Cerastecins inhibit membrane lipooligosaccharide transport in drug-resistant Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii infections have limited treatment options. Synthesis, transport and placement of lipopolysaccharide or lipooligosaccharide (LOS) in the outer membrane of Gram-negative bacteria are important for bacterial virulence and survival. Here we describe the cerastecins, inhibitors of the A. baumannii transporter MsbA, an LOS flippase. These molecules are potent and bactericidal against A. baumannii, including clinical carbapenem-resistant Acinetobacter baumannii isolates. Using cryo-electron microscopy and biochemical analysis, we show that the cerastecins adopt a serpentine configuration in the central vault of the MsbA dimer, stalling the enzyme and uncoupling ATP hydrolysis from substrate flipping. A derivative with optimized potency and pharmacokinetic properties showed efficacy in murine models of bloodstream or pulmonary A. baumannii infection. While resistance development is inevitable, targeting a clinically unexploited mechanism avoids existing antibiotic resistance mechanisms. Although clinical validation of LOS transport remains undetermined, the cerastecins may open a path to narrow-spectrum treatment modalities for important nosocomial infections.

Invention of MK-7845, a SARS-CoV-2 3CL Protease Inhibitor Employing a Novel Difluorinated Glutamine Mimic.

As SARS-CoV-2 continues to circulate, antiviral treatments are needed to complement vaccines. The virus's main protease, 3CLPro, is an attractive drug target in part because it recognizes a unique cleavage site, which features a glutamine residue at the P1 position and is not utilized by human proteases. Herein, we report the invention of MK-7845, a novel reversible covalent 3CLPro inhibitor. While most covalent inhibitors of SARS-CoV-2 3CLPro reported to date contain an amide as a Gln mimic at P1, MK-7845 bears a difluorobutyl substituent at this position. SAR analysis and X-ray crystallographic studies indicate that this group interacts with His163, the same residue that forms a hydrogen bond with the amide substituents typically found at P1. In addition to promising in vivo efficacy and an acceptable projected human dose with unboosted pharmacokinetics, MK-7845 exhibits favorable properties for both solubility and absorption that may be attributable to the unusual difluorobutyl substituent.

Current Practices, Gap Analysis, and Proposed Workflows for PBPK Modeling of Cytochrome P450 Induction: An Industry Perspective.

The International Consortium for Innovation and Quality (IQ) Physiologically Based Pharmacokinetic (PBPK) Modeling Induction Working Group (IWG) conducted a survey across participating companies around general strategies for PBPK modeling of induction, including experience with its utility to address various questions, regulatory interactions, and regulatory acceptance. The results highlight areas where PBPK modeling is used with high confidence and identifies opportunities where confidence is lower and further evaluation is needed. To enhance the survey results, the PBPK-IWG also collected case studies and analyzed recent literature examples where PBPK models were applied to predict CYP3A induction-mediated drug-drug interactions. PBPK modeling of induction has evolved and progressed significantly, proving to have great potential to accelerate drug discovery and development. With the aim of enabling optimal use for new molecular entities that are either substrates and/or inducers of CYP3A, the PBPK-IWG proposes initial workflows for PBPK application, discusses future trends, and identifies gaps that need to be addressed.

Discovery of novel N-1 substituted pyrazolopyrimidinones as potent, selective PDE2 inhibitors.

A focused SAR study was conducted on a series of N1-substituted pyrazolopyrimidinone PDE2 inhibitors to reveal compounds with excellent potency and selectivity. The series was derived from previously identified internal leads and designed to enhance steric interactions with key amino acids in the PDE2 binding pocket. Compound 26 was identified as a lead compound with excellent PDE2 selectivity and good physicochemical properties.

Physiologically Based Pharmacokinetic Modeling of Doravirine and Its Major Metabolite to Support Dose Adjustment With Rifabutin.

Doravirine, a novel nonnucleoside reverse transcriptase inhibitor for the treatment of human immunodeficiency virus 1 (HIV-1), is predominantly cleared by cytochrome P450 (CYP) 3A4 and metabolized to an oxidative metabolite (M9). Coadministration with rifabutin, a moderate CYP3A4 inducer, decreased doravirine exposure. Based on nonparametric superposition modeling, a doravirine dose adjustment from 100 mg once daily to 100 mg twice daily during rifabutin coadministration was proposed. However, M9 exposure may also be impacted by induction, in addition to the dose adjustment. As M9 concentrations have not been quantified in previous clinical studies, a physiologically based pharmacokinetic model was developed to investigate the change in M9 exposure when doravirine is coadministered with CYP3A inducers. Simulations demonstrated that although CYP3A induction increases doravirine clearance by up to 4.4-fold, M9 exposure is increased by only 1.2-fold relative to exposures for doravirine 100 mg once daily in the absence of CYP3A induction. Thus, a 2.4-fold increase in M9 exposure relative to the clinical dose of doravirine is anticipated when doravirine 100 mg twice daily is coadministered with rifabutin. In a subsequent clinical trial, doravirine and M9 exposures, when doravirine 100 mg twice daily was coadministered with rifabutin, were found to be consistent with model predictions using rifampin and efavirenz as representative inducers. These findings support the dose adjustment to doravirine 100 mg twice daily when coadministered with rifabutin.

Application of a Rat Liver Drug Bioactivation Transcriptional Response Assay Early in Drug Development That Informs Chemically Reactive Metabolite Formation and Potential for Drug-induced Liver Injury.

Drug-induced liver injury is a major reason for drug candidate attrition from development, denied commercialization, market withdrawal, and restricted prescribing of pharmaceuticals. The metabolic bioactivation of drugs to chemically reactive metabolites (CRMs) contribute to liver-associated adverse drug reactions in humans that often goes undetected in conventional animal toxicology studies. A challenge for pharmaceutical drug discovery has been reliably selecting drug candidates with a low liability of forming CRM and reduced drug-induced liver injury potential, at projected therapeutic doses, without falsely restricting the development of safe drugs. We have developed an in vivo rat liver transcriptional signature biomarker reflecting the cellular response to drug bioactivation. Measurement of transcriptional activation of integrated nuclear factor erythroid 2-related factor 2 (NRF2)/Kelch-like ECH-associated protein 1 (KEAP1) electrophilic stress, and nuclear factor erythroid 2-related factor 1 (NRF1) proteasomal endoplasmic reticulum (ER) stress responses, is described for discerning estimated clinical doses of drugs with potential for bioactivation-mediated hepatotoxicity. The approach was established using well benchmarked CRM forming test agents from our company. This was subsequently tested using curated lists of commercial drugs and internal compounds, anchored in the clinical experience with human hepatotoxicity, while agnostic to mechanism. Based on results with 116 compounds in short-term rat studies, with consideration of the maximum recommended daily clinical dose, this CRM mechanism-based approach yielded 32% sensitivity and 92% specificity for discriminating safe from hepatotoxic drugs. The approach adds new information for guiding early candidate selection and informs structure activity relationships (SAR) thus enabling lead optimization and mechanistic problem solving. Additional refinement of the model is ongoing. Case examples are provided describing the strengths and limitations of the approach.

Recommendations for the Design of Clinical Drug-Drug Interaction Studies With Itraconazole Using a Mechanistic Physiologically-Based Pharmacokinetic Model.

Regulatory agencies currently recommend itraconazole (ITZ) as a strong cytochrome P450 3A (CYP3A) inhibitor for clinical drug-drug interaction (DDI) studies. This work by an International Consortium for Innovation and Quality in Pharmaceutical Development working group (WG) is to develop and verify a mechanistic ITZ physiologically-based pharmacokinetic model and provide recommendations for optimal DDI study design based on model simulations. To support model development and verification, in vitro and clinical PK data for ITZ and its metabolites were collected from WG member companies. The model predictions of ITZ DDIs with seven different CYP3A substrates were within the guest criteria for 92% of area under the concentration-time curve ratios and 95% of maximum plasma concentration ratios, thus verifying the model for DDI predictions. The verified model was used to simulate various clinical DDI study scenarios considering formulation, duration of dosing, dose regimen, and food status to recommend the optimal design for maximal inhibitory effect by ITZ.

Application of Physiologically Based Pharmacokinetic Modeling to Predict Pharmacokinetics in Healthy Japanese Subjects.

Pharmacokinetics (PKs) in Japanese healthy subjects were simulated for nine compounds using physiologically based PK (PBPK) models parameterized with physicochemical properties, preclinical absorption, distribution, metabolism, and excretion (ADME) data, and clinical PK data from non-Japanese subjects. For each dosing regimen, 100 virtual trials were simulated and predicted/observed ratios for peak plasma concentration (Cmax ) and area under the curve (AUC) were calculated. As qualification criteria, it was prespecified that >80% of simulated trials should demonstrate ratios to observed data ranging from 0.5-2.0. Across all compounds and dose regimens studied, 93% of simulated Cmax values in Japanese subjects fulfilled the criteria. Similarly, for AUC, 77% of single-dosing regimens and 100% of multiple-dosing regimens fulfilled the criteria. In summary, mechanistically incorporating the appropriate ADME properties into PBPK models, followed by qualification using non-Japanese clinical data, can predict PKs in the Japanese population and lead to efficient trial design and conduct of Japanese phase I studies.

Safety, Tolerability, and Pharmacokinetics of Suvorexant: A Randomized Rising-Dose Trial in Healthy Men.

BACKGROUND AND OBJECTIVES: Suvorexant (MK-4305) is an orexin receptor antagonist approved for the treatment of insomnia in the USA and other regions. This randomized, double-blind, placebo-controlled, sequential-panel, Phase 1 trial assessed the safety, tolerability, and pharmacokinetic data following single and multiple dosing of suvorexant in healthy men (aged 18-45 years). METHODS: Within allocated panels, subjects (n = 8) were randomized to receive nightly doses of suvorexant (10, 20, 40, 80, and 100 mg) administered orally for 14 days, or placebo. Safety assessments included daily adverse event (AE) monitoring; pharmacokinetic data were obtained through periodic sampling. RESULTS: Of 40 subjects randomized, 39 completed the trial. The incidence of any AEs in the 10 and 20 mg groups was 67 and 83%, respectively, while 100% of subjects reported AEs in the dose groups of 40, 80, and 100 mg and the placebo group. The most frequently reported AEs were somnolence (n = 19 subjects), fatigue (n = 17), and headache (n = 15). Following single and multiple dosing, median time to reach maximum observed concentration ranged from 1.5 to 4.0 h and the apparent terminal half-life ranged from 7.7 to 14.5 h. Across the investigated doses, accumulation ratios for the area under the concentration-time curve and the maximum observed concentration were independent of dose and ranged from 1.21 to 1.60 and 1.00 to 1.46, respectively. CONCLUSIONS: Suvorexant was generally well tolerated after single and multiple dosing for 14 days. The findings support the once-nightly dosing regimen.

Evaluation of Model-Based Prediction of Pharmacokinetics in the Renal Impairment Population.

Dose recommendations for specific populations are not always provided and, when available, typically rely on empirical derivation from a small fraction of the general population. In this study, a prediction/confirmation framework was applied to 2 model-based methods, physiologically based pharmacokinetics (PBPK) and a static model, to evaluate their ability to predict clearance in mild, moderate, and severe renal impairment populations and to inform dosing recommendations in these populations. Simulated renal impairment/healthy subject AUC ratios (AUCRs) from PBPK and static models were compared with observed AUCRs from dedicated clinical studies in renal impairment subjects for 7 drugs eliminated primarily by renal clearance. Both PBPK and static model predictions were within 2-fold of observed AUCRs for most compounds across all renal impairment categories. Predictions were generally more accurate for the mild and moderate renal impairment populations, with the majority of AUCR predictions within 80% to 125% of observed values for both methods. However, the accuracy of predictions was lower for the severe renal impairment population using the PBPK method. Given the accuracy observed, both methods may be suitable for prospective predictions for early decision-making, but are likely not sufficient sole justification for dose recommendations. There is a need to assess a larger database of compounds to enhance the predictive power of currently available tools.

The identification of a novel lead class for phosphodiesterase 2 inhibition by fragment-based drug design.

We have identified a novel PDE2 inhibitor series using fragment-based screening. Pyrazolopyrimidine fragment 1, while possessing weak potency (Ki = 22.4 µM), exhibited good binding efficiencies (LBE = 0.49, LLE = 4.48) to serve as a start for structure-based drug design. With the assistance of molecular modeling and X-ray crystallography, this fragment was developed into a series of potent PDE2 inhibitors with good physicochemical properties. Compound 16, a PDE2 selective inhibitor, was identified that exhibited favorable rat pharmacokinetic properties.

Design, synthesis and SAR of substituted indoles as selective TrkA inhibitors.

A series of substituted indoles were examined as selective inhibitors of tropomyosin-related kinase receptor A (TrkA), a therapeutic target for the treatment of pain. An SAR optimization campaign based on ALIS screening lead compound 1 is reported.

Investigation of orexin-2 selective receptor antagonists: Structural modifications resulting in dual orexin receptor antagonists.

In an ongoing effort to explore the use of orexin receptor antagonists for the treatment of insomnia, dual orexin receptor antagonists (DORAs) were structurally modified, resulting in compounds selective for the OX2R subtype and culminating in the discovery of 23, a highly potent, OX2R-selective molecule that exhibited a promising in vivo profile. Further structural modification led to an unexpected restoration of OX1R antagonism. Herein, these changes are discussed and a rationale for selectivity based on computational modeling is proposed.

Discovery of highly potent and selective orexin 1 receptor antagonists (1-SORAs) suitable for in vivo interrogation of orexin 1 receptor pharmacology.

While a correlation between blockade of the orexin 2 receptor (OX2R) with either a dual orexin receptor antagonist (DORA) or a selective orexin 2 receptor antagonist (2-SORA) and a decrease of wakefulness is well established, less is known about selective blockade of the orexin 1 receptor (OX1R). Therefore, a highly selective orexin 1 antagonist (1-SORA) with suitable properties to allow in vivo interrogation of OX1R specific pharmacology in preclinical species remains an attractive target. Herein, we describe the discovery of an optimized 1-SORA series in the piperidine ether class. Notably, a 4,4-difluoropiperidine core coupled with a 2-quinoline ether linkage provides OX1R selective compounds. The combination with an azabenzimidazole or imidazopyridine amide substituent leads to analogs 47 and 51 with >625-fold functional selectivity for OX1R over OX2R in rat. Compounds 47 and 51 possess clean off-target profiles and the required pharmacokinetic and physical properties to be useful as 1-SORA tool compounds.

In vitro and in vivo characterisation of the metabolism and disposition of suvorexant in humans.

1. Suvorexant (MK-4305, Belsomra®) is a first-in-class dual orexin receptor antagonist approved in the USA and Japan for the treatment of insomnia. The current studies describe suvorexant's absorption, disposition and potential for CYP-mediated drug interactions in humans. 2. Following single oral administration of [(14)C]suvorexant to healthy human subjects, 90% of the radioactivity was recovered (66% in faeces, 23% in urine), primarily as oxidative metabolites. 3. In plasma, suvorexant and M9 were predominant, accounting for 30 and 37% of the total radioactivity, respectively. Metabolite M17 became more prominent (approaching 10%) following multiple daily doses of unlabelled suvorexant. M9 and M17 are not expected to contribute to the pharmacological activity of suvorexant due to reduced orexin receptor binding affinity and limited brain penetration. 4. CYP3A was determined to be the predominant enzyme mediating suvorexant oxidation. In vitro, suvorexant demonstrated reversible inhibition of CYP3A4 and 2C19 (IC50 ∼ 4-5 µM), and weak time-dependent inhibition of CYP3A4 (KI = 12 µM, kinact = 0.14 min(-1)). Suvorexant was also a weak inducer of CYP3A4, 1A2 and 2B6. Given the low plasma concentrations at clinical doses, suvorexant was not anticipated to cause significant drug interactions via inhibition and/or induction of major CYPs in vivo.

Identification of MK-8133: An orexin-2 selective receptor antagonist with favorable development properties.

Antagonism of orexin receptors has shown clinical efficacy as a novel paradigm for the treatment of insomnia and related disorders. Herein, molecules related to the dual orexin receptor antagonist filorexant were transformed into compounds that were selective for the OX2R subtype. Judicious selection of the substituents on the pyridine ring and benzamide groups led to 6b; which was highly potent, OX2R selective, and exhibited excellent development properties.

Discovery of piperidine ethers as selective orexin receptor antagonists (SORAs) inspired by filorexant.

Highly selective orexin receptor antagonists (SORAs) of the orexin 2 receptor (OX2R) have become attractive targets both as potential therapeutics for insomnia as well as biological tools to help further elucidate the underlying pharmacology of the orexin signaling pathway. Herein, we describe the discovery of a novel piperidine ether 2-SORA class identified by systematic lead optimization beginning with filorexant, a dual orexin receptor antagonist (DORA) that recently completed Phase 2 clinical trials. Changes to the ether linkage and pendant heterocycle of filorexant were found to impart significant selectivity for OX2R, culminating in lead compound PE-6. PE-6 displays sub-nanomolar binding affinity and functional potency on OX2R while maintaining >1600-fold binding selectivity and >200-fold functional selectivity versus the orexin 1 receptor (OX1R). PE-6 bears a clean off-target profile, a good overall preclinical pharmacokinetic (PK) profile, and reduces wakefulness with increased NREM and REM sleep when evaluated in vivo in a rat sleep study. Importantly, subtle structural changes to the piperidine ether class impart dramatic changes in receptor selectivity. To this end, our laboratories have identified multiple piperidine ether 2-SORAs, 1-SORAs, and DORAs, providing access to a number of important biological tool compounds from a single structural class.

Discovery of diazepane amide DORAs and 2-SORAs enabled by exploration of isosteric quinazoline replacements.

Dual orexin receptor antagonists (DORAs), or orexin 1 (OX1) and orexin 2 (OX2) receptor antagonists, have demonstrated clinical utility for the treatment of insomnia. Medicinal chemistry efforts focused on the reduction of bioactivation potential of diazepane amide 1 through the modification of the Western heterocycle resulted in the discovery of suvorexant, a DORA recently approved by the FDA for the treatment of insomnia. A second strategy towards reducing bioactivation risk is presented herein through the exploration of monocyclic quinazoline isosteres, namely substituted pyrimidines. These studies afforded potent DORAs with significantly reduced bioactivation risk and efficacy in rodent sleep models. Surprisingly, side products from the chemistry used to produce these DORAs yielded isomeric pyrimidine-containing diazepane amides possessing selective OX2R antagonist (2-SORA) profiles. Additional exploration of these isomeric pyrimidines uncovered potent 2-SORA diazepane amides with sleep efficacy in mouse EEG studies.

Discovery of MK-3697: a selective orexin 2 receptor antagonist (2-SORA) for the treatment of insomnia.

Orexin receptor antagonists have demonstrated clinical utility for the treatment of insomnia. The majority of clinical efforts to date have focused on the development of dual orexin receptor antagonists (DORAs), small molecules that antagonize both the orexin 1 and orexin 2 receptors. Our group has recently disclosed medicinal chemistry efforts to identify highly potent, orally bioavailable selective orexin 2 receptor antagonists (2-SORAs) that possess acceptable profiles for clinical development. Herein we report additional SAR studies within the 'triaryl' amide 2-SORA series focused on improvements in compound stability in acidic media and time-dependent inhibition of CYP3A4. These studies resulted in the discovery of 2,5-disubstituted isonicotinamide 2-SORAs such as compound 24 that demonstrated improved stability and TDI profiles as well as excellent sleep efficacy across species.

Discovery of dual orexin receptor antagonists with rat sleep efficacy enabled by expansion of the acetonitrile-assisted/diphosgene-mediated 2,4-dichloropyrimidine synthesis.

Recent clinical studies have demonstrated that dual orexin receptor antagonists (OX1R and OX2R antagonists or DORAs) represent a novel treatment option for insomnia patients. Previously we have disclosed several compounds in the diazepane amide DORA series with excellent potency and both preclinical and clinical sleep efficacy. Additional SAR studies in this series were enabled by the expansion of the acetonitrile-assisted, diphosgene-mediated 2,4-dichloropyrimidine synthesis to novel substrates providing an array of Western heterocycles. These heterocycles were utilized to synthesize analogs in short order with high levels of potency on orexin 1 and orexin 2 receptors as well as in vivo sleep efficacy in the rat.