Potent and selective CYP11B1 inhibitors could be promising therapeutics for the treatment of Cushing's syndrome. Optimization of Ref 1 (5-((1H-imidazol-1-yl)methyl)-2-phenylpyridine) led to compound 44 (5-((5-methylpyridin-3-yl)methyl)-2-phenylpyridine) with a 50-fold improved IC50 value of 2 nM toward human CYP11B1 and an enhanced inhibition of the rat enzyme (IC50 = 2440 nM) compared to Ref 1 (IC50 > 10000 nM). Furthermore, selectivities over CYP11B2, CYP17, and CYP19 were observed, as well as satisfying metabolic stability not only in human and rat plasma but also in liver S9 fraction. Investigation of cytotoxicity and inhibition of hepatic CYP2A6 and CYP3A4 showed that 44 fulfills first safety criteria and can be considered for further in vivo evaluation in rats.
Pathologically, high levels of aldosterone are associated with severe cardiovascular diseases such as congestive heart failure, hypertension, and myocardial fibrosis. The inhibition of aldosterone synthase (CYP11B2) to reduce aldosterone levels has been proposed as a promising treatment for diseases related to CYP11B2 because it is the crucial enzyme in the biosynthesis of aldosterone. A series of novel pyridyl- or isoquinolinyl-substituted indolines and indoles was designed via a ligand-based approach. The synthesized compounds were tested and found to be strong CYP11B2 inhibitors. The most potent ones showed IC50 values of less than 3 nM, being similarly potent as fadrozole and LCI699. Among them, compounds 14 and 23 showed good selectivity over the highly homologous CYP11B1, with selectivity factors (SF = IC50 CYP11B1/IC50 CYP11B2) around 170; thus, they are superior to fadrozole and LCI699 (SFs < 15). These potent CYP11B2 inhibitors exhibited no inhibition (IC50 > 50 μM) of a panel of hepatic CYP enzymes including CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 and the crucial steroidogenic enzymes, CYP17 and CYP19. Because of these advantageous profiles, compounds 14 and 23 are considered to be candidates for further in vivo evaluation.
Cushing's disease, characterized by elevated plasma cortisol levels, can be controlled by inhibition of 11β-hydroxylase (CYP11B1). The previously identified selective and potent CYP11B1 inhibitor 5-((5-methylpyridin-3-yl)methyl)-2-phenylpyridine Ref 7 (IC= 2 nM) exhibited promutagenic potential as well as very low oral bioavailability in rats (F = 2%) and was therefore modified to overcome these drawbacks. Successful lead optimization resulted in similarly potent and selective 5-((5-methoxypyridin-3-yl)methyl)-3-phenylisoxazole 25 (IC = 2 nM, 14-fold selectivity over CYP11B2), exhibiting a superior pharmacological profile with no mutagenic potential. Furthermore, compound 25 inhibited rat CYP11B1 (IC = 2 μM) and showed a high oral bioavailability (F = 50%) and sufficient plasma concentrations in rats, providing an excellent starting point for a proof-of-principle study.
Here we report the preclinical profile of REDX08608 our novel, potent and selective, reversible BTK inhibitor that is equipotent against wild-type and mutant C481S BTK. Bruton's tyrosine kinase (BTK) is a member of the src-related Tec family of cytoplasmic tyrosine kinases and plays a key role in the BCR signaling pathway, which is required for the development, activation and survival of B-cells. BTK is a clinically validated target to treat B-cell malignancies that are dependent on BCR signaling i.e.CLL and NHL with ibrutinib approved for the treatment of CLL, MCL and WM. Irreversible and covalent reversible BTK inhibitors such as ibrutinib, acalabrutinib and GS-4059 specifically target a cysteine residue C481 within BTK and mutations at this site clearly interfere with covalent drug binding. C481S, C481Y, C481R, C481F mutations have been reported and linked to cases of resistance that have emerged in patients with CLL progression following treatment with ibrutinib (Byrd2016, Inhye2016, Maddocks2015, Woaych2014). Redx reversible BTK inhibitor, REDX08608, aims to overcome this resistance mechanism by targeting both wild type and C481-mutated BTK. Redx have recently presented REDX06961 our BTK probe (Guisot2016, AACR#4795) and, following lead optimization, we are now disclosing REDX08608, our lead compound, a potent, reversible and selective BTK inhibitor, which displays an improved profile including superior pharmacokinetics. REDX08608, reversibly, inhibits WT and C481S BTK and displays nanomolar binding affinity and potency in biochemical and cellular-based assays. REDX08608 inhibits BTK signaling and growth in cell lines dependent on the BTK pathway such the OCI-LY10 ABC-DLBCL cell line. Importantly, REDX08608 also inhibits BTK signaling in primary CLL cells. In human whole blood and isolated human PBMCs, REDX08608 inhibits activation of B-cells at nanomolar concentrations measured by inhibition of immunoglobulin-induced CD69 in CD19+cells. REDX08608 is highly selective when tested against a panel of 468 kinases and exhibits improved target specificity with >100-fold selectivity against other Tec and Src kinase family members (ITK, TXK, BMX, TEC, BLK, CSK, FYN, HCK, LCK, SRC) and >400-fold selectivity against EGFR. REDX08608 was fully profiled through DMPK in vitro assays including metabolic stability, plasma stability, cytochrome P450 inhibition, PXR activation/cytochrome P450 activity, time dependent inhibition and cytochrome P450 reaction phenotyping. REDX08608 was shown to have an acceptable metabolic and plasma stability profile across species (mouse, rat, dog, monkey and human). REDX08608 displayed no evidence of PXR activation or time dependent inhibition. IC50s were determined for human cytochrome P450s (1A2, 2D6, 2C9, 2C19 and 3A4) and were all >10 µM. Good exposure, oral bioavailability and half-life were demonstrated for REDX08608 in mouse, rat and dog, with dose linearity assessment performed in mouse (F = 73-100%, CL= 11% liver blood flow in mice; F = 55-84%, CL = 28% liver blood flow in rat; F = 85%, CL= 10% liver blood flow in dog). In vivo efficacy studies in preclinical models will also be disclosed. In conclusion, REDX08608 is a potent and selective, reversible BTK inhibitor with efficacy in lymphoma cell lines that offers the potential to target both wild-type BTK and an important emerging resistance mechanism in patients with CLL progression following ibrutinib-treatment. Disclosures Guisot: Redx Oncology Ltd - Redx Pharma Plc: Employment. Best:Redx Oncology Ltd - Redx Pharma Plc: Employment. Wright:Redx Oncology Ltd - Redx Pharma Plc: Employment. Thomason:Redx Oncology Ltd - Redx Pharma Plc: Employment. Woyach:Acerta: Research Funding; Karyopharm: Research Funding; Morphosys: Research Funding. Abet:Redx Oncology Ltd - Redx Pharma Plc: Employment. Castagna:Redx Oncology Ltd - Redx Pharma Plc: Employment. Cousin:Redx Oncology Ltd - Redx Pharma Plc: Employment. Emmerich:Redx Oncology Ltd - Redx Pharma Plc: Employment. Ho:Redx Oncology Ltd - Redx Pharma Plc: Employment. Kelly:Redx Oncology Ltd - Redx Pharma Plc: Employment. King-Tours:Redx Oncology Ltd - Redx Pharma Plc: Employment. Lyons:Redx Oncology Ltd - Redx Pharma Plc: Employment. Muller:Redx Oncology Ltd - Redx Pharma Plc: Employment. Refuerzo:Redx Oncology Ltd - Redx Pharma Plc: Employment. Sargent:Redx Oncology Ltd - Redx Pharma Plc: Employment. Talab:Redx Oncology Ltd - Redx Pharma Plc: Employment. Bingham:Redx Oncology Ltd - Redx Pharma Plc: Employment. Phillips:Redx Oncology Ltd - Redx Pharma Plc: Employment. Armer:Redx Oncology Ltd - Redx Pharma Plc: Employment.
The B-cell receptor (BCR) signaling pathway is required for the survival, activation, proliferation and differentiation of B-cells. Bruton's Tyrosine Kinase (BTK) is a member of the Tec protein tyrosine kinase family that has emerged as an attractive target for the treatment of B-cell malignancies due to the critical role it plays in BCR signaling. Redx Oncology has developed novel differentiated small molecule inhibitors of BTK, combining current best-in-class potency with distinct selectivity profiles, which are suitable for oral once daily dosing. Here we present REDX05194, the result of a successful lead optimization of our proprietary BTK inhibitor series. REDX05194 is a highly selective, covalent BTK inhibitor displaying subnanomolar binding affinity for BTK (0.39 nM) and nanomolar potency towards BTK in a biochemical assay (3.67 nM). In cell proliferation assays, REDX05194 showed significant in vitro potency against ABC-DLBCL cell lines inhibiting the growth of both TMD-8 (0.89 nM) and OCI-Ly10 (1.36 nM) cells. Analysis of BCR signaling in several lymphoma cell lines, including cell lines of ABC-DLBCL, MCL and FL origin, revealed that treatment with REDX05194 inhibits BTK autophosphorylation and downstream activation of PLCγ2. In human PBMCs, REDX05194 inhibited anti-IgM stimulated upregulation of the CD69 activation marker in CD19 positive B-cells. In addition, using a fluorescent probe that binds to BTK, occupancy of BTK in PBMCs has been demonstrated in response to increasing concentrations of REDX05194. To assess selectivity, 456 kinases were screened at 1 μM, confirming that REDX05194 does not significantly inhibit other kinases involved in BCR signaling (e.g. Syk, Lyn). Furthermore, REDX05194 was shown to have high selectivity versus structurally related cysteine-containing kinases such as ITK in binding assays, and EGFR as demonstrated in both binding and cellular assays. REDX05194 also has a favorable in vitro safety profile and drug-like properties, displaying an improved CYP profile and solubility compared to competitor compounds. REDX05194 demonstrated in vivo efficacy in a mouse collagen-induced arthritis (CIA) model. At 10 mg/kg and 30 mg/kg QD, REDX05194 significantly improved all clinical readouts, including disease severity, compared to the vehicle group. Histological data showed that approximately 1/3 of the mice had no or minimal pannus infiltration and no bone resorption, or had bone resorption restricted to small areas. These findings demonstrate potential clinical efficacy and a dose response. In conclusion, REDX05194 is a highly selective and potent BTK inhibitor with proven efficacy in several lymphoma cell lines and human PBMCs and in vivo efficacy demonstrated in a mouse CIA model. Disclosures No relevant conflicts of interest to declare.
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