Bicyclic Ligand-Biased Agonists of S1P1: Exploring Side Chain Modifications to Modulate the PK, PD, and Safety Profiles.

Sphingosine-1-phosphate (S1P) binds to a family of sphingosine-1-phosphate G-protein-coupled receptors (S1P1-5). The interaction of S1P with these S1P receptors has a fundamental role in many physiological processes in the vascular and immune systems. Agonist-induced functional antagonism of S1P1 has been shown to result in lymphopenia. As a result, agonists of this type hold promise as therapeutics for autoimmune disorders. The previously disclosed differentiated S1P1 modulator BMS-986104 (1) exhibited improved preclinical cardiovascular and pulmonary safety profiles as compared to earlier full agonists of S1P1; however, it demonstrated a long pharmacokinetic half-life (T1/2 18 days) in the clinic and limited formation of the desired active phosphate metabolite. Optimization of this series through incorporation of olefins, ethers, thioethers, and glycols into the alkyl side chain afforded an opportunity to reduce the projected human T1/2 and improve the formation of the active phosphate metabolite while maintaining efficacy as well as the improved safety profile. These efforts led to the discovery of 12 and 24, each of which are highly potent, biased agonists of S1P1. These compounds not only exhibited shorter in vivo T1/2 in multiple species but are also projected to have significantly shorter T1/2 values in humans when compared to our first clinical candidate. In models of arthritis, treatment with 12 and 24 demonstrated robust efficacy.

Aryl Ether-Derived Sphingosine-1-Phosphate Receptor (S1P1) Modulators: Optimization of the PK, PD, and Safety Profiles.

Efforts aimed at increasing the in vivo potency and reducing the elimination half-life of 1 and 2 led to the identification of aryl ether and thioether-derived bicyclic S1P1 differentiated modulators 3-6. The effects of analogs 3-6 on lymphocyte reduction in the rat (desired pharmacology) along with pulmonary- and cardiovascular-related effects (undesired pharmacology) are described. Optimization of the overall properties in the aryl ether series yielded 3d, and the predicted margin of safety against the cardiovascular effects of 3d would be large enough for human studies. Importantly, compared to 1 and 2, compound 3d had a better profile in both potency (ED50 < 0.05 mg/kg) and predicted human half-life (t 1/2 ∼ 5 days).

LC/MS/MS-based quantitation of pig and human S100A1 protein in cardiac tissues: Application to gene therapy.

The S100A1 protein is a target of interest for the treatment of heart failure as it has been previously reported to be depleted in failing cardiomyocytes. A gene therapy approach leading to increased expression levels of the protein directly in the heart could potentially lead to restoration of contractile function and improve overall cell survival. S100A1 is a relatively small soluble protein that is extremely well conserved across species with only a single amino acid difference between the sequences in human and pig, a commonly used pre-clinical model for evaluation of efficacy, biodistribution and safety for cardiac-directed gene therapy approaches. This high homology presents a bioanalytical challenge for the accurate detection and quantitation of both endogenous (pig) and exogenous (human) transduced S100A1 proteins post treatment using a human S100A1 gene therapy in pigs. Here we present a sensitive and selective LC-MS/MS approach that can easily differentiate and simultaneously quantitate both human and pig S100A1 proteins. Additionally, we report on a detailed profiling of S100A1 protein in various pig tissues, a comprehensive evaluation of S100A1 distribution in pig hearts and a comparison to S100A1 levels in human cardiac samples.

Identification and Preclinical Pharmacology of ((1 R,3 S)-1-Amino-3-(( S)-6-(2-methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol (BMS-986166): A Differentiated Sphingosine-1-phosphate Receptor 1 (S1P1) Modulator Advanced into Clinical Trials.

Recently, our research group reported the identification of BMS-986104 (2) as a differentiated S1P1 receptor modulator. In comparison to fingolimod (1), a full agonist of S1P1 currently marketed for the treatment of relapse remitting multiple sclerosis (RRMS), 2 offers several potential advantages having demonstrated improved safety multiples in preclinical evaluations against undesired pulmonary and cardiovascular effects. In clinical trials, 2 was found to exhibit a pharmacokinetic half-life ( T1/2) longer than that of 1, as well as a reduced formation of the phosphate metabolite that is required for activity against S1P1. Herein, we describe our efforts to discover highly potent, partial agonists of S1P1 with a shorter T1/2 and increased in vivo phosphate metabolite formation. These efforts culminated in the discovery of BMS-986166 (14a), which was advanced to human clinical evaluation. The pharmacokinetic/pharmacodynamic (PK/PD) relationship as well as pulmonary and cardiovascular safety assessments are discussed. Furthermore, efficacy of 14a in multiple preclinical models of autoimmune diseases are presented.

Development of a Chemiluminescent ELISA Method for the Detection of Total Anti-Adeno Associated Virus Serotype 9 (AAV9) Antibodies.

Recombinant adeno associated viruses (rAAV) have become an important tool for the delivery of gene therapeutics due to long-standing safety and success in clinical trials. Since humans often become exposed to AAVs and develop anti-AAV antibodies (Abs), a potential impediment to the success of gene therapeutics is neutralization of the viral particle before it has had a chance to bind and enter target cells to release the transgene. Identification of subjects with preexisting Abs having neutralizing potential, and exclusion of such subjects from clinical studies is expected to enhance drug efficacy. In vitro cell-based reporter assays are most often employed to determine the level of neutralizing antibodies in a given population. Such assays measure the ability of the Abs to prevent viral binding and entry into cells by engaging epitopes on the viral capsid involved in host cell receptor binding. In general, cell-based assays are low throughput and labor intensive and may suffer from high variability and low sensitivity issues. In contrast, enzyme-linked immunosorbent assays (ELISAs) are simpler, less variable, and have higher throughput. Demonstrating a correlation between neutralizing Abs assessed by a cell-based assay and total binding Abs measured in an ELISA will enable the use and substitution of the latter for screening and exclusion of subjects. In this work, we describe the development of a highly sensitive, specific, robust, and reproducible chemiluminescent ELISA method for the detection of total anti-AAV9 Abs. Using this method, we analyzed the prevalence of preexisting anti-AAV9 Abs in 100 serum samples from heart disease patients. Analysis of neutralizing Abs in the same samples using an in vitro cell-based assay showed a strong correlation between total anti-AAV9 Abs and neutralizing Abs, indicating the feasibility of using the total Ab ELISA in the future for patient screening and exclusion.

Identification of a Potent, Selective, and Efficacious Phosphatidylinositol 3-Kinase δ (PI3Kδ) Inhibitor for the Treatment of Immunological Disorders.

PI3Kδ plays an important role controlling immune cell function and has therefore been identified as a potential target for the treatment of immunological disorders. This article highlights our work toward the identification of a potent, selective, and efficacious PI3Kδ inhibitor. Through careful SAR, the successful replacement of a polar pyrazole group by a simple chloro or trifluoromethyl group led to improved Caco-2 permeability, reduced Caco-2 efflux, reduced hERG PC activity, and increased selectivity profile while maintaining potency in the CD69 hWB assay. The optimization of the aryl substitution then identified a 4'-CN group that improved the human/rodent correlation in microsomal metabolic stability. Our lead molecule is very potent in PK/PD assays and highly efficacious in a mouse collagen-induced arthritis model.

Identification of potent tricyclic prodrug S1P1 receptor modulators.

Recently, our research group reported the identification of prodrug amino-alcohol 2 as a potent and efficacious S1P1 receptor modulator. This molecule is differentiated preclinically over the marketed drug fingolimod (Gilenya 1), whose active phosphate metabolite is an S1P1 full agonist, in terms of pulmonary and cardiovascular safety. S1P1 partial agonist 2, however, has a long half-life in rodents and was projected to have a long half-life in humans. The purpose of this communication is to disclose highly potent partial agonists of S1P1 with shorter half-lives relative to the clinical compound 2. PK/PD relationships as well as their preclinical pulmonary and cardiovascular safety assessment are discussed.

Structure-activity relationships of 4-hydroxy-4-biaryl-proline acylsulfonamide tripeptides: A series of potent NS3 protease inhibitors for the treatment of hepatitis C virus.

The design and synthesis of a series of tripeptide acylsulfonamides as potent inhibitors of the HCV NS3/4A serine protease is described. These analogues house a C4 aryl, C4 hydroxy-proline at the S2 position of the tripeptide scaffold. Information relating to structure-activity relationships as well as the pharmacokinetic and cardiovascular profiles of these analogues is provided.

Discovery of a Potent Acyclic, Tripeptidic, Acyl Sulfonamide Inhibitor of Hepatitis C Virus NS3 Protease as a Back-up to Asunaprevir with the Potential for Once-Daily Dosing.

The discovery of a back-up to the hepatitis C virus NS3 protease inhibitor asunaprevir (2) is described. The objective of this work was the identification of a drug with antiviral properties and toxicology parameters similar to 2, but with a preclinical pharmacokinetic (PK) profile that was predictive of once-daily dosing. Critical to this discovery process was the employment of an ex vivo cardiovascular (CV) model which served to identify compounds that, like 2, were free of the CV liabilities that resulted in the discontinuation of BMS-605339 (1) from clinical trials. Structure-activity relationships (SARs) at each of the structural subsites in 2 were explored with substantial improvement in PK through modifications at the P1 site, while potency gains were found with small, but rationally designed structural changes to P4. Additional modifications at P3 were required to optimize the CV profile, and these combined SARs led to the discovery of BMS-890068 (29).

Discovery and Structure-Activity Relationship (SAR) of a Series of Ethanolamine-Based Direct-Acting Agonists of Sphingosine-1-phosphate (S1P1).

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates a multitude of physiological processes such as lymphocyte trafficking, cardiac function, vascular development, and inflammation. Because of the ability of S1P1 receptor agonists to suppress lymphocyte egress, they have great potential as therapeutic agents in a variety of autoimmune diseases. In this article, the discovery of selective, direct acting S1P1 agonists utilizing an ethanolamine scaffold containing a terminal carboxylic acid is described. Potent S1P1 agonists such as compounds 18a and 19a which have greater than 1000-fold selectivity over S1P3 are described. These compounds efficiently reduce blood lymphocyte counts in rats through 24 h after single doses of 1 and 0.3 mpk, respectively. Pharmacodynamic properties of both compounds are discussed. Compound 19a was further studied in two preclinical models of disease, exhibiting good efficacy in both the rat adjuvant arthritis model (AA) and the mouse experimental autoimmune encephalomyelitis model (EAE).

Evaluation of cardiac function in unrestrained dogs and monkeys using left ventricular dP/dt.

INTRODUCTION: Preclinical assessment for alterations in cardiac ventricular function for drug candidates has not been a focus of ICH S7b guidelines for cardiovascular safety studies, but there is growing interest given that the cardiovascular risk is associated with positive and negative inotropes. METHODS: From 2003 through 2013, 163 telemetry studies with left-ventricular function analyses were conducted in dogs and monkeys at Bristol Myers Squibb (BMS) in support for drug development programs. The ability of the telemetry system to detect changes in cardiac contractility was verified with positive control agents pimobendan and atenolol. Control data from a subset of studies were analyzed to determine dP/dt reference range values, and minimum detectable mean differences (control vs. treated) for statistical significance. RESULTS: Median minimum detectable differences for dogs ranged from 14 to 21% for positive dP/dt and 11 to 21% for negative dP/dt. For monkeys, median minimum detectable differences were 25 and 14% for positive and negative dP/dt, respectively. For BMS programs, 15 drug candidates were identified that produced primary effects on contractility. Changes in contractility that were associated with, and potentially secondary to, drug-related effects on heart rate or systemic blood pressure were observed with an additional 29 drug candidates. DISCUSSION: Changes in contractility have been observed in large animals during drug development studies at BMS over the past 10years. Model sensitivity has been demonstrated and a dP/dt beat-to-beat cloud analysis tool has been developed to help distinguish primary effects from those potentially secondary to systemic hemodynamic changes.

Identification and Preclinical Pharmacology of BMS-986104: A Differentiated S1P1 Receptor Modulator in Clinical Trials.

Clinical validation of S1P receptor modulation therapy was achieved with the approval of fingolimod (Gilenya, 1) as the first oral therapy for relapsing remitting multiple sclerosis. However, 1 causes a dose-dependent reduction in the heart rate (bradycardia), which occurs within hours after first dose. We disclose the identification of clinical compound BMS-986104 (3d), a novel S1P1 receptor modulator, which demonstrates ligand-biased signaling and differentiates from 1 in terms of cardiovascular and pulmonary safety based on preclinical pharmacology while showing equivalent efficacy in a T-cell transfer colitis model.

The discovery of asunaprevir (BMS-650032), an orally efficacious NS3 protease inhibitor for the treatment of hepatitis C virus infection.

The discovery of asunaprevir (BMS-650032, 24) is described. This tripeptidic acylsulfonamide inhibitor of the NS3/4A enzyme is currently in phase III clinical trials for the treatment of hepatitis C virus infection. The discovery of 24 was enabled by employing an isolated rabbit heart model to screen for the cardiovascular (CV) liabilities (changes to HR and SNRT) that were responsible for the discontinuation of an earlier lead from this chemical series, BMS-605339 (1), from clinical trials. The structure-activity relationships (SARs) developed with respect to CV effects established that small structural changes to the P2* subsite of the molecule had a significant impact on the CV profile of a given compound. The antiviral activity, preclincial PK profile, and toxicology studies in rat and dog supported clinical development of BMS-650032 (24).

Discovery and early clinical evaluation of BMS-605339, a potent and orally efficacious tripeptidic acylsulfonamide NS3 protease inhibitor for the treatment of hepatitis C virus infection.

The discovery of BMS-605339 (35), a tripeptidic inhibitor of the NS3/4A enzyme, is described. This compound incorporates a cyclopropylacylsulfonamide moiety that was designed to improve the potency of carboxylic acid prototypes through the introduction of favorable nonbonding interactions within the S1' site of the protease. The identification of 35 was enabled through the optimization and balance of critical properties including potency and pharmacokinetics (PK). This was achieved through modulation of the P2* subsite of the inhibitor which identified the isoquinoline ring system as a key template for improving PK properties with further optimization achieved through functionalization. A methoxy moiety at the C6 position of this isoquinoline ring system proved to be optimal with respect to potency and PK, thus providing the clinical compound 35 which demonstrated antiviral activity in HCV-infected patients.

Identification of 1-{2-[4-chloro-1'-(2,2-dimethylpropyl)-7-hydroxy-1,2-dihydrospiro[indole-3,4'-piperidine]-1-yl]phenyl}-3-{5-chloro-[1,3]thiazolo[5,4-b]pyridin-2-yl}urea, a potent, efficacious and orally bioavailable P2Y(1) antagonist as an antiplatelet agent.

Spiropiperidine indoline-substituted diaryl ureas had been identified as antagonists of the P2Y1 receptor. Enhancements in potency were realized through the introduction of a 7-hydroxyl substitution on the spiropiperidinylindoline chemotype. SAR studies were conducted to improve PK and potency, resulting in the identification of compound 3e, a potent, orally bioavailable P2Y1 antagonist with a suitable PK profile in preclinical species. Compound 3e demonstrated a robust antithrombotic effect in vivo and improved bleeding risk profile compared to the P2Y12 antagonist clopidogrel in rat efficacy/bleeding models.

Role of T-type calcium channel subunits in post-myocardial infarction remodelling probed with genetically engineered mice.

AIMS: Previous studies suggested that T-type Ca(2+)-current (I(CaT))-blockers improve cardiac remodelling, but all available I(CaT)-blockers have non-specific actions on other currents and/or functions. To clarify the role of I(CaT) in cardiac remodelling, we studied mice with either of the principal cardiac I(CaT)-subunits (Cav3.1 or Cav3.2) knocked out. METHODS AND RESULTS: Adult male Cav3.1- or Cav3.2-knockout (Cav3.1(-/-), Cav3.2(-/-)) mice and respective wild-type (WT) littermate controls were subjected to left anterior descending coronary artery ligation to create myocardial infarction (MI). Echocardiography and programmed electrical stimulation were performed at baseline and 4 weeks post-MI. At baseline, Cav3.1(-/-) mice had slowed heart rates and longer PR intervals vs. WT, but no other electrophysiological and no haemodynamic differences. Cav3.2(-/-) showed no differences vs. WT. Contractile indices (left ventricular fractional shortening and ejection fraction) decreased more post-MI in Cav3.1(-/-) mice than in Cav3.1(+/+) (e.g. by 34 and 29% for WT; 50 and 45% for Cav3.1(-/-), respectively; P < 0.05 for each). Cav3.1(-/-) mice had increased ventricular tachycardia (VT) inducibility post-MI (9 of 11, 82%) vs. WT (3 of 10, 30%; P < 0.05). Cav3.2(-/-) mice were not different in cardiac function or VT inducibility vs. WT. Quantitative polymerase chain reaction showed that Cav3.1 is the major I(CaT)-subunit and that no compensatory Cav3.2 up-regulation occurs in Cav3.1(-/-) mice. Cav3.1(-/-) and Cav3.2(-/-) mice had no mRNA expression for the knocked-out gene, at baseline or post-MI. CONCLUSION: Our findings suggest that, contrary to suggestions from previous studies with (imperfectly selective) pharmacological agents having T-type Ca(2+)-channel-blocking actions, elimination of Cav3.1 expression leads to impaired cardiac function and enhanced arrhythmia vulnerability post-MI, whereas Cav3.2 elimination has no effect.

Drug safety is a barrier to the discovery and development of new androgen receptor antagonists.

BACKGROUND: Androgen receptor (AR) antagonists are part of the standard of care for prostate cancer. Despite the almost inevitable development of resistance in prostate tumors to AR antagonists, no new AR antagonists have been approved for over a decade. Treatment failure is due in part to mutations that increase activity of AR in response to lower ligand concentrations as well as to mutations that result in AR response to a broader range of ligands. The failure to discover new AR antagonists has occurred in the face of continued research; to enable progress, a clear understanding of the reasons for failure is required. METHODS: Non-clinical drug safety studies and safety pharmacology assays were performed on previously approved AR antagonists (bicalutamide, flutamide, nilutamide), next generation antagonists in clinical testing (MDV3100, BMS-641988), and a pre-clinical drug candidate (BMS-501949). In addition, non-clinical studies with AR mutant mice, and EEG recordings in rats were performed. Non-clinical findings are compared to disclosures of clinical trial results. RESULTS: As a drug class, AR antagonists cause seizure in animals by an off-target mechanism and are found in vitro to inhibit GABA-A currents. Clinical trials of candidate next generation AR antagonists identify seizure as a clinical safety risk. CONCLUSIONS: Non-clinical drug safety profiles of the AR antagonist drug class create a significant barrier to the identification of next generation AR antagonists. GABA-A inhibition is a common off-target activity of approved and next generation AR antagonists potentially explaining some side effects and safety hazards of this class of drugs.

Design and SAR of selective T-type calcium channel antagonists containing a biaryl sulfonamide core.

T-type calcium channel antagonists were designed using a protocol involving the program SPROUT and constrained by a ComFA-based pharmacophore model. Scaffolds generated by SPROUT were evaluated based on their ability to be translated into structures that were synthetically tractable. From this exercise, a novel series of potent and selective T-type channel antagonists containing a biaryl sulfonamide core were discovered.

Tetrahydronaphthalene-derived amino alcohols and amino ketones as potent and selective inhibitors of the delayed rectifier potassium current IKs.

Class III anti-arrhythmic drugs (e.g., dofetilide) prolong cardiac action potential duration (APD) by blocking the fast component of the delayed rectifier potassium current (I(Kr)). The block of I(Kr) can result in life threatening ventricular arrhythmias (i.e., torsades de pointes). Unlike I(Kr), the role of the slow component of the delayed rectifier potassium current (I(Ks)) becomes significant only at faster heart rate. Therefore selective blockers of I(Ks) could prolong APD with a reduced propensity to cause pro-arrhythmic side effects. This report describes structure-activity relationships (SARs) of a series of I(Ks) inhibitors derived from 6-alkoxytetralones with good in vitro activity (IC(50) > or =30 nM) and up to 40-fold I(Ks)/I(Kr) selectivity.

Expression and characterization of human transient receptor potential melastatin 3 (hTRPM3).

Transient receptor potential (TRP) cation-selective channels are an emerging class of proteins that are involved in a variety of important biological functions including pain transduction, thermosensation, mechanoregulation, and vasorelaxation. Utilizing a bioinformatics approach, we have identified the full-length human TRPM3 (hTRPM3) as a member of the TRP family. The hTRPM3 gene is comprised of 24 exons and maps to human chromosome 9q-21.12. hTRPM3 is composed of 1555 amino acids and possesses the characteristic six-transmembrane domain of the TRP family. hTRPM3 is expressed primarily in kidney and, at lesser levels, in brain, testis, and spinal cord as demonstrated by quantitative RT-PCR and Northern blotting. In situ hybridization in human kidney demonstrated that hTRPM3 mRNA expression is predominantly found in the collecting tubular epithelium. Heterologous expression of hTRPM3 in human embryonic kidney cells (HEK 293) showed that hTRPM3 is localized to the cell membrane. hTRPM3-expressing cells exhibited Ca2+ concentration-dependent Ca2+ entry. Depletion of intracellular Ca2+ stores by lowering extracellular Ca2+ concentration and treatment with the Ca2+-ATPase inhibitor thapsigargin or the muscarinic receptor agonist carbachol further augmented hTRPM3-mediated Ca2+ entry. The nonselective Ca2+ channel blocker, lanthanide gadolinium (Gd3+), partially inhibited hTRPM3-mediated Ca2+ entry. These results are consistent with the hypothesis that hTRPM3 mediates a Ca2+ entry pathway that apparently is distinct from the endogenous Ca2+ entry pathways present in HEK 293 cells.