Steve Friedman

Discovery and Pharmacological Characterization of a Novel Rodent-Active CCR2 Antagonist, INCB3344

This report describes the characterization of INCB3344, a novel, potent and selective small molecule antagonist of the mouse CCR2 receptor. The lack of rodent cross-reactivity inherent in the small molecule CCR2 antagonists discovered to date has precluded pharmacological studies of antagonists of this receptor and its therapeutic relevance. In vitro, INCB3344 inhibits the binding of CCL2 to mouse monocytes with nanomolar potency (IC50 = 10 nM) and displays dose-dependent inhibition of CCL2-mediated functional responses such as ERK phosphorylation and chemotaxis with similar potency. Against a panel of G protein-coupled receptors that includes other CC chemokine receptors, INCB3344 is at least 100-fold selective for CCR2. INCB3344 possesses good oral bioavailability and systemic exposure in rodents that allows in vivo pharmacological studies. INCB3344 treatment results in a dose-dependent inhibition of macrophage influx in a mouse model of delayed-type hypersensitivity. The histopathological analysis of tissues from the delayed-type hypersensitivity model demonstrates that inhibition of CCR2 leads to a substantial reduction in tissue inflammation, suggesting that macrophages play an orchestrating role in immune-based inflammatory reactions. These results led to the investigation of INCB3344 in inflammatory disease models. We demonstrate that therapeutic dosing of INCB3344 significantly reduces disease in mice subjected to experimental autoimmune encephalomyelitis, a model of multiple sclerosis, as well as a rat model of inflammatory arthritis. In summary, we present the first report on the pharmacological characterization of a selective, potent and rodent-active small molecule CCR2 antagonist. These data support targeting this receptor for the treatment of chronic inflammatory diseases.

ADAM proteases, ErbB pathways and cancer

A disintegrin and metalloproteases (ADAMs) are zinc-dependent trans-membrane metalloproteases that shed the extracellular domains of membrane-bound growth factors, cytokines and receptors. Key functions of ADAMs have emerged in ErbB signalling pathways as being sheddases for multiple ErbB ligands. As the ErbB pathway is a validated target for anti-cancer drugs, the upstream activators of ErbB ligands, their sheddases, now enter the spotlight as new drug targets in the ErbB pathway. ADAMs are involved not only in tumour cell proliferation but also in angiogenesis and metastasis. Therefore, strategies targeting ADAMs might be an important complement to existing anti-ErbB approaches.

Clinical Benefit of INCB7839, a Potent and Selective Inhibitor of ADAM10 and ADAM17, in Combination with Trastuzumab in Metastatic HER2 Positive Breast Cancer Patients.

Background: In HER2 over-expressing breast cancer cells, the HER2 protein can be cleaved by a metalloproteinase, ADAM10. While the extracellular domain (ECD) of HER2 is released into the serum, the truncated HER2 receptor fragment, termed p95, remains in the tumor cell membrane as a constitutively active receptor tyrosine kinase. Previous studies have shown that the presence of p95 in tumor cells is associated with poor clinical outcomes in patients with metastatic HER2 positive breast cancer and it was recently shown that patients with p95+ HER2 positive breast cancer are resistant to trastuzumab-based therapy. Therefore, inhibition of HER2 cleavage by the ADAM10/ADAM17 inhibitor, INCB7839, which reduces the formation of soluble HER2 ECD as well as p95 levels in the tumor, may enhance the clinical efficacy of trastuzumab in HER2+ breast cancer patients. Materials and Methods: This study is a single arm modified dose escalation open label trial of INCB7839 + trastuzumab in women with HER2+ metastatic breast cancer, naive to chemotherapy. Three doses of INCB7839 were studied (100 mg, 200 mg, 300 mg BID) with 6 patients/cohort and an expansion arm at the 300mg BID dose. Herceptin was administered on a Q3 week schedule. Pharmacokinetics, plasma HER2 ECD levels and p95 expression in tumor tissue were assessed in addition to clinical response and safety. Results: 39 patients have been enrolled in the study and assessment of HER2 ECD levels, p95 status and best clinical response completed on 30 patients. INCB7839 administration results in a dose-dependent reduction in the elevated levels of circulating HER2 ECD with a mean of ∼80% inhibition achieved at the highest dose tested (300 mg BID). At the 300mg BID dose, the current overall response rate is 40% (6/15 evaluable patients) with a higher response rate (6/11 or 55%) observed in patients with average plasma concentrations of INCB7839 above the IC50 for reduction of ECD levels, 320nM. Importantly, INCB7839 increases the clinical response rate in p95+ patients, with equivalent responses observed in the p95+ and p95- patients treated with INCB7839 + trastuzumab. The combination has been generally safe and well tolerated. Discussion: Proteolytic cleavage of the HER2 receptor by ADAM proteases results in the formation of a cytoplasmic fragment (p95) that possesses constitutive kinase activity with the release of ECD. Importantly, elevated levels of ECD and/or p95 have been associated with poor prognosis and clinical data suggest that p95 affords resistance to trastuzumab. Biomarker and clinical data from this trial demonstrate that INCB7839 can markedly reduce HER2 cleavage in patients with HER2+ breast cancer, and suggests that INCB7839, by inhibiting the HER2 shedding process, can render a subpopulation of HER2+ patients (as defined by the presence of p95) that would be predicted to be trastuzumab resistant clinically responsive to trastuzumab therapy. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5056.

Identification and Characterization of INCB9471, an Allosteric Noncompetitive Small-Molecule Antagonist of C-C Chemokine Receptor 5 with Potent Inhibitory Activity against Monocyte Migration and HIV-1 Infection

C-C chemokine receptor 5 (CCR5) is a clinically proven target for inhibition of HIV-1 infection and a potential target for various inflammatory diseases. In this article, we describe 5-[(4-{(3S)-4-[(1R,2R)-2-ethoxy-5-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-3-methylpiperazin-1-yl}-4-methylpiperidin-1-yl)carbonyl]-4,6-dimethylpyrimidine dihydrochloride (INCB9471), a potent and specific inhibitor of human CCR5 that has been proven to be safe and efficacious in viral load reduction in phase I and II human clinical trails. INCB9471 was identified using a primary human monocyte-based radioligand competition binding assay. It potently inhibited macrophage inflammatory protein-1β-induced monocyte migration and infection of peripheral blood mononuclear cells by a panel of R5-HIV-1 strains. The results from binding and signaling studies using incremental amounts of INCB9471 demonstrated INCB9471 as a noncompetitive CCR5 inhibitor. The CCR5 residues that are essential for interaction with INCB9471 were identified by site-specific mutagenesis studies. INCB9471 rapidly associates with but slowly dissociates from CCR5. When INCB9471 was compared with three CCR5 antagonists that had been tested in clinical trials, the potency of INCB9471 in blocking CCR5 ligand binding was similar to those of 4,6-dimethyl-5-{[4-methyl-4-((3S)-3-methyl-4-{(1R0–2-(methyloxy)-1-[4-(trifluoromethyl) phenyl]ethyl}-1-piperazingyl)-1-piperidinyl]carbonyl}pyrimidine (SCH-D; vicriviroc), 4-{[4-({(3R)-1-butyl-3-[(R)-cyclohexyl(hydroxyl)methyl]-2, 5-dioxo-1,4,9-triazaspiro[5.5]undec-9-yl}methyl)phenyl]oxy}benzoic acid hydrochloride (873140; aplaviroc), and 4,4-difluoro-N-((1S)-3-{(3-endo)-3-[3-methyl-5-(1-methylethyl)-4H-1,2,4-triazol-4-yl]-8-azabicyclo[3.2.1]oct-8-yl}-1-phenylpropyl)cyclohexanecarboxamide (UK427857; maraviroc). Its inhibitory activity against CCR5-mediated Ca2+ mobilization was also similar to those of SCH-D and 873140. Further analysis suggested that INCB9471 and UK427857 may have different binding sites on CCR5. The significance of two CCR5 antagonists with different binding sites is discussed in the context of potentially overcoming drug-resistant HIV-1 strains.

Discovery of novel selective HER-2 sheddase inhibitors through optimization of P1 moiety.

A novel series of carbamates was discovered as potent and selective HER-2 sheddase inhibitors. Significant enhancement in potency and selectivity was achieved through attenuating the P1 moiety, which was conventionally believed to be exposed to solvent.

An Inhibitor of ADAMs (a Distintegrin and Metalloproteinase) Overcomes HER3-Mediated Resistance to Trastuzumab and Lapatinib.

Background: Inhibitors of HER-2/neu and EGF receptors such as trastuzumab, lapatinib, and erlotinib have demonstrated clinical efficacy but not all HER-2/neu or EGFR positive tumors respond and many that initially respond develop resistance. Ligand mediated HER-3 signaling results in tumor growth and survival and is a proposed mechanism of resistance to trastuzumab and lapatinib. Proteolytic cleavage of both ErbB ligands and receptors has been shown to be a critical event that results in ErbB pathway activation. Cleavage is necessary for the generation of soluble, functionally active forms of ErbB ligands and in the case of HER-2/neu, cleavage results in a shed extracellular domain (ECD) and a membrane bound fragment (p95) that contains a kinase domain with significant constitutive activity. In addition, it has been shown that the preferential association between HER3 and p95 can further activate the pathway. Both ErbB ligand and HER-2/neu cleavage are mediated by the ADAM family of proteases. Further, we have previously shown that the ADAM protease inhibitor, INCB7839, provides synergistic inhibition of HER2 + breast cancer cell line growth when combined with either trastuzumab or lapatinib. Materials and Methods: The HER-2 overexpressing BT-474 human breast cancer cell line was treated with either lapatinib or trastuzumab in the presence or absence of the HER-3 ligand, heregulin, and the effects on cell growth measured. The effects of the ADAM protease inhibitor INCB7839 in this system were also examined. Results: The addition of heregulin overcame the anti-proliferative effects of both lapatinib and trastuzumab on the growth of the BT-474 cell line in vitro. Addition of INCB7839 synergized with lapatinib or trastuzumab and importantly, restored the anti-proliferative effects of these agents in the presence of heregulin. Further, pretreatment of BT-474 cells with INCB7839 for 6 days, which we have previously shown completely eliminates the presence of the p95 form of HER2, amplified these effects, presumably by eliminating p95 before stimulating the cells with heregulin. Discussion: Together, these results confirm that heregulin can overcome the anti-proliferative effects of both trastuzumab and lapatinib as previously reported. Prevention of p95 formation by ADAM protease inhibitors appears to restore the anti-proliferative effects of both trastuzumab and lapatinib when heregulin/HER3 signaling occurs, negating a proposed mechanism of resistance to these agents in the clinic. These results suggest that combining an ADAM inhibitor with targeted inhibitors of the ErbB family can overcome HER-3-mediated resistance and enhance the clinical efficacy of approved HER2-targeted agents in the clinical setting. INCB7839 is currently under clinical investigation in combination with trastuzumab for women with metastatic breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3138.