Small molecule phagocytosis inhibitors for immune cytopenias

Small molecule phagocytosis inhibitors for immune cytopenias

AUTREV-01874; No of Pages 5 Autoimmunity Reviews xxx (2016) xxx–xxx Contents lists available at ScienceDirect Autoimmunity Reviews journal homepage:...

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AUTREV-01874; No of Pages 5 Autoimmunity Reviews xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Autoimmunity Reviews journal homepage: www.elsevier.com/locate/autrev

Review

Small molecule phagocytosis inhibitors for immune cytopenias Anton Neschadim a, Lakshmi P. Kotra b,c,d,e, Donald R. Branch a,f,g,h,⁎ a

Centre for Innovation, Canadian Blood Services, Toronto, ON, Canada Center for Molecular Design and Preformulations, University Health Network, Toronto, ON, Canada c Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada d McLaughlin Center for Molecular Medicine, University of Toronto, Toronto, ON, Canada e Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada f Department of Medicine, University of Toronto, Toronto, ON, Canada g Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada h Division of Advanced Diagnostics, Infection and Immunity Group, Toronto General Hospital Research Institute, Toronto, ON, Canada b

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Article history: Received 22 May 2016 Accepted 7 June 2016 Available online xxxx Keywords: Immune cytopenias Thrombocytopenia ITP Phagocytosis Inhibitor Small molecule drugs

a b s t r a c t Immune cytopenias are conditions characterized by low blood cell counts, such as platelets in immune thrombocytopenia (ITP) and red blood cells in autoimmune hemolytic anemia (AIHA). Chronic ITP affects approximately 4 in 100,000 adults annually while AIHA is much less common. Extravascular phagocytosis and massive destruction of autoantibody-opsonized blood cells by macrophages in the spleen and liver are the hallmark of these conditions. Current treatment modalities for ITP and AIHA include the first-line use of corticosteroids; whereas, IVIg shows efficacy in ITP but not AIHA. One main mechanism of action by which IVIg treatment leads to the reduction in platelet destruction rates in ITP is thought to involve Fcγ receptor (FcγR) blockade, ultimately leading to the inhibition of extravascular platelet phagocytosis. IVIg, which is manufactured from the human plasma of thousands of donors, is a limited resource, and alternative treatments, particularly those based on bioavailable small molecules, are needed. In this review, we overview the pathophysiology of ITP, the role of Fcγ receptors, and the mechanisms of action of IVIg in treating ITP, and outline the efforts and progress towards developing novel, first-in-class inhibitors of phagocytosis as synthetic, small molecule substitutes for IVIg in ITP and other conditions where the pathobiology of the disease involves phagocytosis. © 2016 Published by Elsevier B.V.

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Immune thrombocytopenia (ITP) . . . . . . . . . . . . . . Role of phagocytosis in ITP . . . . . . . . . . . . . . . . . Intravenous immunoglobulin and other ITP treatment modalities Development of first-in-class small molecule inhibitors of phagocytosis for use in ITP . . . . . . . . . . . . . . . . . Take-home messages . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Immune thrombocytopenia (ITP)

⁎ Corresponding author at: Canadian Blood Services, 67 College St., Toronto, ON M5G 2M1, Canada. Tel.: +1 4163134458; fax: +1 4169749757. E-mail address: [email protected] (D.R. Branch).

Immune thrombocytopenia (ITP) is an autoimmune cytopenia characterized by a low platelet count in the absence of bone marrow-related or other abnormalities [1]. Patients suffering from ITP have an increased tendency to bleed, which can affect the skin and, in more severe cases,

http://dx.doi.org/10.1016/j.autrev.2016.06.004 1568-9972/© 2016 Published by Elsevier B.V.

Please cite this article as: Neschadim A, et al, Small molecule phagocytosis inhibitors for immune cytopenias, Autoimmun Rev (2016), http:// dx.doi.org/10.1016/j.autrev.2016.06.004

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A. Neschadim et al. / Autoimmunity Reviews xxx (2016) xxx–xxx

can result in bleeding from various mucous membranes. Internal bleeding complication can prove severe and sometimes fatal [2]. Acute ITP is common in pediatric populations but usually resolves spontaneously in less than a few months. Chronic ITP, which primarily affects adults, can last for 6 months or longer. Overall incidence of chronic ITP in the population is estimated at approximately 4 in 100,000 persons annually [3]. Generation of autoantibodies against various platelet antigens is a hallmark of ITP [4,5]. The mechanisms leading to the breakdown of tolerance in ITP are not entirely clear, but involve B cells, T cells and antigen-presenting cells [6]. Platelet opsonization by anti-platelet antibodies facilitates their extravascular destruction via antibodymediated phagocytosis by the splenic macrophages in the spleen or by Kupffer macrophages in the liver [7,8]. Massive and ongoing loss of platelets underlies the resulting pathology. ITP autoantibodies against the collagen receptor (glycoproteins Ia/IIa or CD49b/CD29 or integrin α2β1) [9], von Willebrand factor and thrombin receptor (glycoproteins Ib/IX or CD42a–d) [10], and fibrinogen receptor (glycoproteins IIb/IIIa or CD41/CD61) [11] have been documented. In the vast majority of cases, platelet autoantibodies bind either the CD41 or CD42 complexes [10,11]. 2. Role of phagocytosis in ITP Although some platelet destruction in ITP has been suggested to involve complement, the main mechanism of platelet destruction is phagocytosis that is mediated by mononuclear phagocytes. Fcγ receptors (FcγRs) can be both activating (FcγRI, FcγRIIA, FcγRIII) and inhibitory (FcγRIIB). Signaling by Fcγ receptors is mediated by immunoreceptor tyrosine-based activating (ITAM) or inhibitory (ITIM) motifs. Engagement of activating Fcγ receptors leads to receptor aggregation, phosphorylation of the ITAMs in the cytoplasmic tail of the receptor or in an associated adaptor protein by the Srcfamily tyrosine kinases, Fyn, c-Src and c-Yes, which then activate the spleen tyrosine kinase (Syk). This activation of intracellular signaling cascades in macrophages triggers phagocytic function [12–14]. The ITIM motif of the inhibitory FcγR, FcγRIIB, is associated with the tyrosine phosphatases, SHP-1 and SHIP-1, which downregulate the ITAM phosphorylation resulting in inhibition of the phagocytic signal [15]. A variety of the activating and inhibitory receptors expressed on phagocytes in the spleen (and liver) could be involved in the platelet destruction in ITP [16]. These FcγRs recognize autoantibodies, primarily IgG1 subclass [17], that are coating platelets in the affected individuals, which results in their phagocytosis and intracellular degradation in phagolysosomes. While increased platelet production can compensate for platelet destruction in ITP, the massive rate of platelet loss eventually overcomes compensatory megakaryopoiesis, which can also be affected in ITP [18], and results in the severely low platelet counts that are the hallmark of this disorder. 3. Intravenous immunoglobulin and other ITP treatment modalities Treatment of ITP aims to promptly restore platelet counts in patients. Intravenous immunoglobulin (IVIg) and corticosteroids are standard first-line treatments for chronic ITP. IVIg is a purified immunoglobulin product, primarily consisting of IgG monomers, manufactured from pooled human plasma of thousands of donors [19]. IVIg was first used for the treatment of ITP in 1981 [20]. Clinic response rates to IVIg and corticosteroids are approximately 70% [21,22]. Thrombopoietin (TPO) mimetics and TPO receptor antagonists are used to maintain platelet levels in affected patients over the long term [23]. However, less than 15% of patients remain in remission. Laparascopic splenectomy can be used in these chronic ITP patients with curative outcomes, but a third of the patients still relapse [24]. Only some patients are able to discontinue TPO receptor antagonists and remain in remission [25].

A number of other treatment options are available for ITP, including general immunosuppression (cyclosporine), anti-Rh(D) antibodies, anti-CD40L, and FcγR-blocking monoclonal antibodies [26]. More treatments appear valuable but are still being evaluated, including rituximab (a B-cell-targeted anti-CD20 chimeric monoclonal antibody); however, despite good response rates, remission and infusion reactions associated with its use are still very common [27]. Despite new treatment modalities, the use of IVIg for ITP continues to be quite widespread. Based on one European retrospective analysis, IVIg is used in more than 50% of ITP cases, and in more than 12% first-line therapies [28]. Utilization of IVIg in ITP is projected to remain high and costs will remain significant. In Canada, the annual total cost of IVIg use per patient is estimated to exceed $100,000 [29], and the cost of IVIg use for ITP in the province of Ontario is projected at $5 million annually [30]. IVIg use in ITP in Canada only accounts for 8–17% of its use in all indications [30,31]. Finally, while IVIg use has a long clinical history and an excellent safety record, its use is not without side-effects, which could include, in mild cases, transient side-effects such as headache, nausea, fever, vomiting, cough, malaise, muscle, join and abdominal pain, flushing, urticarial lesions, and variations in heart rate and blood pressure, and in rare cases, leukopenia, neutropenia, and monocytopenia [32]. Severe side-effects following IVIg treatment are rare but have also been documented, and include aseptic meningitis, acute renal failure, stroke, exacerbation of pre-existing congestive heart failure, infections, life threatening hemolysis [33], deep venous thrombosis and pulmonary embolism [34], and anaphylactic shock [32]. The pursuit of alternatives to IVIg is thus of great interest. While a number of recombinant products are being evaluated as IVIg alternatives [35], the development of small molecule-based inhibitors stands to offer the greatest advantages with respect to both cost savings in manufacture and ease of administration. 4. Development of first-in-class small molecule inhibitors of phagocytosis for use in ITP Because a number of mechanisms for IVIg activity have been suggested to play a role in the vast number of indications where it is utilized, developing a small-molecule alternative for IVIg is a disease-specific endeavor and necessitates the clear understanding of the IVIg mechanistic axis that is being tackled. Different mechanisms have been suggested to explain the efficacy of IVIg in ITP, but a dominant mechanism involves FcγR blockade [36,37]. Infused IVIg inhibits Fcγ receptors on mononuclear phagocytes, thereby interfering with their ability to phagocytose autoantibody-opsonized platelets. Even if the mechanism of IVIg in ITP is not FcγR blockade, the known pathophysiology of ITP (as for all immune cytopenias) involves phagocytosis as a major mechanism driving the destruction of platelets and provides strong rationale for the treatment of ITP via inhibition of phagocytosis. Thus, development of a small molecule inhibitor of phagocytosis is a viable strategy to replace or augment the use IVIg in ITP; and, could be applied for the treatment of other immune cytopenias. While Fcγ receptors are promising targets in ITP, it is yet unclear which Fcγ receptor family should be optimally targeted to inhibit phagocytosis and treat human ITP. Some studies indicate that the shift in the balance of expression of various Fcγ receptors on patient monocytes towards inhibitory FcγR, as a result of treatment or infection, could have a significant effect on ITP outcomes [38,39]. Thus, both the inhibition of activating Fcγ receptors and activation of the inhibitory Fcγ receptors, or the underlying intracellular pathways, are attractive mechanistic targets. Fig. 1 outlines the potential modes of action of small molecule inhibitors of phagocytosis that could substitute for IVIg activity in ITP. Early evidence that sulfhydryl and disulfide chemical groups were important for phagocytosis came from phagocytosis inhibition studies

Please cite this article as: Neschadim A, et al, Small molecule phagocytosis inhibitors for immune cytopenias, Autoimmun Rev (2016), http:// dx.doi.org/10.1016/j.autrev.2016.06.004

A. Neschadim et al. / Autoimmunity Reviews xxx (2016) xxx–xxx

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Fig. 1. Mechanisms of IVIg activity in ITP via Fc blockade and potential modes of action of small molecule inhibitors of phagocytosis that could substitute for IVIg. (A) In ITP, spleen and/or liver macrophages recognize autoantibody-opsonized platelets, with the Fc portion of the autoantibody engaging and aggregating Fc receptors on the surface of the phagocyte, which triggers platelet phagocytosis and degradation. (B) Infused IVIg can saturate Fc receptors on macrophages, blocking their activation by autoantibody-coated platelets [12]. (C) Small molecule inhibitors of phagocytosis [46,47] could mimic IVIg-mediated Fc blockade by interacting with and inhibiting the activating Fc receptors on macrophages, or, alternatively, by inhibiting downstream signaling pathways driving Fc-mediated phagocytosis.

with sodium iodoacetate, a chemical capable of irreversibly reacting with free sulfhydryl groups [40,41]. Additional studies demonstrated the importance of sulfhydryl and disulfide groups in phagocytosis [42, 43]. Based on these insights, our laboratory has explored a number of compounds capable of interacting with sulfhydryl and disulfide groups on the cell surface of monocytic phagocytes in an in vitro phagocytosis assay, the monocyte monolayer assay (MMA) [44,45]. In the MMA, Rh+ red blood cells are opsonized with an anti-D antibody and are phagocytosed by adherent monocytes isolated from peripheral blood mononuclear cells (PBMCs). Isolated adherent monolayers of monocytes can be incubated with a test treatment prior to the addition of antibody-opsonized red blood cells. Treatment of the adherent monocytes with IVIg inhibits red blood cell phagocytosis. Various degrees of phagocytosis can be accurately quantified with an MMA to generate a phagocytic index. Several mercaptans tested showed inhibitory effects on the ability of human macrophages to phagocytose antibody-coated red blood cells, including 4-benzoyl-5-mercapto-3-methyl-1-phenyl pyrazole and a number of disulfide-containing compounds [44,45]. These studies also suggested, using benzoylmethyl methyl disulfide, that the presence of the disulfide in the compounds was necessary, as the sulfhydryl counterpart (benzoylmethyl mercaptan) was not active [45]. Most compounds tested did not significantly affect the viability of the monocytes. We have thus embarked on making and iteratively exploring the structure–activity relationships of several sulfhydryl- and disulfide-containing derivatives of the pyrazole-containing lead identified in earlier studies using the MMA [46]. Five leads were identified, which were only active as disulfide-bridged dimers and not in their monomeric form, with phagocytosis inhibitory activity in low-μM concentration range. The most promising lead, a disulfide-bridged derivative, 1,2bis(3-methyl-1-phenyl-1H-pyrazol-5-yl)disulfane, inhibited phagocytosis in vitro with an IC50 of 100 nM [46]. Our subsequent work with this

compound led us to explore derivatives with the disulfide bond removed, to minimize loss of the drug due to oxidation/reduction reactions with non-targeted proteins, identifying several leads with more favorable drug-like properties and phagocytosis inhibition activities of up to 58% at test concentrations of 5 μM [47]. Concomitant evaluation of cytotoxicity revealed that the majority of the active compounds did not increase the apoptotic index of treated monocytes or PBMCs. One of the leads, 3-methyl-1-((3-methyl-1-phenyl-1H-pyrazol-5-yl)methyl)-1Hpyrazol-5-ol, demonstrated an IC50 of 14 μM at inhibiting phagocytosis in vitro [47], and has undergone preliminary evaluations in our passive antibody-transfer mouse model of ITP in vivo [48], where it demonstrates some activity in restoring platelet counts and synergizes with IVIg (unpublished). The compounds we developed represent first-in-class small molecule inhibitors of phagocytosis, potentially capable of reversing ITP and embodying a first step towards developing a synthetic, small molecule replacement for IVIg. Because these molecules inhibit phagocytosis, they should have efficacy in all conditions where phagocytosis is involved in the pathophysiology of the disease. These conditions would include all immune cytopenias, including AIHA, hemolytic transfusion reactions (HTRs) both acute and delayed, and hemolytic disease of the fetus and newborn (HDFN). In addition, small molecule phagocytosis inhibitors may be useful in treatment of rheumatoid arthritis (RA) and AIHA, which we will be testing using mouse models of these diseases [49,50]. Current work is ongoing to improve upon the activity and solubility of the current leads and evaluate promising compounds in vivo in mouse models of ITP, RA and AIHA. Although we suspect that the mechanism of action of these various pyrazole derivatives involves the inhibition of Fcγ receptors on macrophages, the studies to understand the precise mechanism of action of these compounds and identify their molecular targets are currently ongoing.

Please cite this article as: Neschadim A, et al, Small molecule phagocytosis inhibitors for immune cytopenias, Autoimmun Rev (2016), http:// dx.doi.org/10.1016/j.autrev.2016.06.004

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Take-home messages • Extravascular phagocytosis drives blood cell destruction in immune cytopenias, and inhibition of phagocytosis is a viable therapeutic strategy to restore normal blood cell levels. • FcγR blockade and inhibition of platelet phagocytosis are the principal mechanisms of action mediating the efficacy of IVIg in ITP. • IVIg, manufactured from human plasma from thousands of donors, is a limited resource, and its replacement with small molecule alternatives will increase cost effectiveness and ease of delivery. • Several pyrazole-containing derivatives that inhibit phagocytosis by human monocytes have strong potential to become first-in-class small-molecule substitutes for IVIg in the treatment of ITP and other immune cytopenias.

Acknowledgments DRB and LPK acknowledge the financial support from the Canadian Blood Services/Canadian Institutes of Health Research Partnership (Grant #XT00104). LPK acknowledges the generous support of Canada Foundation for Innovation (John R. Evans Leaders Fund; Grant #32350). This research also had support from the Centre for Innovation of the Canadian Blood Services (Grant #XT00655), using general resources provided in part by Health Canada, a department of the federal government of Canada, to DRB. As a condition of funding, this report must contain the statement, “The views expressed herein do not necessarily represent the view of the federal government of Canada.”

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Please cite this article as: Neschadim A, et al, Small molecule phagocytosis inhibitors for immune cytopenias, Autoimmun Rev (2016), http:// dx.doi.org/10.1016/j.autrev.2016.06.004