Soluble guanylate cyclase stimulators for the treatment of hypertension: Discovery of MK-2947.

The NO-sGC-cGMP signaling pathway plays an important role in the cardiovascular system. Loss of nitric oxide tone or impaired signaling has been associated with cardiovascular diseases, such as hypertension, pulmonary hypertension and heart failure. Direct activation of sGC enzyme independent of NO represents a novel approach for modulating NO signaling with tremendous therapeutic potential. Herein, we describe the design of a structurally novel class of heme-dependent sGC stimulators containing the 3,3-dimethylpyrrolidin-2-one moiety which resulted in the identification of the potent, selective stimulator 30 (MK-2947) for the treatment of hypertension.

Assessing the Utility of In Vitro Screening Tools for Predicting Bio-Performance of Oral Peptide Delivery.

PURPOSE: In this study we evaluated the utility of in-vitro screening tools for predicting the in-vivo behavior of six cyclic peptides with different solubility and permeability properties (BCS class II and III), intended for oral delivery in presence of permeation enhancer Labrasol. METHODS: An in vitro flux assay was used to assess peptide permeation across a biomimetic, lipid-based membrane and in vivo studies in rats were used to determine oral peptide bioavailability in the presence of Labrasol. RESULTS: The in vitro flux was significantly increased for BCS class III peptides, while it significantly decreased or remained unchanged for BCS class II peptides with increasing Labrasol concentrations. The different flux responses were attributed to the combination of reduced effective free peptide concentration and increased membrane permeability in the presence of Labrasol. In vivo studies in male Wistar-Hans rats indicated improved oral bioavailability at different extents for all peptides in presence of Labrasol. On comparing the in vitro and in vivo data, a potential direct correlation for BCS class III peptides was seen but not for BCS class II peptides, due to lower free concentrations of peptides in this class. CONCLUSION: This study assessed the utility of in vitro screening tools for selecting peptides and permeation excipients early in drug product development. Graphical Abstract Graphical Abstract and Figure 1 contains small text.Graphical Abstract text is made larger. The Figure 1 text cannot be made larger.

Discovery of indazole aldosterone synthase (CYP11B2) inhibitors as potential treatments for hypertension.

We report the discovery and hit-to-lead optimization of a structurally novel indazole series of CYP11B2 inhibitors. Benchmark compound 34 from this series displays potent inhibition of CYP11B2, high selectivity versus related steroidal and hepatic CYP targets, and lead-like physical and pharmacokinetic properties. On the basis of these and other data, the indazole series was progressed to lead optimization for further refinement.

Investigation of piperazine benzamides as human ß3 adrenergic receptor agonists for the treatment of overactive bladder.

The synthesis of a novel class of piperazine benzamide (reverse amides) targeting the human ß3-adrenergic receptor for the treatment of overactive bladder (OAB) is described. The SAR studies directed towards maintaining well established ß3 potency and selectivities while improving the overall pharmacokinetic profile in the reverse amide class will be evaluated. The results and consequences associated with functional activity at the norepinephrine transporter (NET) will also be discussed.

Discovery of phenyl acetamides as potent and selective GPR119 agonists.

The paper describes the SAR/SPR studies that led to the discovery of phenoxy cyclopropyl phenyl acetamide derivatives as potent and selective GPR119 agonists. Based on a cis cyclopropane scaffold discovered previously, phenyl acetamides such as compound 17 were found to have excellent GPR119 potency and improved physicochemical properties. Pharmacokinetic data of compound 17 in rat, dog and rhesus will be described. Compound 17 was suitable for QD dosing based on its predicted human half-life, and its projected human dose was much lower than that of the recently reported structurally-related benzyloxy compound 2. Compound 17 was selected as a tool compound candidate for NHP (Non-Human Primate) efficacy studies.

Development of a Convenient In Vitro Gel Diffusion Model for Predicting the In Vivo Performance of Subcutaneous Parenteral Formulations of Large and Small Molecules.

Parenteral delivery remains a compelling drug delivery route for both large- and small-molecule drugs and can bypass issues encountered with oral absorption. For injectable drug products, there is a strong patient preference for subcutaneous administration due to its convenience over intravenous infusion. However, in subcutaneous injection, in contrast to intravenous administration, the formulation is in contact with an extracellular matrix environment that behaves more like a gel than a fluid. This can impact the expected performance of a formulation. Since typical bulk fluid dissolution studies do not accurately simulate the subcutaneous environment, improved in vitro models to help better predict the behavior of the formulation are critical. Herein, we detail the development of a new model system consisting of a more physiologically relevant gel phase to simulate the rate of drug release and diffusion from a subcutaneous injection site using agarose hydrogels as a tissue mimic. This is coupled with continuous real-time data collection to accurately monitor drug diffusion. We show how this in vitro model can be used as an in vivo performance differentiator for different formulations of both large and small molecules. Thus, this model system can be used to improve optimization and understanding of new parenteral drug formulations in a rapid and convenient manner.

Evaluation of CETP activity in vivo under non-steady-state conditions: influence of anacetrapib on HDL-TG flux.

Studies in lipoprotein kinetics almost exclusively rely on steady-state approaches to modeling. Herein, we have used a non-steady-state experimental design to examine the role of cholesteryl ester transfer protein (CETP) in mediating HDL-TG flux in vivo in rhesus macaques, and therefore, we developed an alternative strategy to model the data. Two isotopomers ([(2)H11] and [(13)C18]) of oleic acid were administered (orally and intravenously, respectively) to serve as precursors for labeling TGs in apoB-containing lipoproteins. The flux of a specific TG (52:2) from these donor lipoproteins to HDL was used as the measure of CETP activity; calculations are also presented to estimate total HDL-TG flux. Based on our data, we estimate that the peak total postprandial TG flux to HDL via CETP is ∼ 13 mg · h(-1) · kg(-1) and show that this transfer was inhibited by 97% following anacetrapib treatment. Collectively, these data demonstrate that HDL TG flux can be used as a measure of CETP activity in vivo. The fact that the donor lipoproteins can be labeled in situ using well-established stable isotope tracer techniques suggests ways to measure this activity for native lipoproteins in free-living subjects under any physiological conditions.

Discovery of benzamides as potent human ß3 adrenergic receptor agonists.

The paper will describe the synthesis and SAR studies that led to the discovery of benzamide (reverse amide) as potent and selective human ß3-adrenergic receptor agonist. Based on conformationally restricted pyrrolidine scaffold we discovered earlier, pyrrolidine benzoic acid intermediate 22 was synthesized. From library synthesis and further optimization efforts, several structurally diverse reverse amides such as 24c and 24i were found to have excellent human ß3-adrenergic potency and good selectivity over the ß1 and ß2 receptors. In addition to human ß1, ß2, ß3 and hERG data, PK of selected compounds will be described.

Evaluation of cynomolgus monkeys for the identification of endogenous biomarkers for hepatic transporter inhibition and as a translatable model to predict pharmacokinetic interactions with statins in humans.

Inhibition of hepatic transporters such as organic anion transporting polypeptides (OATPs) 1B can cause drug-drug interactions (DDIs). Determining the impact of perpetrator drugs on the plasma exposure of endogenous substrates for OATP1B could be valuable to assess the risk for DDIs early in drug development. As OATP1B orthologs are well conserved between human and monkey, we assessed in cynomolgus monkeys the endogenous OATP1B substrates that are potentially suitable to assess DDI risk in humans. The effect of rifampin (RIF), a potent inhibitor for OATP1B, on plasma exposure of endogenous substrates of hepatic transporters was measured. From the 18 biomarkers tested, RIF (18 mg/kg, oral) caused significant elevation of plasma unconjugated and conjugated bilirubin, which may be attributed to inhibition of cOATP1B1 and cOATP1B3 based on in vitro to in vivo extrapolation analysis. To further evaluate whether cynomolgus monkeys are a suitable translational model to study OATP1B-mediated DDIs, we determined the inhibitory effect of RIF on in vitro transport and pharmacokinetics of rosuvastatin (RSV) and atorvastatin (ATV). RIF strongly inhibited the uptake of RSV and ATV by cOATP1B1 and cOATP1B3 in vitro. In agreement with clinical observations, RIF (18 mg/kg, oral) significantly decreased plasma clearance and increased the area under the plasma concentration curve (AUC) of intravenously administered RSV by 2.8- and 2.7-fold, and increased the AUC and maximum plasma concentration of orally administered RSV by 6- and 10.3-fold, respectively. In contrast to clinical findings, RIF did not significantly increase plasma exposure of either intravenous or orally administered ATV, indicating species differences in the rate-limiting elimination pathways.

A novel series of indazole-/indole-based glucagon receptor antagonists.

A novel, potent series of glucagon receptor antagonists (GRAs) was discovered. These indazole- and indole-based compounds were designed on an earlier pyrazole-based GRA lead MK-0893. Structure-activity relationship (SAR) studies were focused on the C3 and C6 positions of the indazole core, as well as the benzylic position on the N-1 of indazole. Multiple potent GRAs were identified with excellent in vitro profiles and good pharmacokinetics in rat. Among them, GRA 16d was found to be orally active in blunting glucagon induced glucose excursion in an acute glucagon challenge model in glucagon receptor humanized (hGCGR) mice at 1, 3 and 10mg/kg (mpk), and significantly lowered acute glucose levels in hGCGR ob/ob mice at 3 mpk dose.

Modulating Drug Release and Enhancing the Oral Bioavailability of Torcetrapib with Solid Lipid Dispersion Formulations.

The development of drug dispersions using solid lipids is a novel formulation strategy that can help address the challenges of poor drug solubility and systemic exposure after oral administration. The highly lipophilic and poorly water-soluble drug torcetrapib could be effectively formulated into solid lipid microparticles (SLMs) using an anti-solvent precipitation strategy. Acoustic milling was subsequently used to obtain solid lipid nanoparticles (SLNs). Torcetrapib was successfully incorporated into the lipid matrix in an amorphous state. Spherical SLMs with mean particle size of approximately 15-18 µm were produced with high drug encapsulation efficiency (>96%) while SLNs were produced with a mean particle size of 155 nm and excellent colloidal stability. The in vitro drug release and the in vivo absorption of the solid lipid micro- and nanoparticles after oral dosing in rats were evaluated against conventional crystalline drug powders as well as a spray dried amorphous polymer dispersion formulation. Interestingly, the in vitro drug release rate from the lipid particles could be tuned for immediate or extended release by controlling either the particle size or the precipitation temperature used when forming the drug-lipid particles. This change in the rate of drug release was manifested in vivo with changes in Tmax as well. In addition, in vivo pharmacokinetic studies revealed a significant increase (∼6 to 11-fold) in oral bioavailability in rats dosed with the SLMs and SLNs compared to conventional drug powders. Importantly, this formulation approach can be performed rapidly on a small scale, making it ideal as a formulation technology for use early in the drug discovery timeframe.

Antidiabetic and antisteatotic effects of the selective fatty acid synthase (FAS) inhibitor platensimycin in mouse models of diabetes.

Platensimycin (PTM) is a recently discovered broad-spectrum antibiotic produced by Streptomyces platensis. It acts by selectively inhibiting the elongation-condensing enzyme FabF of the fatty acid biosynthesis pathway in bacteria. We report here that PTM is also a potent and highly selective inhibitor of mammalian fatty acid synthase. In contrast to two agents, C75 and cerulenin, that are widely used as inhibitors of mammalian fatty acid synthase, platensimycin specifically inhibits fatty acid synthesis but not sterol synthesis in rat primary hepatocytes. PTM preferentially concentrates in liver when administered orally to mice and potently inhibits hepatic de novo lipogenesis, reduces fatty acid oxidation, and increases glucose oxidation. Chronic administration of platensimycin led to a net reduction in liver triglyceride levels and improved insulin sensitivity in db/+ mice fed a high-fructose diet. PTM also reduced ambient glucose levels in db/db mice. These results provide pharmacological proof of concept of inhibiting fatty acid synthase for the treatment of diabetes and related metabolic disorders in animal models.

Novel tetrahydropyran analogs as dipeptidyl peptidase IV inhibitors: Profile of clinical candidate (2R,3S,5R)-2- (2,5-difluorophenyl)-5-(4,6-dihydropyrrolo [3,4-c]pyrazol-5-(1H)-yl)tetrahydro-2H-pyran-3-amine (23) [corrected].

A series of novel tri-2,3,5-substituted tetrahydropyran analogs were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-4) for the treatment of type 2 diabetes. Optimization of the series provided inhibitors with good DPP-4 potency and selectivity over other peptidases (QPP, DPP8, and FAP). Compound 23, which is very potent, selective, efficacious in the diabetes PD model, and has an excellent pharmacokinetic profile, is selected as a clinical candidate.

Ten-Year Follow-up of 9-Valent Human Papillomavirus Vaccine: Immunogenicity, Effectiveness, and Safety.

BACKGROUND AND OBJECTIVES: The 9-valent human papillomavirus (9vHPV) vaccine Phase III immunogenicity study in 9- to 15-year-old boys and girls was extended to assess immunogenicity and effectiveness through 10 years after the last vaccine dose (NCT00943722). METHODS: Boys (n = 301) and girls (n = 971) who received three 9vHPV vaccine doses in the base study (day 1, months 2 and 6) enrolled in the extension. Serum was collected through month 126 for antibody assessments by competitive Luminex immunoassay and immunoglobulin G-Luminex immunoassay. For effectiveness analysis starting at age 16 years, genital swabs were collected (to assess HPV DNA by polymerase chain reaction) and external genital examinations conducted every 6 months. Primary analyses were conducted in per-protocol populations. RESULTS: Geometric mean antibody titers peaked around month 7, decreased sharply between months 7 and 12, then gradually through month 126. Seropositivity rates remained ≥81% by competitive Luminex immunoassay and ≥95% by immunoglobin G-Luminex immunoassay at month 126 for each 9vHPV vaccine type. After up to 11.0 (median 10.0) years of follow-up postdose 3, there were no cases of HPV6/11/16/18/31/33/45/52/58-related high-grade intraepithelial neoplasia or condyloma in males or females. Incidence rates of HPV6/11/16/18/31/33/45/52/58-related 6-month persistent infection in males and females were low (54.6 and 52.4 per 10000 person-years, respectively) and within ranges expected in vaccinated cohorts, based on previous human papillomavirus vaccine efficacy trials. CONCLUSIONS: The 9vHPV vaccine demonstrated sustained immunogenicity and effectiveness through ∼10 years post 3 doses of 9vHPV vaccination of boys and girls aged 9 to 15 years.

Prediction of frozen virus stability based on degradation mechanisms, real-time data and modeling.

Aim: Critical virus reagents in regulated bioanalytical assays require stability monitoring. Although stability at ultra-low frozen temperatures is generally assumed, published data are limited and real-time studies are time consuming. Materials & methods: The authors reviewed literature data, typical mechanisms of molecular degradation, glass transition temperatures of commonly used buffers and available real-time storage data to model frozen virus reagent stability. Results: Storage at ultra-low temperatures below the glass transition temperature was critical for virus stability. Modeling of real-time data suggested that virus potency remained within 0.5 log10 of its starting potency at a probability of >99, 90 and 73% after 10, 20 and 30 years, respectively. Conclusion: The study supports the practice of virus storage at -70°C or below for 20-30 years.

3-Substituted 3-(4-aryloxyaryl)-propanoic acids as GPR40 agonists.

The design, synthesis, and structure-activity relationship (SAR) for a series of ß-substituted 3-(4-aryloxyaryl)propanoic acid GPR40 agonists is described. Systematic replacement of the pendant aryloxy group led to identification of potent GPR40 agonists. In order to identify candidates suitable for in vivo validation of the target, serum shifted potency and pharmacokinetic properties were determined for several compounds. Finally, further profiling of compound 7 is presented, including demonstration of enhanced glucose tolerance in an in vivo mouse model.

Design of a novel pyrrolidine scaffold utilized in the discovery of potent and selective human ß3 adrenergic receptor agonists.

A novel class of human ß(3)-adrenergic receptor agonists was designed in effort to improve selectivity and metabolic stability versus previous disclosed ß(3)-AR agonists. As observed, many of the ß(3)-AR agonists seem to need the acyclic ethanolamine core for agonist activity. We have synthesized derivatives that constrained this moiety by introduction of a pyrrolidine. This unique modification maintains human ß(3) functional potency with improved selectivity versus ancillary targets and also eliminates the possibility of the same oxidative metabolites formed from cleavage of the N-C bond of the ethanolamine. Compound 39 exhibited excellent functional ß(3) agonist potency across species with good pharmacokinetic properties in rat, dog, and rhesus monkeys. Early de-risking of this novel pyrrolidine core (44) via full AMES study supports further research into various new ß(3)-AR agonists containing the pyrrolidine moiety.

Discovery of N-aryl-2-acylindole human glucagon receptor antagonists.

A novel class of N-aryl-2-acylindole human glucagon receptor (hGCGR) antagonists is reported. These compounds demonstrate good pharmacokinetic profiles in multiple preclinical species. One compound from this series, indole 33, is orally active in a transgenic murine pharmacodynamic model. Furthermore, a 1mg/kg oral dose of indole 33 lowers ambient glucose levels in an ob/ob/hGCGR transgenic murine diabetes model. This compound was deemed suitable for preclinical safety studies and was found to be well tolerated in an 8-day experimental rodent tolerability study. The combination of preclinical efficacy and safety observed with compound 33 highlights the potential of this class as a treatment for type 2 diabetes.

Discovery of cyclic guanidines as potent, orally active, human glucagon receptor antagonists.

In the course of the development of an aminobenzimidazole class of human glucagon receptor (hGCGR) antagonists, a novel class of cyclic guanidine hGCGR antagonists was discovered. Rapid N-dealkylation resulted in poor pharmacokinetic profiles for the benchmark compound in this series. A strategy aimed at blocking oxidative dealkylation led to a series of compounds with improved rodent pharmacokinetic profiles. One compound was orally efficacious in a murine glucagon challenge pharmacodynamic model and also significantly lowered glucose levels in a murine diabetes model.

Small molecule insulin mimetics reduce food intake and body weight and prevent development of obesity.

Obesity and insulin resistance are major risk factors for a number of metabolic disorders, such as type 2 diabetes mellitus. Insulin has been suggested to function as one of the adiposity signals to the brain for modulation of energy balance. Administration of insulin into the brain reduces food intake and body weight, and mice with a genetic deletion of neuronal insulin receptors are hyperphagic and obese. However, insulin is also an anabolic factor; when administered systemically, pharmacological levels of insulin are associated with body weight gain in patients. In this study, we investigated the efficacy and feasibility of small molecule insulin mimetic compounds to regulate key parameters of energy homeostasis. Central intracerebroventricular (i.c.v.) administration of an insulin mimetic resulted in a dose-dependent reduction of food intake and body weight in rats, and altered the expression of hypothalamic genes known to regulate food intake and body weight. Oral administration of a mimetic in a mouse model of high-fat diet-induced obesity reduced body weight gain, adiposity and insulin resistance. Thus, insulin mimetics have a unique advantage over insulin in the control of body weight and hold potential as a novel anti-obesity treatment.