Yvonne Lo

Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms

Constitutive JAK2 activation in hematopoietic cells by the JAK2V617F mutation recapitulates myeloproliferative neoplasm (MPN) phenotypes in mice, establishing JAK2 inhibition as a potential therapeutic strategy. Although most polycythemia vera patients carry the JAK2V617F mutation, half of those with essential thrombocythemia or primary myelofibrosis do not, suggesting alternative mechanisms for constitutive JAK-STAT signaling in MPNs. Most patients with primary myelofibrosis have elevated levels of JAK-dependent proinflammatory cytokines (eg, interleukin-6) consistent with our observation of JAK1 hyperactivation. Accordingly, we evaluated the effectiveness of selective JAK1/2 inhibition in experimental models relevant to MPNs and report on the effects of INCB018424, the first potent, selective, oral JAK1/JAK2 inhibitor to enter the clinic. INCB018424 inhibited interleukin-6 signaling (50% inhibitory concentration [IC(50)] = 281nM), and proliferation of JAK2V617F(+) Ba/F3 cells (IC(50) = 127nM). In primary cultures, INCB018424 preferentially suppressed erythroid progenitor colony formation from JAK2V617F(+) polycythemia vera patients (IC(50) = 67nM) versus healthy donors (IC(50) > 400nM). In a mouse model of JAK2V617F(+) MPN, oral INCB018424 markedly reduced splenomegaly and circulating levels of inflammatory cytokines, and preferentially eliminated neoplastic cells, resulting in significantly prolonged survival without myelosuppressive or immunosuppressive effects. Preliminary clinical results support these preclinical data and establish INCB018424 as a promising oral agent for the treatment of MPNs.

Selective Inhibition of ADAM Metalloproteases as a Novel Approach for Modulating ErbB Pathways in Cancer

Purpose: ErbB receptor signaling pathways are important regulators of cell fate, and their dysregulation, through (epi)genetic alterations, plays an etiologic role in multiple cancers. ErbB ligands are synthesized as membrane-bound precursors that are cleaved by members of the ADAM family of zinc-dependent metalloproteases. This processing, termed ectodomain shedding, is essential for the functional activation of ErbB ligands. Recent studies suggest that elevated levels of ErbB ligands may circumvent the effectiveness of ErbB-targeted therapeutics. Here, we describe the discovery and preclinical development of potent, selective inhibitors of ErbB ligand shedding. Experimental Design: A series of biochemical and cell-based assays were established to identify selective inhibitors of ErbB ligand shedding. The therapeutic potential of these compounds was assessed in multiple in vivo models of cancer and matrix metalloprotease–related toxicity. Results: INCB3619 was identified as a representative selective, potent, orally bioavailable small-molecule inhibitor of a subset of ADAM proteases that block shedding of ErbB ligands. Administration of INCB3619 to tumor-bearing mice reduced ErbB ligand shedding in vivo and inhibited ErbB pathway signaling (e.g., phosphorylation of Akt), tumor cell proliferation, and survival. Further, INCB3619 synergized with clinically relevant cancer therapeutics and showed no overt or compounding toxicities, including fibroplasia, the dose-limiting toxicity associated with broad-spectrum matrix metalloprotease inhibitors. Conclusions: Inhibition of ErbB ligand shedding offers a potentially novel and well-tolerated therapeutic strategy for the treatment of human cancers and is currently being evaluated in the clinic.

The Pharmacokinetics, Pharmacodynamics, and Safety of Orally Dosed INCB018424 Phosphate in Healthy Volunteers

INCB018424 phosphate, a potent inhibitor of JAK enzymes with selectivity for JAK1&2, is in development for the treatment of myelofibrosis (MF). The oral dose pharmacokinetics, pharmacodynamics, safety, and tolerability of INCB018424 were evaluated in healthy volunteers in 2 double‐blind, randomized, and placebo‐controlled studies. The first study evaluated single ascending doses of 5 to 200 mg INCB018424 and the effect of food, whereas the second study evaluated multiple ascending doses, including both once‐ and twice‐daily dosing for 10 days. As a Biopharmaceutical Classification System class I drug, INCB018424 exhibited good oral bioavailability and dose‐proportional systemic exposures. INCB018424 showed low oral dose clearance and a small volume of distribution, with an approximate 3‐hour plasma half‐life and insignificant accumulation following repeat dosing. A high‐fat meal reduced INCB018424 Cmax by 24% but had little effect on INCB018424 AUC. INCB018424 was cleared primarily by metabolism with negligible renal excretion. The pharmacodynamics of INCB018424, evaluated by the inhibition of phosphorylated STAT3 following cytokine stimulation in whole blood, showed good correlation with INCB018424 plasma concentrations. INCB018424 was generally safe and well tolerated, with 25 mg bid and 100 mg qd established as the maximum tolerated doses in healthy volunteers.

The Effect of CYP3A4 Inhibition or Induction on the Pharmacokinetics and Pharmacodynamics of Orally Administered Ruxolitinib (INCB018424 Phosphate) in Healthy Volunteers

Ruxolitinib, a selective Janus kinase (JAK) 1&2 inhibitor in development for the treatment of myeloproliferative neoplasms, is primarily metabolized by CYP3A4. The effects of inhibition or induction of CYP3A4 on single oral dose ruxolitinib pharmacokinetics (PK) and pharmacodynamics (PD) were evaluated in healthy volunteers. Coadministration of ketoconazole (a potent CYP3A4 inhibitor) and erythromycin (a moderate CYP3A4 inhibitor) increased total ruxolitinib plasma exposure (AUC0‐∞) by 91% and 27%, respectively, and ruxolitinib PD, as measured by the inhibition of interleukin (IL)–6‐stimulated STAT3 phosphorylation in whole blood, was generally consistent with the PK observed. Pretreatment with rifampin, a potent CYP3A4 inducer, decreased ruxolitinib AUC0‐∞ by 71% while resulting in only a 10% decrease in the overall PD activity. This apparent PK/PD discrepancy may be explained, in part, by an increase in the relative abundance of ruxolitinib active metabolites with the rifampin coadministration. The collective PK/PD data suggest that starting doses of ruxolitinib should be reduced by 50% if coadministered with a potent CYP3A4 inhibitor, whereas adjustments in ruxolitinib starting doses may not be needed when coadministered with inducers or mild/moderate inhibitors of CYP3A4. All study doses of ruxolitinib were generally safe and well tolerated when given alone and in combination with ketoconazole, erythromycin, or rifampin.

The pharmacokinetics, pharmacodynamics, and safety of baricitinib, an oral JAK 1/2 inhibitor, in healthy volunteers.

Baricitinib (also known as LY3009104 or INCB028050), a novel and potent small molecule inhibitor of Janus kinase family of enzymes (JAKs) with selectivity for JAK1 and JAK2, is currently in clinical development for the treatment of rheumatoid arthritis (RA) and other inflammatory disorders. Two double‐blind, randomized, and placebo‐controlled studies were conducted to evaluate single ascending doses of 1–20 mg and multiple ascending doses of 2–20 mg QD and 5 mg BID for 10 or 28 days in healthy volunteers. Following oral administration, baricitinib plasma concentration typically attains its peak value within 1.5 hours postdose and subsequently declines in a bi‐exponential fashion. Baricitinib demonstrates dose‐linear and time‐invariant pharmacokinetics, with low oral‐dose clearance (17 L/h) and minimal systemic accumulation observed following repeat dosing. The mean renal clearance of baricitinib was determined to be ∼12 L/h. [Correction added after publication 12 November 2014: in the preceding sentence, “2 L/h” was changed to “12 L/h.”] The effect of a high‐fat meal on baricitinib pharmacokinetics was insignificant. The pharmacodynamics of baricitinib, evaluated by the inhibition of STAT3 phosphorylation following cytokine stimulation in the whole blood ex vivo, was well correlated with baricitinib plasma concentrations. Baricitinib was generally safe and well tolerated, with no serious treatment‐related adverse events (AEs) reported from either of the studies. An expected rapidly reversible, dose‐related decline in absolute neutrophil count was seen with baricitinib.

Pharmacological characterization of INCB3344, a small molecule antagonist of human CCR2

The chemokine receptor 2 (CCR2) directs migration of monocytes and has been proposed to be a drug target for chronic inflammatory diseases. INCB3344 was first published as a small molecule nanomolar inhibitor of rodent CCR2. Here, we show that INCB3344 can also bind human CCR2 (hCCR2) with high affinity, having a dissociation constant (K(d)) of approximately 5nM. The binding of the compound to the receptor is rapid and reversible. INCB3344 potently inhibits hCCR2 binding of monocyte chemoattractant protein-1 (MCP-1) and MCP-1-induced signaling and function in hCCR2-expressing cells, including ERK phosphorylation and chemotaxis, and is competitive against MCP-1 in vitro. INCB3344 also blocks MCP-1 binding to monocytes in human whole blood, with potency consistent with in vitro studies. The whole blood binding assay described here can be used for monitoring pharmacodynamic activity of CCR2 antagonists in both preclinical models and in the clinic.

Downmodulation of key inflammatory cell markers with a topical Janus kinase 1/2 inhibitor.

INCB018424 is a novel, potent Janus kinase (JAK)1/JAK2 inhibitor that blocks signal transduction of multiple proinflammatory cytokines.

Pharmacokinetics and pharmacodynamics of orally administered ruxolitinib (INCB018424 phosphate) in renal and hepatic impairment patients

Hepatic and renal impairment studies were conducted with ruxolitinib, a JAK1&2 inhibitor that is cleared predominantly by metabolism. Both studies were open label, single‐dose studies. Ruxolitinib area under the curve (AUC) was increased by 87%, 28%, and 65%, respectively, in subjects with mild, moderate, and severe hepatic impairment compared to healthy subjects with no correlation between exposure of ruxolitinib and the degree of hepatic impairment. The pharmacodynamics (PD) data were consistent with ruxolitinib pharmacokinetics (PK). The renal impairment study showed a surprising finding. While there was no change in ruxolitinib PK with varying degrees of renal impairment, the PD showed increasing pharmacological activity with increased severity of renal impairment. Analysis of the metabolite exposures revealed that active metabolites contributed to the observed incremental increase in PD activity. The recovery of ruxolitinib in dialysate was negligible. The starting dose of ruxolitinib in subjects with any hepatic impairment or moderate or severe renal impairment should be decreased to 10 mg twice daily (BID) if their platelet counts are between 100 × 109/L and 150 × 109/L. Subjects on dialysis should initiate dosing with a single dose of 15 or 20 mg, based on platelet counts, with dosing only on the days of dialysis.

Abstract 4712: Discovery of INCB059872, a novel FAD-directed LSD1 inhibitor that is effective in preclinical models of human and murine AML

Acute myeloid leukemia (AML) is a disease characterized by the expansion of a hematopoietic stem cell like population caused in part by a block of myeloid differentiation. In AML, an altered epigenetic landscape, often arising from genetic lesions in epigenetic regulators, enforces an oncogenic expression profile and suppresses myeloid differentiation. Lysine specific demethylase 1 (LSD1) catalyzes the demethylation of lysine 4 and 9 of histone H3 through an FAD-dependent redox process. Aberrant LSD1 activity has been proposed to maintain oncogenic programs and prevent differentiation of multiple subtypes of AML. Here, we describe INCB059872, a potent, selective and orally bioavailable inhibitor of LSD1 that achieves inhibitory activity through the formation of covalent FAD-adducts. INCB059872 inhibited cellular proliferation and induced cellular differentiation as measured by induction of CD86 and CD11b myeloid differentiation markers in a panel of human AML cell lines and primary human AML cells ex vivo. In vivo, pharmacodynamic (PD) assays confirmed the sustained induction of CD86 in human AML xenograft models, consistent with the mechanism of FAD-directed inhibition of LSD1. Oral administration of INCB059872 significantly inhibited tumor growth of human AML xenograft models as a single agent at doses exhibiting significant PD effects in vivo. Efficacy was further evaluated in the murine retroviral MLL-AF9 disseminated leukemia model that recapitulates hallmarks of human AML. INCB059872 significantly prolonged the median survival of MLL-AF9 expressing leukemic mice compared with vehicle treated animals. Mechanistic studies demonstrated that INCB059872 induced cell differentiation of murine blast cells, reduced blast colonies, and normalized clinical hematological parameters to those of non-leukemic mice. Notably, in both human AML xenografts and the murine MLL-AF9 leukemic model, maximal efficacy could be achieved with both daily (QD) and alternative-day (QoD) dosing regimens of INCB059872, consistent with the prolonged PD effects observed. Collectively, these studies demonstrate the key role that LSD1 activity can play in preventing leukemic cell differentiation, and support the therapeutic potential of INCB059872 in the treatment of human AML. Citation Format: Sang Hyun Lee, Matthew Stubbs, Xuesong Mike Liu, Melody Diamond, Valerie Dostalik, Min Ye, Yvonne Lo, Margaret Favata, Gengjie Yang, Karen Gallagher, Lynn Leffet, Chunhong He, Liangxing Wu, Alexander Margulis, Maryanne Covington, Richard Wynn, Wenqing Yao, Gregory Hollis, Reid Huber, Bruce Ruggeri, Peggy Scherle. Discovery of INCB059872, a novel FAD-directed LSD1 inhibitor that is effective in preclinical models of human and murine AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4712.

Abstract 4702: Combination of BET inhibitor INCB054329 and LSD1 inhibitor INCB059872 is synergistic for the treatment of AML in vitro and in vivo

Acute myeloid leukemia (AML) is a disease characterized by the expansion of a hematopoietic stem cell like population caused in part by a block of myeloid differentiation. In AML an altered epigenetic landscape, often arising from genetic lesions in epigenetic regulators, enforces an oncogenic expression profile and suppresses myeloid differentiation. Indeed, a screen for essential genes in the murine MLL-AF9 retroviral model of leukemia identified the epigenetic regulators BRD4 and LSD1 as potential therapeutic targets. Furthermore, independent studies have demonstrated that inhibition of BRD4 or LSD1 by small molecules induced myeloid differentiation and suppressed leukemic stem cell phenotype in AML models. Recently it has been demonstrated that combinations of inhibitors that target distinct epigenetic regulators can exhibit synergistic effects on target gene transcription and cancer cell growth. Therefore we investigated the potential combinatorial effects of a novel FAD-directed LSD1 inhibitor, INCB059872, together with the BET inhibitor, INCB054329. The effects of single agent LSD1 and BET inhibitors or their combination were assessed using human AML models in vitro and in vivo. INCB054329 as monotherapy inhibited cell proliferation and induced apoptosis in human AML cell lines, while INCB059872 induced cellular differentiation in these cell lines as determined by the induction of myeloid differentiation markers, CD86 and CD11b. The combination of INCB054329 and INCB059872 enhanced myeloid differentiation and apoptosis in human AML cell lines compared with the single agents. Interestingly, c-myc expression was down-regulated to greater extent with the combination of both compounds compared to either agent alone. Enhanced anti-tumor efficacy with favorable tolerability was observed in human AML xenograft models when both agents were administered simultaneously or sequentially, with INCB059872 dosing regimens preceding INCB054329 administration demonstrating the greatest efficacy. These ongoing studies demonstrate that concurrent inhibition of two distinct families of epigenetic regulators, BET and LSD1, is active in preclinical AML models, and provide a rationale for the clinical evaluation of this novel, epigenetic doublet therapy in AML. Citation Format: Xuesong Liu, Matthew Stubbs, Min Ye, Roberts Collins, Margaret Favata, Gengjie Yang, Melody Diamond, Valerie Dostalik, Yvonne Lo, Chunhong He, Liangxing Wu, Andrew Combs, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri, Phillip Liu, Sang Hyun Lee. Combination of BET inhibitor INCB054329 and LSD1 inhibitor INCB059872 is synergistic for the treatment of AML in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4702.