Patients with myelofibrosis (MF) often develop anemia and frequently become dependent on red blood cell transfusions. Results from a phase 2 study for the treatment of MF with the Janus kinase 1/2 ...(JAK1/2) inhibitor momelotinib (MMB) demonstrated that MMB treatment ameliorated anemia, which was unexpected for a JAK1/2 inhibitor, because erythropoietin-mediated JAK2 signaling is essential for erythropoiesis. Using a rat model of anemia of chronic disease, we demonstrated that MMB treatment can normalize hemoglobin and red blood cell numbers. We found that this positive effect is driven by direct inhibition of the bone morphogenic protein receptor kinase activin A receptor, type I (ACVR1), and the subsequent reduction of hepatocyte hepcidin production. Of note, ruxolitinib, a JAK1/2 inhibitor approved for the treatment of MF, had no inhibitory activity on this pathway. Further, we demonstrated the effect of MMB is not mediated by direct inhibition of JAK2-mediated ferroportin (FPN1) degradation, because neither MMB treatment nor myeloid-specific deletion of JAK2 affected FPN1 expression. Our data support the hypothesis that the improvement of inflammatory anemia by MMB results from inhibition of ACVR1-mediated hepcidin expression in the liver, which leads to increased mobilization of sequestered iron from cellular stores and subsequent stimulation of erythropoiesis.
•MMB ameliorates anemia in a rodent anemia of chronic disease model by inhibiting activin receptor-like kinase-2 activity.•Hepcidin-dependent ferroportin degradation is independent of JAK2 phosphorylation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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SAR studies focused on improving the pharmacokinetic (PK) properties of the previously reported potent and selective Btk inhibitor CGI-1746 (1) resulted in the clinical candidate ...GDC-0834 (2), which retained the potency and selectivity of CGI-1746, but with much improved PK in preclinical animal models. Structure based design efforts drove this work as modifications to 1 were investigated at both the solvent exposed region as well as ‘H3 binding pocket’. However, in vitro metabolic evaluation of 2 revealed a non CYP-mediated metabolic process that was more prevalent in human than preclinical species (mouse, rat, dog, cyno), leading to a high-level of uncertainly in predicting human pharmacokinetics. Due to its promising potency, selectivity, and preclinical efficacy, a single dose IND was filed and 2 was taken in to a single dose phase I trial in healthy volunteers to quickly evaluate the human pharmacokinetics. In human, 2 was found to be highly labile at the exo-cyclic amide bond that links the tetrahydrobenzothiophene moiety to the central aniline ring, resulting in insufficient parent drug exposure. This information informed the back-up program and discovery of improved inhibitors.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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Patients with myelofibrosis (MF) often develop anemia and frequently become dependent on red blood cell transfusions. Elevated levels of hepcidin in MF patients suggest that deregulated iron ...homeostasis is a driver of anemia in MF (Pardanani et al., Am J Hematol, 2013). A phase 2 study in MF patients demonstrated that momelotinib (MMB) treatment resulted in improvement of anemia (Pardanani et al., Blood, 2013, ASH Suppl). We have demonstrated that in addition to inhibiting JAK1/2, MMB also inhibits the bone morphogenetic protein receptor kinase, activin A receptor type 1 (ACVR1), a key regulator of hepcidin production in hepatocytes (Asshoff et al., Blood, 2015, ASH Suppl). This work supports a model by which the improvement of anemia observed in MF patients with MMB treatment results from the inhibition of ACVR1-mediated hepcidin expression in the liver, increased mobilization of sequestered iron from cellular stores and subsequent stimulation of erythropoiesis.
Here we demonstrate that MMB treatment in anemic rats resulted in a reduction of hepcidin and amelioration of anemia; whereas ruxolitinib dosing affected neither hepcidin nor anemia. Short-term MMB oral treatment in anemic rats resulted in a dose responsive inhibition of serum hepcidin induction with complete suppression at 25 mg/kg QD, 82% inhibition at 10 mg/kg QD, and 55% inhibition at 5 mg/kg QD (Table 1). Interestingly, only the two higher doses show significant time above EC50 levels, whereas the 5 mg/kg dose (which most closely approximates MMB human clinical exposures) only shows significant time above EC20. This suggests that systemic drug concentrations alone may not accurately predict activity in the model as 5 mg/kg demonstrated a 55% reduction in serum hepcidin. In a rat quantitative whole body autoradiography tissue distribution study, MMB levels have been demonstrated to be 3-4 fold higher in the liver relative to blood (Figure 1). Thus, the significant inhibiton of hepcidin levels in animals dosed with 5 mg/kg MMB may be due to higher concentrations in the liver. MMB concentrations would be expected to be higher in the liver relative to blood in humans as well.
Our results provide preclinical evidence for a direct role of MMB in regulating iron homeostasis through the ACVR1/hepcidin axis. Evaluation of the activity of MMB on the ACVR1/hepcidin axis is being done in ongoing MMB clinical studies in patients with MF.
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Warr:Gilead Sciences: Employment, Equity Ownership. Zheng:Gilead Sciences: Employment, Equity Ownership. Sharma:Gilead Sciences: Employment, Equity Ownership. Maciejewski:Gilead Sciences: Employment, Equity Ownership. Theurl:Gilead Sciences: Research Funding. Whitney:Gilead Sciences: Employment, Equity Ownership.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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BTK inhibitor GDC-0834 (1) was found to be rapidly metabolized in human studies, resulting in a suspension of clinical trials. The primary route of metabolism was through cleavage of ...the acyclic amide bond connecting the terminal tetrahydrobenzothiophene with the central linker aryl ring. SAR studies were focused on reducing metabolic cleavage of this amide, and resulted in the identification of several central aryl linker substituents that conferred improved stability. The most promising substituted aryl linkers were then incorporated into an optimized pyridazinone scaffold, resulting in the identification of lead analog 23, possessing improved potency, metabolic stability and preclinical properties.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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Many patients with myelofibrosis (MF) develop anemia and are, or become, dependent on frequent red blood cell transfusions 1. Results from the phase 2 studies for the treatment of myelofibrosis ...(MF) with the Jak1/2 inhibitor momelotinib (MMB) demonstrated that MMB treatment provided an anemia benefit, in addition to providing a durable spleen response and improvement in constitutional symptoms in MF patients 2. MMB's anemia benefit was an unexpected outcome as erythropoietin (EPO)-mediated JAK2 signaling is essential for erythropoiesis 3, 4. Systemic iron homeostasis is controlled by the peptide hormone hepcidin produced by the liver. Hepcidin reduces iron export from duodenal enterocytes and splenic and hepatic macrophages by binding to and down-regulating the iron exporter ferroportin 5-7. In chronic disease, there is a significant increase in hepcidin levels which can result in severe anemia; this pathologic condition is termed anemia of chronic disease (ACD). Recently, MF patients have been shown to have elevated serum hepcidin levels and this increase is associated with inferior overall survival in these patients 8.
This study aimed to determine whether MMB's clinical anemia benefit is driven by direct activity of MMB on the hepcidin pathway. We assayed MMB inhibitory activity on the BMP-receptor kinase pathway (the central driver of hepcidin transcription in hepatocytes) and assessed the activity of MMB in a rodent model of anemia of chronic disease (ACD). We demonstrate that MMB inhibits BMP6-induced in vitro production of hepcidin in cultured hepatocytes (HepG2 cells) with an EC50 = 651 +/- 203 nM (n=3). This inhibitory activity is mediated by direct suppresion of the BMP-receptor kinase Alk2 as MMB inhibits Alk2 enzymatic activity with an IC50 = 8.4 +/- 1.5 nM (n=3). Ruxolitinib has no activity on either Alk2 or the BMP-receptor kinase pathway. To understand whether MMB could modulate hepcidin levels in vivo and ameliorate anemia in vivo we assessed the effect of MMB in a peptidoglycan-polysaccharide fragment (PG-APS)-induced rat ACD model. Treatment with clinically relevant exposure levels of MMB for 3 days resulted in a dose dependent reduction in both liver RNA and serum protein hepcidin levels and caused an increase in serum iron. Furthermore, long-term treatment with MMB for 21 days increased the numbers and percent of reticulocytes and mature red blood cells in the bone marrow and increased Hgb and hematocrit to normal levels in the blood.
Our data suggest that MMB's clinical anemia benefit results from inhibition of ALK2-mediated expression of hepcidin in the liver, which results in increased release of iron from sequestered cellular stores and enhanced erythropoiesis. A phase 2 translational study in anemic subjects with MPNs is scheduled to confirm this mechanism. The Alk2-mediated activity on iron metabolism through hepcidin could prove beneficial in a number of additional indications, and facilitate the combination of MMB with myelosuppressive agents.
1. Tefferi, A., et al., One thousand patients with primary myelofibrosis: the mayo clinic experience. Mayo Clin Proc, 2012. 87 (1): p. 25-33.
2. Pardanani, A., et al., Update On The Long-Term Efficacy and Safety Of Momelotinib, a JAK1 and JAK2 Inhibitor, For The Treatment Of Myelofibrosis. Vol. 122. 2013. 108-108.
3. Verstovsek, S., et al., Safety and efficacy of INCB018424, a JAK1 and JAK2 inhibitor, in myelofibrosis. N Engl J Med, 2010. 363 (12): p. 1117-27.
4. Verstovsek, S., et al., A double-blind, placebo-controlled trial of ruxolitinib for myelofibrosis. N Engl J Med, 2012. 366 (9): p. 799-807.
5. Nemeth, E., et al., Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science, 2004. 306 (5704): p. 2090-3.
6. Theurl, I., et al., Dysregulated monocyte iron homeostasis and erythropoietin formation in patients with anemia of chronic disease. Blood, 2006. 107 (10): p. 4142-8.
7. Theurl, I., et al., Autocrine formation of hepcidin induces iron retention in human monocytes. Blood, 2008. 111 (4): p. 2392-9.
8. Pardanani, A., et al., Associations and prognostic interactions between circulating levels of hepcidin, ferritin and inflammatory cytokines in primary myelofibrosis. Am J Hematol, 2013. 88 (4): p. 312-6.
Warr:Gilead Science: Employment. Maciejewski:Gilead Science: Employment. Fowles:Gilead Science: Employment. Whitney:Gilead Sciences: Employment. Theurl:Gilead Science: Research Funding.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In our continued effort to discover and develop best-in-class Bruton’s tyrosine kinase (Btk) inhibitors for the treatment of B-cell lymphomas, rheumatoid arthritis, and systemic lupus erythematosus, ...we devised a series of novel tricyclic compounds that improved upon the druglike properties of our previous chemical matter. Compounds exemplified by G-744 are highly potent, selective for Btk, metabolically stable, well tolerated, and efficacious in an animal model of arthritis.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant ...IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, UL, UM, UPUK
Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are known driver mutations in acute myeloid leukemia (AML) and other cancer types. Patient outcomes in AML have remained poor, especially for ...patients above 60 years of age who typically do not tolerate high dose chemotherapy and stem cell transplantation, leading to cure rates below 20%. The development of novel targeted therapies for defined AML subtypes is urgently desired. Inhibitors of mutants of the closely related IDH2 gene as well as IDH1 have recently been described and show promising pre-clinical and early phase clinical activity. However, the specific molecular and functional effects of IDH1 inhibitors in AML, including in primary patients' cells, have not been reported yet.
Here, we report the development of novel allosteric inhibitors of mutant IDH1 for differentiation therapy of acute myeloid leukemia. A high-throughput biochemical screen targeting an IDH1 heterodimer composed of R132H and WT IDH1 led to the identification of a tetrahydropyrazolopyridine series of inhibitors. Structural and biochemical analyses revealed that these novel compounds bind to an allosteric site that does not contact any of the mutant residues in the enzymes active site and inhibit enzymatic turnover. The enzyme complex locked in the catalytically inactive conformation inhibits the production of the oncometabolite 2-hydroxyglutarate (2-HG). In biochemical studies, we observed potent inhibition of several different clinically relevant R132 mutants in the presence or absence of the cofactor NADPH, accompanied by significant decrease in H3K9me2 levels.
Allosteric inhibitor treatment of primary AML patients' cells with different clinically relevant R132 mutants of IDH1 ex vivo uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block, increased cell death and induction of differentiation both at the level of leukemic blasts and immature stem-like cells. Allosteric inhibition of IDH1 also led to a decrease in blasts in an in vivo xenotransplantation model. At the molecular level, enhanced reduced representation bisulfite sequencing showed that treatment with allosteric IDH1 inhibitors led to a significant reversal of the DNA cytosine hypermethylation pattern induced by mutant IDH1, accompanied by gene expression changes of key sets of genes and pathways, including "Cell Cycle", "G1/S transition", "Cellular growth and proliferation", and "Cell death and survival".
Taken together, our findings provide novel insight into the cellular and molecular effects of inhibition of mutant IDH1 in primary AML patients' cells. Furthermore, our study provides proof-of-concept for the molecular and biological activity of novel allosteric inhibitors for targeting of different mutant forms of IDH1 in leukemia, and opens new avenues for future investigations with these and other allosteric inhibitors for targeting mutant IDH1 in leukemia and other cancers.
Gao:GlaxoSmithKline: Employment. Pietrak:GlaxoSmithKline: Employment. Rendina:GlaxoSmithKline: Employment. Rominger:GlaxoSmithKline: Employment. Quinn:GlaxoSmithKline: Employment. Smallwood:GlaxoSmithKline: Employment. Wiggall:GlaxoSmithKline: Employment. Reif:GlaxoSmithKline: Employment. Schmidt:GlaxoSmithKline: Employment. Qi:GlaxoSmithKline: Employment. Zhao:GlaxoSmithKline: Employment. Joberty:GlaxoSmithKline: Employment. Faelth-Savitski:GlaxoSmithKline: Employment. Bantscheff:GlaxoSmithKline: Employment. Drewes:GlaxoSmithKline: Employment. Duraiswami:GlaxoSmithKline: Employment. Brady:GlaxoSmithKline: Employment. Concha:GlaxoSmithKline: Employment. Adams:GlaxoSmithKline: Employment. Schwartz:GlaxoSmithKline: Employment. McCabe:GlaxoSmithKline: Employment.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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