly reduced disease severity in tissue-specific and systemic animal models of autoimmunity. Bcl-2 family antagonism by ABT-737 was efficacious in treating animal models of arthritis and lupus. Our results suggest that treatment with a Bcl-2 family antagonist represents a novel and potentially attractive therapeutic approach for the clinical treatment of autoimmunity.

Role of α7 nicotinic acetylcholine receptors in regulating tumor necrosis factor-α (TNF-α) as revealed by subtype selective agonists

Immunological responses to protect against excessive inflammation can be regulated by the central nervous system through the cholinergic anti-inflammatory pathway wherein acetylcholine released from vagus nerves can inhibit inflammatory cytokines. Although a role for the α7 nicotinic acetylcholine receptor (α7 nAChR) in mediating this pathway has been suggested, pharmacological modulation of the pathway by selective agonists remains to be further elucidated. In this study, the role of α7 nAChRs in the regulation of TNF-α release was investigated using high affinity and selective α7 nAChR agonists in mouse peritoneal macrophage and human whole blood in vitro, and in mouse serum in vivo. In mouse peritoneal macrophages, LPS-induced TNF-α release in vitro was inhibited by a selective α7 nAChR agonist, A-833834 (5-[6-(5-Methyl-hexahydro-pyrrolo[3,4-c]pyrrol-2-yl)-pyridazin-3-yl]-1H-indole), and that effect was attenuated by α7 nAChR antagonist methyllycaconitine. The inhibitory effect of A-833834 on LPS-induced TNF-α release was also observed in human whole blood in vitro. I.v. LPS-induced TNF-α release in mouse serum was attenuated following i.p. administration of A-833834. Similarly, i.v. LPS-induced TNF-α release in mouse serum was also attenuated following i.p. administration of A-585539, another α7 nAChR agonist with limited brain penetration, suggesting that these effects are mediated by peripheral α7 nAChRs. A-833834 was also efficacious in suppressing TNF-α release in mouse serum following oral administration in zymosan-induced peritonitis. These studies collectively demonstrate that selectively targeting α7 nAChRs could offer a novel therapeutic modality to treat acute and chronic inflammatory disease states.

Etanercept ameliorates inflammation and pain in a novel mono-arthritic multi-flare model of streptococcal cell wall induced arthritis

BackgroundThe impact of anti-TNF, corticosteroid and analgesic therapy on inflammation and pain was evaluated in a novel mono-arthritic multi-flare rat Streptococcal Cell Wall (SCW) model using Etanercept, Dexamethasone and Buprenorphine.MethodsMultiple flares of arthritis were induced with an intra-articular injection of SCW in the hind ankle on day 1, followed by intravenous challenges on days 21 and 42. Inflammation and pain were monitored in the hind paws. Cytokine profiling, cell phenotyping, bioluminescence imaging and histopathological evaluation were also performed.ResultsLocal injection of SCW caused a rapid onset of inflammation and pain in the injected ankle which resolved within 4 days (Flare 1). Intravenous injection 20 days after sensitization resulted in an increase in ankle diameter and pain, which partially resolved in 8 days (Flare 2). The subsequent intra-venous injection in the same animals 14 days after resulted in a more chronic disease with inflammation and pain persisting over a period of 10 days (Flare 3). In Flare 2, therapeutic administration of Dexamethasone inhibited paw swelling (95%; P<0.001) and pain (55%; P<0.05). Therapeutic administration of Buprenorphine inhibited pain (80%; P<0.001) without affecting paw swelling (0%). Prophylactic administration of Etanercept in Flare 2 inhibited paw swelling (≥60%; P<0.001) and pain by ≥30%. Expression of IL-1β, IL-6, MCP-1 and CINC was reduced by >50% (P<0.001). Treatment with Etanercept in Flare 3 inhibited paw swelling by 60% (P<0.001) and pain by 25%. Prior treatment with Etanercept in Flare 2 followed by re-administration in Flare 3 led to a complete loss in the efficacy of Etanercept. Systemic exposure of Etanercept corroborated with lack of efficacy. Dexamethasone inhibited inflammation and pain in both Flares 2 and 3 (P<0.001).ConclusionsWe established a novel multi-flare SCW arthritis model enabling drug intervention in different stages of disease. We show for the first time the evaluation of inflammation and pain simultaneously in this model. Etanercept and Dexamethasone inhibited inflammation, pain and proinflammatory cytokines in this model. Taken together, this model facilitates the assessment of anti-rheumatic agents targeting inflammation and pain in the multiple flare paradigm and offers a powerful tool for drug discovery.

Structure activity optimization of 6H-pyrrolo[2,3-e][1,2,4]triazolo[4,3-a]pyrazines as Jak1 kinase inhibitors

Previous work investigating tricyclic pyrrolopyrazines as kinase cores led to the discovery that 1-cyclohexyl-6H-pyrrolo[2,3-e][1,2,4]triazolo[4,3-a]pyrazine (12) had Jak inhibitory activity. Herein we describe our initial efforts to develop orally bioavailable analogs of 12 with improved selectivity of Jak1 over Jak2.

Abstract A23: Development of a novel preclinical model to delineate the role of tumor microenvironment on mechanism of action and efficacy of PD-1 checkpoint blockade

Background: The cellular environment in which the tumor exists, also known as tumor microenvironment, includes surrounding blood vessels, immune cells, fibroblasts, signaling molecules, and the extracellular matrix. This microenvironment can contribute to the growth and metastases of tumors, potentially influencing the efficacy of therapeutic agents. Thus, understanding the contribution of the microenvironment on tumorigenesis may enable us to investigate biology and kinetics between tumor and surrounding environment and improve target selection for standalone or combination therapies. Methods: To interrogate the role of the microenvironment we developed a tumor model in which the tumors can grow inside an air-pouch created in the dorsal part of the mouse. The air-pouch serves as the local microenvironment, which can be modulated by pro or anti-inflammatory stimuli to study the impact of local cells and cytokines on tumor growth as well as the efficacy of therapeutic agents. Results: Using luciferized mouse colon carcinoma (mc38-LUC2) syngeneic cells, we demonstrate that the kinetics of tumor growth within the air-pouch is similar to standard subcutaneously induced tumor models and was quantified via bioluminescence imaging. Additionally, further characterization of the tumor microenvironment was performed using MR and CT imaging, cytokine expression, and cell phenotyping. Furthermore, we also evaluated checkpoint inhibitor agent targeting the PD-1pathway to evaluate its effect on tumor growth and impact on cellular and cytokine microenvironment. Conclusions: Taken together, our novel model can facilitate innovative assessment of the mechanism of action of immunomodulatory agents across multiple oncological malignancies and evaluation of next generation therapeutics. Citation Format: Kalyan Chakravarthy, Raquel Sevilla, Michael Caniga, Weisheng Zhang, Anwar Murtaza, Lily Moy, Milenko Cicmil. Development of a novel preclinical model to delineate the role of tumor microenvironment on mechanism of action and efficacy of PD-1 checkpoint blockade. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A23.

AB0130 Novel Mono-Arthritic Multi-Flare Model of Streptococcal Cell Wall Induced Arthritis: Pharmacological Evaluation with Etanercept

Background To assess the efficacy of novel anti-rheumatic agents on inflammation and pain we developed a novel mono-arthritic multi-flare rat Streptococcal Cell Wall (SCW) model which captures the remitting and chronic phases of arthritis similar to Rheumatoid Arthritis (RA). We have evaluated the impact of anti-TNF and steroid therapy using Etanercept and Dexamethasone to further understand the translatability of this model to human disease. Objectives To assess the effects of Etanercept and Dexamethasone in multiple flares of a novel model of SCW induced arthritis in rats. Methods SCW arthritis was induced in 6-8 week old female Lewis rats with an intra-articular injection in the hind ankle joint on day 1 followed by two intravenous challenges on days 21 and 42 of SCW extract PG-PS 100p. The contralateral paw was used as a negative control. Local inflammation and pain were monitored through the course of the study in the hind paws by measuring paw swelling and withdrawal threshold respectively. In addition, cytokine profiling, cell phenotyping, bioluminescence imaging and histopathological evaluation were also performed in the local joint. Then we assessed the efficacy of Etanercept and Dexamethasone in this model. Results Local injection of SCW caused a rapid onset of inflammation and pain in the injected ankle joint which resolved in 4 days (Flare 1). Systemic intravenous injection 20 days after sensitization resulted in a profound increase in ankle diameter and pain, which resolved in 8 days (Flare 2). A subsequent systemic challenge in the same animals on day 42 resulted in a chronic disease phenotype with inflammation and pain that continued to persist over the period of 10 days (Flare 3). Prophylactic administration of Etanercept in Flare 2 significantly inhibited paw swelling by 60% (p<0.001) and partially inhibited pain by 30% respectively. Production of proinflammatory cytokines IL-1β, IL-6, MCP-1 and CINC was reduced by >70% (p<0.001). Histopathological analysis corroborated with our efficacy data. Furthermore, prophylactic treatment with Etanercept in Flare 3 inhibited paw swelling by 60% (p<0.001) and partially inhibited pain by 25%. Interestingly, prior treatment with Etanercept in Flare 2 followed by a wash out period of 14 days and re-administration in Flare 3 led to a complete loss in efficacy, which could be due to potential immunogenicity. Serum exposure levels of Etanercept corroborated well with the lack of efficacy. Positive control Dexamethasone significantly inhibited inflammation and pain in both Flares 2 and 3 (p<0.001). Conclusions This is the first report to demonstrate a novel multi-flare extended paradigm of the SCW model. This model exhibits certain aspects of the human disease such as reactivating remission and exacerbation flares enabling investigation of RA pathogenesis and drug intervention in different stages of disease progression. We also report that the model can be used to evaluate clinically relevant parameters of inflammation and pain simultaneously. Our data shows that Etanercept and Dexamethasone inhibit inflammation, pain and relevant cytokines in this model. Taken together our novel model can facilitate innovative assessment of anti-rheumatic agents in multiple flares and offers a powerful tool for drug discovery. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2161