Comprehensive genotoxicity and carcinogenicity assessment of molnupiravir.

Molnupiravir is registered or authorized in several countries as a 5-day oral COVID-19 treatment for adults. Molnupiravir is a prodrug of the antiviral ribonucleoside ß-D-N4-hydroxycytidine (NHC) that distributes into cells, where it is phosphorylated to its pharmacologically active ribonucleoside triphosphate (NHC-TP) form. NHC-TP incorporates into SARS-CoV-2 RNA by the viral RNA-dependent RNA polymerase, resulting in an accumulation of errors in the viral genome, leading to inhibition of viral replication and loss of infectivity. The potential of molnupiravir to induce genomic mutations and DNA damage was comprehensively assessed in several in vitro and in vivo genotoxicity assays and a carcinogenicity study, in accordance with international guideline recommendations and expert opinion. Molnupiravir and NHC induced mutations in vitro in bacteria and mammalian cells but did not induce chromosome damage in in vitro or in vivo assays. The in vivo mutagenic and carcinogenic potential of molnupiravir was tested in a series of in vivo mutagenicity studies in somatic and germ cells (Pig-a Assay and Big Blue® TGR Mutation Assay) and in a carcinogenicity study (transgenic rasH2-Tg mouse), using durations of exposure and doses exceeding those used in clinical therapy. In vitro genotoxicity results are superseded by robustly conducted in vivo studies. Molnupiravir did not increase mutations in somatic or germ cells in the in vivo animal studies and was negative in the carcinogenicity study. The interpretation criteria for each study followed established regulatory guidelines. Taken together, these data indicate that molnupiravir use does not present a genotoxicity or carcinogenicity risk for patients.

Structure Guided Discovery of Novel Pan Metallo-ß-Lactamase Inhibitors with Improved Gram-Negative Bacterial Cell Penetration.

The use of ß-lactam (BL) and ß-lactamase inhibitor combination to overcome BL antibiotic resistance has been validated through clinically approved drug products. However, unmet medical needs still exist for the treatment of infections caused by Gram-negative (GN) bacteria expressing metallo-ß-lactamases. Previously, we reported our effort to discover pan inhibitors of three main families in this class: IMP, VIM, and NDM. Herein, we describe our work to improve the GN coverage spectrum in combination with imipenem and relebactam. This was achieved through structure- and property-based optimization to tackle the GN cell penetration and efflux challenges. A significant discovery was made that inhibition of both VIM alleles, VIM-1 and VIM-2, is essential for broad GN coverage, especially against VIM-producing P. aeruginosa. In addition, pharmacokinetics and nonclinical safety profiles were investigated for select compounds. Key findings from this drug discovery campaign laid the foundation for further lead optimization toward identification of preclinical candidates.

Early-Onset albuminuria and Associated Renal Pathology in Leucine-Rich Repeat Kinase 2 Knockout Rats.

Activating mutations of the leucine-rich repeat kinase 2 (LRRK2) gene are associated with Parkinson disease (PD), prompting development of LRRK2 inhibitors as potential treatment for PD. However, kidney safety concerns have surfaced from LRRK2 knockout (KO) mice and rats and from repeat-dose studies in rodents administered LRRK2 inhibitors. To support drug development of this therapeutic target, we conducted a study of 26 weeks' duration in 2-month-old wild-type and LRRK2 KO Long-Evans Hooded rats to systematically examine the performance of urinary safety biomarkers and to characterize the nature of the morphological changes in the kidneys by light microscopy and by ultrastructural evaluation. Our data reveal the time course of early-onset albuminuria at 3 and 4 months in LRRK2 KO female and male rats, respectively. The increases in urine albumin were not accompanied by concurrent increases in serum creatinine, blood urea nitrogen, or renal safety biomarkers such as kidney injury molecule 1 or clusterin, although morphological alterations in both glomerular and tubular structure were identified by light and transmission electron microscopy at 8 months of age. Diet optimization with controlled food intake attenuated the progression of albuminuria and associated renal changes.

Nonclinical Cardiovascular Assessment of the Soluble Guanylate Cyclase Stimulator Vericiguat.

Vericiguat and its metabolite M-1 were assessed for proarrhythmic risk in nonclinical in vitro and in vivo studies. In vitro manual voltage-clamp recordings at room temperature determined the effect of vericiguat on human Ether-a-go-go Related Gene (hERG) K+ channels. Effects of vericiguat and M-1 on hERG K+, Nav1.5, hCav1.2, hKvLQT1/1minK, and hKv4.3 channels were investigated via automated voltage-clamp recordings at ambient temperature. Effects of vericiguat and M-1 on hERG K+ and Nav1.5 channels at pathophysiological conditions were explored via manual voltage-clamp recordings at physiologic temperature. Single oral doses of vericiguat (0.6, 2.0, and 6.0 mg/kg) were assessed for in vivo proarrhythmic risk via administration to conscious telemetered dogs; electrocardiogram (ECG) and hemodynamic parameters were monitored. ECG recordings were included in 4- and 39-week dog toxicity studies. In manual voltage-clamp recordings, vericiguat inhibited hERG K+-mediated tail currents in a concentration-dependent manner (20% threshold inhibitory concentration ∼1.9 µM). In automated voltage-clamp recordings, neither vericiguat nor M-1 were associated with biologically relevant inhibition (>20%) of hNav1.5, hCav1.2, hKvLQT1, and hKv4.3. No clinically relevant observations were made for hNav1.5 and hKvLQT1 under simulated pathophysiological conditions. Vericiguat was associated with expected mode-of-action-related dose-dependent changes in systolic arterial blood pressure (up to -20%) and heart rate (up to +53%). At maximum vericiguat dose, corrected QT (QTc) interval changes from baseline varied slightly (-6 to +1%) depending on correction formula. Toxicity studies confirmed absence of significant QTc interval changes. There was no evidence of an increased proarrhythmic risk from nonclinical studies with vericiguat or M-1. SIGNIFICANCE STATEMENT: There was no evidence of an increased proarrhythmic risk from in vitro and in vivo nonclinical studies with vericiguat or M-1. The integrated risk assessment of these nonclinical data combined with existing clinical data demonstrate administration of vericiguat 10 mg once daily in patients with heart failure with reduced ejection fraction is not associated with a proarrhythmic risk.

Lead Optimization to Advance Protease-Activated Receptor-1 Antagonists in Early Discovery.

Vorapaxar is an approved drug for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. Subsequent to the discovery of Vorapaxar, medicinal chemistry efforts were continued to identify structurally differentiated leads. Toward this goal, extensive structure-activity relationship studies using a C-ring-truncated version of Vorapaxar culminated in the discovery of three leads, represented as 13, 14, and 23. Among these leads, compound 14 possessed favorable pharmacokinetic properties and an off-target profile, which supported additional profiling in an exploratory rat toxicology study.

Guiding Chemically Synthesized Peptide Drug Lead Optimization by Derisking Mast Cell Degranulation-Related Toxicities of a NaV1.7 Peptide Inhibitor.

Studies have shown that some peptides and small molecules can induce non IgE-mediated anaphylactoid reactions through mast cell activation. Upon activation, mast cells degranulate and release vasoactive and proinflammatory mediators, from cytoplasmic granules into the extracellular environment which can induce a cascade of severe adverse reactions. This study describes a lead optimization strategy to select NaV1.7 inhibitor peptides that minimize acute mast cell degranulation (MCD) toxicities. Various in vitro, in vivo, and PKPD models were used to screen candidates and guide peptide chemical modifications to mitigate this risk. Anesthetized rats dosed with peptides demonstrated treatment-related decreases in blood pressure and increases in plasma histamine concentrations which were reversible with a mast cell stabilizer, supporting the MCD mechanism. In vitro testing in rat mast cells with NaV1.7 peptides demonstrated a concentration-dependent increase in histamine. Pharmacodynamic modeling facilitated establishing an in vitro to in vivo correlation for histamine as a biomarker for blood pressure decline via the MCD mechanism. These models enabled assessment of structure-activity relationship (SAR) to identify substructures that contribute to peptide-mediated MCD. Peptides with hydrophobic and cationic characteristics were determined to have an elevated risk for MCD, which could be reduced or avoided by incorporating anionic residues into the protoxin II scaffold. Our analyses support that in vitro MCD assessment in combination with PKPD modeling can guide SAR to improve peptide lead optimization and ensure an acceptable early in vivo tolerability profile with reduced resources, cycle time, and animal use.

Development of ProTx-II Analogues as Highly Selective Peptide Blockers of Nav1.7 for the Treatment of Pain.

Inhibitor cystine knot peptides, derived from venom, have evolved to block ion channel function but are often toxic when dosed at pharmacologically relevant levels in vivo. The article describes the design of analogues of ProTx-II that safely display systemic in vivo blocking of Nav1.7, resulting in a latency of response to thermal stimuli in rodents. The new designs achieve a better in vivo profile by improving ion channel selectivity and limiting the ability of the peptides to cause mast cell degranulation. The design rationale, structural modeling, in vitro profiles, and rat tail flick outcomes are disclosed and discussed.

A Series of Novel, Highly Potent, and Orally Bioavailable Next-Generation Tricyclic Peptide PCSK9 Inhibitors.

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a key regulator of plasma LDL-cholesterol (LDL-C) and a clinically validated target for the treatment of hypercholesterolemia and coronary artery disease. Starting from second-generation lead structures such as 2, we were able to refine these structures to obtain extremely potent bi- and tricyclic PCSK9 inhibitor peptides. Optimized molecules such as 44 demonstrated sufficient oral bioavailability to maintain therapeutic levels in rats and cynomolgus monkeys after dosing with an enabled formulation. We demonstrated target engagement and LDL lowering in cynomolgus monkeys essentially identical to those observed with the clinically approved, parenterally dosed antibodies. These molecules represent the first report of highly potent and orally bioavailable macrocyclic peptide PCSK9 inhibitors with overall profiles favorable for potential development as once-daily oral lipid-lowering agents. In this manuscript, we detail the design criteria and multiparameter optimization of this novel series of PCSK9 inhibitors.

Invention of MK-8262, a Cholesteryl Ester Transfer Protein (CETP) Inhibitor Backup to Anacetrapib with Best-in-Class Properties.

Cholesteryl ester transfer protein (CETP) represents one of the key regulators of the homeostasis of lipid particles, including high-density lipoprotein (HDL) and low-density lipoprotein (LDL) particles. Epidemiological evidence correlates increased HDL and decreased LDL to coronary heart disease (CHD) risk reduction. This relationship is consistent with a clinical outcomes trial of a CETP inhibitor (anacetrapib) combined with standard of care (statin), which led to a 9% additional risk reduction compared to standard of care alone. We discuss here the discovery of MK-8262, a CETP inhibitor with the potential for being the best-in-class molecule. Novel in vitro and in vivo paradigms were integrated to drug discovery to guide optimization informed by a critical understanding of key clinical adverse effect profiles. We present preclinical and clinical evidence of MK-8262 safety and efficacy by means of HDL increase and LDL reduction as biomarkers for reduced CHD risk.

Series of Novel and Highly Potent Cyclic Peptide PCSK9 Inhibitors Derived from an mRNA Display Screen and Optimized via Structure-Based Design.

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a key regulator of plasma LDL-cholesterol (LDL-C) and a clinically validated target for the treatment of hypercholesterolemia and coronary artery disease. In this paper, we describe a series of novel cyclic peptides derived from an mRNA display screen which inhibit the protein-protein interaction between PCSK9 and LDLR. Using a structure-based drug design approach, we were able to modify our original screening lead 2 to optimize the potency and metabolic stability and minimize the molecular weight to provide novel bicyclic next-generation PCSK9 inhibitor peptides such as 78. These next-generation peptides serve as a critical foundation for continued exploration of potential oral, once-a-day PCSK9 therapeutics for the treatment of cardiovascular disease.