Judithann M. Lee

Identification of a Potent, Selective Non-peptide CXCR2 Antagonist That Inhibits Interleukin-8-induced Neutrophil Migration

Interleukin-8 (IL-8) and closely related Glu-Leu-Arg (ELR) containing CXC chemokines, including growth-related oncogene (GRO)α, GROβ, GROγ, and epithelial cell-derived neutrophil-activating peptide-78 (ENA-78), are potent neutrophil chemotactic and activating peptides, which are proposed to be major mediators of inflammation. IL-8 activates neutrophils by binding to two distinct seven-transmembrane (7-TMR) G-protein coupled receptors CXCR1 (IL-8RA) and CXCR2 (IL-8RB), while GROα, GROβ, GROγ, and ENA-78 bind to and activate only CXCR2. A chemical lead, which selectively inhibited CXCR2 was discovered by high throughput screening and chemically optimized. SB 225002 (N-(2-hydroxy-4-nitrophenyl)-N′-(2-bromophenyl)urea) is the first reported potent and selective non-peptide inhibitor of a chemokine receptor. It is an antagonist of 125I-IL-8 binding to CXCR2 with an IC50 = 22 nm. SB 225002 showed >150-fold selectivity over CXCR1 and four other 7-TMRs tested. In vitro, SB 225002 potently inhibited human and rabbit neutrophil chemotaxis induced by both IL-8 and GROα. In vivo, SB 225002 selectively blocked IL-8-induced neutrophil margination in rabbits. The present findings suggest that CXCR2 is responsible for neutrophil chemotaxis and margination induced by IL-8. This selective antagonist will be a useful tool compound to define the role of CXCR2 in inflammatory diseases where neutrophils play a major role.

A Potent and Selective Nonpeptide Antagonist of CXCR2 Inhibits Acute and Chronic Models of Arthritis in the Rabbit

Much evidence implicates IL-8 as a major mediator of inflammation and joint destruction in rheumatoid arthritis. The effects of IL-8 and its related ligands are mediated via two receptors, CXCR1 and CXCR2. In the present study, we demonstrate that a potent and selective nonpeptide antagonist of human CXCR2 potently inhibits 125I-labeled human IL-8 binding to, and human IL-8-induced calcium mobilization mediated by, rabbit CXCR2 (IC50 = 40.5 and 7.7 nM, respectively), but not rabbit CXCR1 (IC50 = >1000 and 2200 nM, respectively). These data suggest that the rabbit is an appropriate species in which to examine the anti-inflammatory effects of a human CXCR2-selective antagonist. In two acute models of arthritis in the rabbit induced by knee joint injection of human IL-8 or LPS, and a chronic Ag (OVA)-induced arthritis model, administration of the antagonist at 25 mg/kg by mouth twice a day significantly reduced synovial fluid neutrophils, monocytes, and lymphocytes. In addition, in the more robust LPS- and OVA-induced arthritis models, which were characterized by increased levels of proinflammatory mediators in the synovial fluid, TNF-α, IL-8, PGE2, leukotriene B4, and leukotriene C4 levels were significantly reduced, as was erythrocyte sedimentation rate, possibly as a result of the observed decreases in serum TNF-α and IL-8 levels. In vitro, the antagonist potently inhibited human IL-8-induced chemotaxis of rabbit neutrophils (IC50 = 0.75 nM), suggesting that inhibition of leukocyte migration into the knee joint is a likely mechanism by which the CXCR2 antagonist modulates disease.

Identification of Potent, Selective Non-peptide CC Chemokine Receptor-3 Antagonist That Inhibits Eotaxin-, Eotaxin-2-, and Monocyte Chemotactic Protein-4-induced Eosinophil Migration

Eosinophils have been implicated in the pathogenesis of asthma and other allergic diseases. Several CC chemokines including eotaxin (CCL-11), eotaxin-2 (CCL-24), RANTES (CCL-5), and monocyte chemotactic protein-3 (MCP-3, CCL-7) and 4 (MCP-4, CCL-13) are potent eosinophil chemotactic and activating peptides acting through CC chemokine receptor-3 (CCR3). Thus, antagonism of CCR3 could have a therapeutic role in asthma and other eosinophil-mediated diseases. A high throughput, cellular functional screen was configured using RBL-2H3 cells stably expressing CCR3 (RBL-2H3-CCR3) to identify non-peptide receptor antagonists. A small molecule CCR3 antagonist was identified, SK&F 45523, and chemical optimization led to the generation of a number of highly potent, selective CCR3 antagonists including SB-297006 and SB-328437. These compounds were further characterized in vitro and demonstrated high affinity, competitive inhibition of125I-eotaxin and 125I-MCP-4 binding to human eosinophils. The compounds were potent inhibitors of eotaxin- and MCP-4-induced Ca2+ mobilization in RBL-2H3-CCR3 cells and eosinophils. Additionally, SB-328437 inhibited eosinophil chemotaxis induced by three ligands that activate CCR3 with similar potencies. Selectivity was affirmed using a panel of 10 seven-transmembrane receptors. This is the first description of a non-peptide CCR3 antagonist, which should be useful in further elucidating the pathophysiological role of CCR3 in allergic inflammatory diseases.

Discovery of potent and selective phenylalanine derived CCR3 receptor antagonists. Part 2.

Highly potent CCR3 antagonists have been developed from a previously reported series of phenylalanine ester-based leads. Solution-phase, parallel synthesis optimization was utilized to identify highly potent, functional CCR3 antagonists.

Discovery of potent and selective phenylalanine derived CCR3 antagonists. Part 1.

The discovery of a series of phenylalanine derived CCR3 antagonists is reported. Parallel, solution-phase library synthesis has been utilized to delineate the structure-activity relationship leading to the synthesis of highly potent, CCR3-selective antagonists.

The role of the anionic groups in the receptor binding of interleukin-8 antagonists

In an effort to determine the role of the acidic group in the receptor binding ofN-(2-hydroxy-4-nitrophenyl)-N′-(phenyl) urea, an interleukin-8B receptor antagonist, its binding and that of several analogs was measured as a function of pH. These titrations indicate that these ureas bind most strongly in their anionic form. Studies of antagonists, with different acidities, demonstrated that the greatest change in binding of each urea occurred around the pK a of the compound being examined. The studies suggest that the increase in binding of the antagonists at higher pH is a result of the increased negative charge on the compounds rather than the effects of pH on the receptor or radioligand.

Combination therapy with ofatumumab and bendamustine in xenograft model of chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia, accounting for c. 30% of all leukaemia diagnoses. The median age at diagnosis is 70 years. Therefore the treatment strategies must be tailored to the age-related reduction in tolerance to toxic chemotherapies (Lin, 2010). For decades, the initial front-line CLL therapy has been a monotherapy with cytotoxic agents. Novel CD20-targeting monoclonal antibody (mAb) therapies were recently approved for use in CLL in Europe and the USA (Hallek, 2009). Ofatumumab is a fully human CD20 mAb approved for treatment of patients with fludarabineand alemtuzumab-refractory CLL (Reagan & Castillo, 2010). Ofatumumab binds a unique membraneproximal epitope encompassing both the smalland large-loop of the CD20 molecule (Tsimberidou, 2010). Its mechanism of action includes complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) (de Haij et al, 2010). Bendamustine (4-[5-[Bis(2-chloroethyl)amino]-1methylbenzimidazol-2methyl]butanoic acid) is a chemotherapeutic that has been used in cancer management for almost 50 years in Europe, and was approved for CLL indication in USA in 2008. It is a bi-functional mechlorethamine derivative that kills cancer cells by inducing DNA breaks as an alkylating agent and by acting as a purine analog (Aivado et al, 2002). A possible synergy was indicated between bendamustine and the chimeric CD20 mAb rituximab in relapsed/ refractory CLL (Hallek, 2009). Here we used preclinical lymphoma and leukaemia models to explore the activity of ofatumumab monotherapy, and to evaluate the effect of ofatumumab in combination with bendamustine in CLL. CD20 expression on Ramos and BC-1 lymphoma cell lines, and JVM-3 CLL cell lines was evaluated by flow cytometry. Staining was performed using ofatumumab, rituximab, and a mouse anti-human CD20 mAb from BD Bioscience (San Jose, CA, USA). All three antibodies were directly labelled with a Zenon fluorescent tag. The expression was highest on Ramos (lymphoma cell line), followed by JVM-3 (CLL cell line). The BC-1 lymphoma cell line was CD20-negative. To study the in vivo effect of ofatumumab, Ramos, JVM-3 and BC-1 cells were subcutaneously injected into C.B-17 severe combined immunodeficiency (SCID) female mice, and treated with an ascending dose of Ofatumumab alone, or in combination with bendamustine in suboptimal doses. Tumour volumes were calculated using formula (1⁄2 · length) · width, and analysed using Prism GraphPad (GraphPad Software, Inc., La Jolla, CA, USA) and one-way analysis of variance (anova) with Bonferroni post-test. Therapy started when mean volumes reached 80–120 mm. Ofatumumab was administered intraperitoneally 2·/week, and bendamustine was injected in a single intravenous dose on day 15. Efficacy was determined by calculating % tumour growth inhibition (TGI = 100 · (1 ) [Vt/Vc]); Vt, Vc = mean V in treated or control group) and tumour growth delay (TGD = 100 · ([Tt ) T])/Tc); Tt, Tc = time to end point in treated or control group). Experiments presented are representative studies that confirmed reproducibility of data. All studies were conducted in accordance with the GlaxoSmithKline Institutional Animal Care and Use Committee and Policy on the Care, Welfare and Treatment of Laboratory Animals. Ofatumumab monotherapy completely arrested (2 mg/kg) or significantly reduced Ramos (1 and 0Æ2 mg/kg) tumour growth. Reduction of Ramos tumour volumes on day 28 was significantly different from controls in all ofatumumab groups (**P < 0Æ01 (2 and 1 mg/kg), and *P < 0Æ05 (0Æ2 mg/kg). Ofatumumab monotherapy in the JVM-3 leukaemia model resulted in a significant TGD at doses 2 mg/kg (*P < 0Æ05) and 4 mg/kg (**P < 0Æ01 and ***P < 0Æ001) (Fig 1). Reduction of tumour volumes on day 29 was significantly different in the ofatumumab groups as compared to the control group (*P < 0Æ05 (2 mg/kg) and **P < 0Æ01 (4 mg/kg). TGI and TGD showed dose-dependent improvement with ofatumumab therapy (Fig 1). Suboptimal doses of ofatumumab (2 mg/kg) and bendamustine (50 mg/kg) were used for combination studies in the JVM-3 s.c. xenograft model. Data

Abstract LB-317: Combination therapy with ofatumumab and bendamustine in xenograft model of chronic lymphocytic leukemia

Ofatumumab (OFA) is a fully human CD20 monoclonal antibody (mAb) recently approved for treatment of patients with fludarabine- and alemtuzumab-refractory chronic lymphocytic leukemia (CLL). OFA binds a unique membrane-proximal epitope encompassing both the small- and large-loop on the CD20 receptor. Its mechanism of action includes complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Combining OFA with cytotoxic chemotherapy may lead to elimination of additional tumor cells by alternate mechanisms such as apoptosis via alkylating agents. We conducted preclinical studies to determine: 1) OFA activity in B-cell lymphoma and leukemia murine xenograft models with differential CD20 expression; and 2) activity of combining OFA with bendamustine in a CLL model. CD20 expression on tumor cell lines was determined by flow cytometry, and lymphoma (Ramos, Daudi) and CLL (JVM-3) lines with differential CD20 expression were selected for in vivo animal model development. CD20-negative BC-1 lymphoma model served as a control. Tumor cells were implanted s.c. into immunodeficient 4-6 weeks old C.B-17 SCID female mice (Taconic), and tumor volumes were determined twice a week based on caliper measurements (tumor volume = width 2 × length/2). Mice were randomized and therapy was initiated when tumors reached mean volume (V) = 80 mm 3 at two weeks after tumor implantation. OFA was administered i.p. twice a week, and bendamustine was injected in a single i.v. dose on day 15 of the study. Efficacy of therapy was determined by evaluating % tumor growth inhibition (TGI=100x(1-(Vt/Vc), where Vt, Vc = mean V in the treated, or control group, respectively) and tumor growth delay (TGD=100x((Tt-Tc)/Tc) where Tt, Tv = time to end-point in the treated, or control group). CD20 expression ranged from highest to lowest on Ramos, JVM-3 and BC-1 (CD20 negative) cells. All cell lines were tumorigenic in C.B-17 SCID mice. OFA monotherapy was effective in the Ramos and JVM-3, but not in the control BC-1 s.c. xenograft model. OFA dose-response was performed and suboptimal doses of OFA (2 mg/kg i.p.) and bendamustine (50 mg/kg i.v.) were used for chemotherapy combination studies in the JVM-3 s.c. xenograft model. Combination therapy resulted in a synergistic anti-tumor activity with TGI = 96% and TGD = 42%, compared to bendamustin (TGI = 9%, TGD = 14%) or OFA alone (TGI = 16%, TGD = 14%) in the CLL model. In conclusion, single-agent therapy with OFA demonstrated significant and dose-dependent activity in preclinical animal xenograft models of human B-cell lymphoma and CLL. The combination of suboptimal OFA and bendamustine doses in the CLL model resulted in significant antitumor activity, compared exposure to either agent alone. These preclinical findings provide a rationale for clinical studies of the combination of OFA and bendamustine in patients with CLL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-317.