Discovery of MDV6058 (PF-06952229), a selective and potent TGFßR1 inhibitor: Design, synthesis and optimization.

Compound 1 is a potent TGF-ß receptor type-1 (TGFßR1 or ALK5) inhibitor but is metabolically unstable. A solvent-exposed part of this molecule was used to analogue and modulate cell activity, liver microsome stability and mouse pharmacokinetics. The evolution of SAR that led to the selection of 2 (MDV6058 / PF-06952229) as a preclinical lead compound is described.

Comparative Analyses of Poly(ADP-Ribose) Polymerase Inhibitors.

Poly(ADP-ribose) polymerase inhibitors (PARPi) are approved as monotherapies in BRCA1/2-mutated (mBRCA1/2) metastatic breast and ovarian cancers, and in advanced pancreatic and metastatic castration-resistant prostate cancers. Differential safety profiles across PARPi necessitate improved mechanistic understanding of inhibitor differences, especially with expansion of PARPi indications and drug combinations. Here, we report in vitro evaluations of PARPi (-/+ PARP trapper temozolomide, TMZ) with reference to total clinical mean concentration average or maximum (tCavg, tCmax), to elucidate contributions of primary pharmacology and structural differences to clinical efficacy and safety. In biochemical assays, rucaparib and niraparib demonstrated off-target secondary pharmacology activities, and in selectivity assays, talazoparib, olaparib, and rucaparib inhibited a broader panel of PARP enzymes. In donor-derived human bone marrow mononuclear cells, only olaparib both increased early apoptosis and decreased the cell viability half inhibitory concentration (IC50) at ≤ tCavg, whereas other PARPi only did so in the presence of TMZ. In cancer cell lines with DNA damage repair mutations, all PARPi decreased cell viability in H1048 but not TK6 cells, and only talazoparib decreased cell growth in DU145 cells at ≤ tCavg concentrations. When combined with low dose TMZ, only talazoparib left-shifted the functional consequences of PARP trapping (S-phase arrest, apoptosis, S-phase double-stranded breaks) and reduced cell viability/growth in TK6 and DU145 cell lines at ≤ tCavg, whereas the other inhibitors required high-dose TMZ. Our study suggests structural differences across PARPi may contribute to differences in PARP selectivity and off-target activities, which along with distinct pharmacokinetic properties, may influence inhibitor-specific toxicities in patients.

Enzalutamide and Apalutamide: In Vitro Chemical Reactivity Studies and Activity in a Mouse Drug Allergy Model.

Enzalutamide and apalutamide are two androgen receptor inhibitors approved for the treatment of castration-resistant prostate cancer (CRPC) and nonmetastatic castration-resistant prostate cancer (nmCRPC), respectively. Apalutamide is associated with an increased incidence of skin rash above the placebo groups in the SPARTAN trial in nmCRPC and in the TITAN trial in metastatic castration-sensitive prostate cancer patients. On the contrary, the rate of skin rash across all clinical trials (including PROSPER [nmCRPC]) for enzalutamide is similar to the placebo. We hypothesized that the apalutamide-associated increased skin rash in patients could be linked to a structural difference. The 2-cyanophenyl and dimethyl moieties in enzalutamide are substituted in apalutamide with 2-cyanopyridine and cyclobutyl, respectively. In our evaluations, the 2-cyanopyridine moiety of apalutamide was chemically reactive with the thiol nucleophile glutathione, resulting in rearranged thiazoline products. Radiolabeled apalutamide, but not radiolabeled enzalutamide, was shown to react with mouse and human plasma proteins. Thiol nucleophiles decreased the extent of covalent binding to the model protein bovine serum albumin, whereas amine and alcohol nucleophiles had no effect, suggesting reactivity with cysteine of proteins. Subcutaneous administration of apalutamide dose dependently increased lymphocyte cellularity in draining lymph nodes in a mouse drug allergy model (MDAM). Enzalutamide, and its known analogue RD162 in which the cyanophenyl was retained but the dimethyl was replaced by cyclobutyl, demonstrated substantially less covalent binding activity and negative results in the MDAM assay. Collectively, these data support the hypothesis that the 2-cyanopyridine moiety in apalutamide may react with cysteine in proteins forming haptens, which may trigger an immune response, as indicated by the activity of apalutamide in the MDAM assay, which in turn may be leading to increased potential for skin rash versus placebo in patients in the SPARTAN and TITAN clinical trials.

Salt Selection Matters: Differential Renal Toxicity With MDV1634.Maleate Versus MDV1634.2HCl.

Salt forms of pharmaceutical compounds can have unique pharmacokinetic and toxicity properties. MDV1634 was evaluated for neurology indication and also demonstrated blood pressure (BP)-lowering effects in nonclinical studies. During the chemistry manufacturing campaign, 2 salt forms, dihydrochloride (2HCl) and maleate (MAL), which improved chemical stability and water solubility of the free base were identified. MDV1634.MAL showed better chemical attributes and was evaluated in toxicology studies for further development. A 28-day oral toxicity study in dogs with MDV1634.MAL demonstrated partially reversible renal toxicity. Although MAL salt is generally regarded as safe, renal toxicity is sometimes observed in rats and dogs. To evaluate contribution of each salt form to renal toxicity and BP lowering, an additional 28-day study was conducted with MDV1634.2HCL and MDV1634.MAL, which included toxicokinetics, continuous BP measurement in a subset of dogs, and sensitive urinary biomarker evaluation for temporal monitorability and reversibility of potential renal findings. In the repeat study, both salt forms showed similar exposures during the dosing period, but renal tubular toxicity was observed only with MDV1634.MAL and not with MDV1634.2HCl. The renal findings with MDV1634.MAL included early urinary biomarker changes (increase in albumin, clusterin, ß2 microglobulin, and neutrophil gelatinase-associated lipocalin); elevations in serum blood urea nitrogen and creatinine; and microscopic findings of partially reversible tubular basophilia, single cell necrosis, pigmentation, and mineralization. The renal findings in contrast to the BP findings were MAL-specific and considered not related to MDV1634, thereby under scoring the importance of salt forms in pharmaceutical development.

In vitro and in vivo evaluation of dasatinib and imatinib on physiological parameters of pulmonary arterial hypertension.

PURPOSE: Pulmonary arterial hypertension (PAH) results from occlusion or vasoconstriction of pulmonary vessels, leading to progressive right ventricular failure. Dasatinib, a BCR-ABL1 tyrosine kinase inhibitor (TKI) approved for the treatment of chronic myelogenous leukemia, has been associated with PAH. In contrast, the BCR-ABL1 TKI imatinib has demonstrated anti-vasoproliferative properties and has been investigated as a potential treatment for PAH. Here we describe studies evaluating the effects of dasatinib and imatinib on cardiovascular and pulmonary functions to understand the reported differential consequences of the two TKIs in a clinical setting. METHODS: The direct effects of dasatinib and imatinib were explored in vivo to investigate possible mechanisms of dasatinib-induced PAH. In addition, effects of dasatinib and imatinib on PAH-related mediators were evaluated in vitro. RESULTS: In rats, both TKIs increased plasma nitric oxide (NO), did not induce PAH-related structural or molecular changes in PA or lungs, and did not alter hemodynamic lung function compared with positive controls. Similarly, in the pulmonary artery endothelial cells and smooth muscle cells co-culture model, imatinib and dasatinib increased NO and decreased endothelin-1 protein and mRNA. CONCLUSIONS: The results of these studies indicated that dasatinib did not induce physiological changes or molecular signatures consistent with PAH when compared to positive controls. Instead, dasatinib induced changes consistent with imatinib. Both dasatinib and imatinib induced biochemical and structural changes consistent with a protective effect for PAH. These data suggest that other factors of unclear etiology contributed to the development of PAH in patients treated with dasatinib.

The impact of dasatinib on pregnancy outcomes.

Prolonged survival in patients with chronic myeloid leukemia treated with BCR-ABL1-targeted tyrosine kinase inhibitors allows consideration of parenthood for patients on chronic therapy, but there are limited data about the effects of dasatinib on pregnancy. Pregnancy-related outcomes in dasatinib-treated patients or their partners reported to Bristol-Myers Squibb from clinical trials or healthcare providers through December 2013 were reviewed. Outcomes were available in 46/78 dasatinib-treated women (59%) and 33/69 partners of dasatinib-treated men (48%). Fifteen women (33%) delivered a normal infant; 18 (39%) and 8 (17%) had an elective or spontaneous abortion; and 5 (11%) had an abnormal pregnancy. There were 7 reports of fetal/infant abnormalities (encephalocele, renal tract abnormalities, and hydrops fetalis). Thirty of 33 (91%) infants fathered by dasatinib-treated men were reported normal at birth. Also, animal studies evaluated the impact of dasatinib on fertility, embryo-fetal toxicity, and development, suggesting that dasatinib may be a selective developmental toxicant. The outcomes of most pregnancies conceived by men treated with dasatinib were normal, but due to the small number of cases, further monitoring is required. Significant effects on pregnancy outcomes in women treated with dasatinib were found, supporting current recommendations that women avoid becoming pregnant during dasatinib treatment and be informed of fetal risks.

Clinical features of pulmonary arterial hypertension in patients receiving dasatinib.

The prognosis of most leukemia patients treated with BCR-ABL tyrosine kinase inhibitors (TKIs) is favorable, and a more precise understanding of serious and potentially irreversible treatment-related toxicities is essential to properly inform treatment choice. Few cases of pulmonary arterial hypertension (PAH) have been reported in patients with leukemia treated with dasatinib, a second-generation BCR-ABL TKI. To better understand characteristics and outcomes of dasatinib-treated patients with PAH, all clinical cases of PAH confirmed by right-heart catheterization in the Bristol-Myers Squibb pharmacovigilance database (N = 41), including 22 previously unpublished cases, were examined for previous treatments for leukemia, patient characteristics, time to PAH onset, and outcomes. Our analysis shows that compared with PAH due to other etiologies, dasatinib-related PAH is atypical, in that it is associated with partial to complete reversibility upon treatment discontinuation. The incidence of dasatinib-related PAH appears to be low. Most PAH cases were observed in patients who had received prior treatments for leukemia. No specific patient attributes appear to be associated with an increased risk of developing PAH while receiving dasatinib. Symptoms of PAH in dasatinib-treated leukemia patients should prompt a thorough workup, including consideration of confirmatory right-heart catheterization. In cases of confirmed PAH, dasatinib should be discontinued.

Improved ruggedness of an ion-pairing liquid chromatography/tandem mass spectrometry assay for the quantitative analysis of the triphosphate metabolite of a nucleoside reverse transcriptase inhibitor in peripheral blood mononuclear cells.

RATIONALE: Nucleotide analogs are highly polar and ionic, which impose great challenges on bioanalysis. Ion-pairing liquid chromatography/tandem mass spectrometry (LC/MS/MS) is the predominant reported approach for such compounds. Assay ruggedness of ion-pairing LC/MS/MS methods was often a challenge due to the potential contamination of the ion source of the mass spectrometer and LC column performance deterioration caused by ion-pairing reagents. METHODS: An ion-pairing reagent was only added to the reconstitution solution to minimize its exposure to the MS ion source. To achieve optimum sensitivity, high pH mobile phases and negative ion ESI were needed for the LC/MS/MS method. However, high pH mobile phases led to the accumulation of ion-pairing reagent on the analytical column, which was washed off with an acidic solution to restore the column performance. In addition, isopropanol was used as a mobile phase modifier to improve peak shape and sensitivity. RESULTS: The limit of detection was established at 1.0 ng/mL in the cell lysate. The calibration curve showed good linearity over the range of 1.0 to 100 ng/mL. The overall accuracy was no less than 87.7% based on four levels of quality control samples. Inter-run precision and intra-run precision across four analytical runs for low, geometric, medium and high QCs were less than 12.9. CONCLUSIONS: By identifying and addressing the root cause of the assay ruggedness problem, we have developed a rugged ion-pairing LC/MS/MS method for a triphosphate (TP) metabolite of BMS-986001 in peripheral blood mononuclear cells. The new method overcame challenges such as a rapid deterioration of the peak shape, increased carryover and extremely poor column life. The peak shape was well maintained throughout multiple analytical runs. This method has been successfully applied to a toxicology study in cynomolgus monkey.

Assessment of biomarkers of drug-induced kidney injury in cynomolgus monkeys treated with a triple reuptake inhibitor.

Drug-induced kidney injury (DIKI) results in attrition during drug development; new DIKI urinary biomarkers offer potential to detect and monitor DIKI progression and regression, but frequently only in rats. The triple reuptake inhibitor (TRI) PRC200-SS represents a new class of antidepressants that elevate synaptic levels of serotonin, norepinephrine, and dopamine and is expected to produce more rapid onset and better antidepressant efficacy than single or dual inhibitors. Although preclinical studies and recent clinical trials lend support to this concept of superior efficacy for TRIs, there is little information on the safety profile of this class of compounds. Using histopathology and DIKI biomarkers, in single- and repeat dose toxicological studies in cynomolgus monkeys, PRC200-SS demonstrated dose-proportional kidney toxicity. Characterization of the histopathological lesions, using a combination of immunohistochemistry (IHC) and urinary biomarker analysis, indicated that the compound is a distal tubule and collecting duct toxicant. Segment specificity for the lesions was shown using a newly developed triple IHC combination method with antibodies against calbindin D28, aquaporin 2, and aquaporin 1. Urinary biomarker analyses, using multiplex immunoassays, confirmed a dose-proportional increase in the excretion of calbindin D28 and clusterin in compound-treated monkeys with levels returning to baseline during the drug-free recovery period. These results constitute the validation of distal nephron DIKI biomarkers in the cynomolgus monkey and demonstrate the utility of calbindin D28 and clusterin to monitor the progression of distal nephron DIKI, representing potential early biomarkers of DIKI for the clinic.

Synthesis and SAR of 2-aryl-3-aminomethylquinolines as agonists of the bile acid receptor TGR5.

Optimization of a screening hit from uHTS led to the discovery of TGR5 agonist 32, which was shown to have activity in a rodent model for diabetes.

Specific down-regulation of connective tissue growth factor attenuates progression of nephropathy in mouse models of type 1 and type 2 diabetes.

Diabetic nephropathy (DN) remains a major complication in both type 1 and type 2 diabetes. Systemic administration of antitransforming growth factor-beta (TGF-beta) antibody has shown some promise in mouse models of DN. However, chronic blockade of the multifunctional TGB-beta could be problematic. Several downstream effects of TGF-beta are mediated by connective tissue growth factor (CTGF), which is up-regulated in several renal cells and secreted in the urine in the diabetic state. Using murine models of DN (type 1 and type 2) and a CTGF antisense oligonucleotide (ASO) of novel chimeric chemistry, we evaluated the specific role of this target in DN. In the type 1 model of DN, C57BL6 mice were made diabetic using streptozotocin injections and hyperglycemic animals were treated with CTGF ASOs (20 mg/kg/2 qw) for 4 months. ASO, but not mismatch control oligonucleotide, -treated animals showed significant reduction in target CTGF expression in the kidney with a concomitant decrease in proteinuria and albuminuria. Treatment with the CTGF ASO for 8 wk reduced serum creatinine and attenuated urinary albuminuria and proteinuria in diabetic db/db mice, a model of type 2 DN. The ASO also reduced expression of genes involved in matrix expansion such as fibronectin and collagen (I and IV) and an inhibitor of matrix degradation, PAI-1, in the renal cortex, contributing to significant reversal of mesangial expansion in both models of DN. Pathway analyses demonstrated that diabetes-induced phosphorylation of p38 MAPK and its downstream target CREB was also inhibited by the ASO. Our results strongly suggest that blocking CTGF using a chimeric ASO holds substantial promise for the treatment of DN.

Inhaled CD86 antisense oligonucleotide suppresses pulmonary inflammation and airway hyper-responsiveness in allergic mice.

The B7-family molecule CD86, expressed on the surface of pulmonary and thoracic lymph node antigen-presenting cells, delivers essential costimulatory signals for T-cell activation in response to inhaled allergens. CD86-CD28 signaling is involved in priming allergen-specific T cells, but it is unclear whether these interactions play a role in coordinating memory T-helper 2 cell responses. In the ovalbumin (OVA)-induced mouse model of asthma, administration of CD86-specific antibody before systemic sensitization suppresses inhaled OVA-induced pulmonary inflammation and airway hyper-responsiveness (AHR). In previously OVA-sensitized mice, systemic and intranasal coadministration of CD86 antibody is required to produce these effects. To directly assess the importance of pulmonary CD86 expression in secondary immune responses to inhaled allergens, mice were sensitized and locally challenged with nebulized OVA before treatment with an inhaled aerosolized CD86 antisense oligonucleotide (ASO). CD86 ASO treatment suppressed OVA-induced up-regulation of CD86 protein expression on pulmonary dendritic cells and macrophages as well as on recruited eosinophils. Suppression of CD86 protein expression correlated with decreased methacholine-induced AHR, airway inflammation, and mucus production following rechallenge with inhaled OVA. CD86 ASO treatment reduced BAL eotaxin levels, but it did not reduce CD86 protein on cells in the draining lymph nodes of the lung, and it had no effect on serum IgE levels, suggesting a local and not a systemic effect. These results demonstrate that CD86 expression on pulmonary antigen-presenting cells plays a vital role in regulating pulmonary secondary immune responses and suggest that treatment with an inhaled CD86 ASO may have utility in asthma and other chronic inflammatory lung conditions.

Anti-inflammatory activity of inhaled IL-4 receptor-alpha antisense oligonucleotide in mice.

The Th2 cytokines IL-4 and IL-13 mediate allergic pulmonary inflammation and airways hyperreactivity (AHR) in asthma models through signaling dependent upon the IL-4 receptor-alpha chain (IL-4Ralpha). IL-13 has been further implicated in the overproduction of mucus by the airway epithelium and in lung remodeling that commonly accompanies chronic inflammation. IL-4Ralpha-deficient mice are resistant to allergen-induced asthma, highlighting the therapeutic promise of selective molecular inhibitors of IL-4Ralpha. We designed a chemically modified IL-4Ralpha antisense oligonucleotide (IL-4Ralpha ASO) that specifically inhibits IL-4Ralpha protein expression in lung eosinophils, macrophages, dendritic cells, and airway epithelium after inhalation in allergen-challenged mice. Inhalation of IL-4Ralpha ASO attenuated allergen-induced AHR, suppressed airway eosinophilia and neutrophilia, and inhibited production of airway Th2 cytokines and chemokines in previously allergen-primed and -challenged mice. Histologic analysis of lungs from these animals demonstrated reduced goblet cell metaplasia and mucus staining that correlated with inhibition of Muc5AC gene expression in lung tissue. Therapeutic administration of inhaled IL-4Ralpha ASO in chronically allergen-challenged mice produced a spectrum of anti-inflammatory activity similar to that of systemically administered Dexamethasone with the added benefit of reduced airway neutrophilia. These data support the potential utility of a dual IL-4 and IL-13 oligonucleotide inhibitor in allergy/asthma, and suggest that local inhibition of IL-4Ralpha in the lung is sufficient to suppress allergen-induced pulmonary inflammation and AHR.

Dexamethasone enhances LPS induction of tissue factor expression in human monocytic cells by increasing tissue factor mRNA stability.

Glucocorticoids, such as dexamethasone (Dex), are used clinically in the treatment of various inflammatory diseases. Dex acts by inhibiting the expression of inflammatory mediators, such as tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant protein-1 (MCP-1). It is surprising that Dex enhances bacterial lipopolysaccharide (LPS) induction of tissue factor (TF) expression in human monocytic cells. TF is a transmembrane glycoprotein that activates the coagulation protease cascade. In this study, we analyze the mechanism by which Dex enhances LPS-induced TF expression in human monocytic cells. We found that Dex reduced LPS-induced TF gene transcription but increased the stability of TF mRNA. Dex decreased the stability of MCP-1 mRNA and did not affect TNF-alpha mRNA stability. Finally, we showed that Dex increased the stability of a transcript consisting of the final 297 nucleotides of the TF mRNA in in vitro decay assays. This region contains AU-rich elements that regulate mRNA stability and may mediate the Dex response. Therefore, despite an inhibition of TF gene transcription, Dex enhances TF expression in human monocytic cells by increasing the stability of TF mRNA.

High glucose-induced expression of proinflammatory cytokine and chemokine genes in monocytic cells.

Monocyte activation and adhesion to the endothelium play important roles in inflammatory and cardiovascular diseases. These processes are further aggravated by hyperglycemia, leading to cardiovascular complications in diabetes. We have previously shown that high glucose (HG) treatment activates monocytes and induces the expression of tumor necrosis factor (TNF)-alpha via oxidant stress and nuclear factor-kB transcription factor. To determine the effects of HG on the expression of other inflammatory genes, in the present study, HG-induced gene profiling was performed in THP-1 monocytes using cytokine gene arrays containing 375 known genes. HG treatment upregulated the expression of 41 genes and downregulated 15 genes that included chemokines, cytokines, chemokines receptors, adhesion molecules, and integrins. RT-PCR analysis further confirmed that HG significantly increased the expression of monocyte chemoattractant protein-1 (MCP-1), TNF-alpha, beta(2)-integrin, interleukin-1beta, and others. HG treatment increased transcription of the MCP-1 gene, MCP-1 protein levels, and adhesion of THP-1 cells to endothelial cells. HG-induced MCP-1 mRNA expression and monocyte adhesion were blocked by specific inhibitors of oxidant stress, protein kinase C, ERK1/2, and p38 mitogen-activated protein kinases. These results show for the first time that multiple inflammatory cytokines and chemokines relevant to the pathogenesis of diabetes complications are induced by HG via key signaling pathways.

Regulation of tissue factor and inflammatory mediators by Egr-1 in a mouse endotoxemia model.

In septic shock, tissue factor (TF) activates blood coagulation, and cytokines and chemokines orchestrate an inflammatory response. In this study, the role of Egr-1 in lipopolysaccharide (LPS) induction of TF and inflammatory mediators in vivo was evaluated using Egr-1(+/+) and Egr-1(-/-) mice. Administration of LPS transiently increased the steady-state levels of Egr-1 mRNA in the kidneys and lungs of Egr-1(+/+) mice with maximal induction at one hour. Egr-1 was expressed in epithelial cells in the kidneys and lungs in untreated and LPS-treated mice. LPS induction of monocyte chemoattractant protein mRNA in the kidneys and lungs of Egr-1(-/-) mice was not affected at 3 hours, but its expression was significantly reduced at 8 hours compared with the expression observed in Egr-1(+/+) mice. Similarly, LPS induction of TF mRNA expression in the kidneys and lungs at 8 hours was reduced in Egr-1(-/-) mice. However, Egr-1 deficiency did not affect plasma levels of tumor necrosis factor alpha in endotoxemic mice. Moreover, Egr-1(+/+) and Egr-1(-/-) mice exhibited similar survival times in a model of acute endotoxemia. These data indicate that Egr-1 does not contribute to the early inflammatory response in the kidneys and lungs or the early systemic inflammatory response in endotoxemic mice. However, Egr-1 does contribute to the sustained expression of inflammatory mediators and to the maximal expression of TF at 8 hours in the kidneys and lungs.

The phosphatidylinositol 3-kinase-Akt pathway limits lipopolysaccharide activation of signaling pathways and expression of inflammatory mediators in human monocytic cells.

Monocytes and macrophages express cytokines and procoagulant molecules in various inflammatory diseases. In sepsis, lipopolysaccharide (LPS) from Gram-negative bacteria induces tumor necrosis factor-alpha (TNF-alpha) and tissue factor (TF) in monocytic cells via the activation of the transcription factors Egr-1, AP-1, and nuclear factor-kappa B. However, the signaling pathways that negatively regulate LPS-induced TNF-alpha and TF expression in monocytic cells are currently unknown. We report that inhibition of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway enhances LPS-induced activation of the mitogen-activated protein kinase pathways (ERK1/2, p38, and JNK) and the downstream targets AP-1 and Egr-1. In addition, inhibition of PI3K-Akt enhanced LPS-induced nuclear translocation of nuclear factor-kappa B and prevented Akt-dependent inactivation of glycogen synthase kinase-beta, which increased the transactivational activity of p65. We propose that the activation of the PI3K-Akt pathway in human monocytes limits the LPS induction of TNF-alpha and TF expression. Our study provides new insight into the inhibitory mechanism by which the PI3K-Akt pathway ensures transient expression of these potent inflammatory mediators.