Orally Bioavailable Macrocyclic Peptide That Inhibits Binding of PCSK9 to the Low Density Lipoprotein Receptor.

BACKGROUND: Inhibition of PCSK9 (proprotein convertase subtilisin/kexin type 9)-low density lipoprotein receptor interaction with injectable monoclonal antibodies or small interfering RNA lowers plasma low density lipoprotein-cholesterol, but despite nearly 2 decades of effort, an oral inhibitor of PCSK9 is not available. Macrocyclic peptides represent a novel approach to target proteins traditionally considered intractable to small-molecule drug design. METHODS: Novel mRNA display screening technology was used to identify lead chemical matter, which was then optimized by applying structure-based drug design enabled by novel synthetic chemistry to identify macrocyclic peptide (MK-0616) with exquisite potency and selectivity for PCSK9. Following completion of nonclinical safety studies, MK-0616 was administered to healthy adult participants in a single rising-dose Phase 1 clinical trial designed to evaluate its safety, pharmacokinetics, and pharmacodynamics. In a multiple-dose trial in participants taking statins, MK-0616 was administered once daily for 14 days to characterize the safety, pharmacokinetics, and pharmacodynamics (change in low density lipoprotein cholesterol). RESULTS: MK-0616 displayed high affinity (Ki = 5pM) for PCSK9 in vitro and sufficient safety and oral bioavailability preclinically to enable advancement into the clinic. In Phase 1 clinical studies in healthy adults, single oral doses of MK-0616 were associated with >93% geometric mean reduction (95% CI, 84-103) of free, unbound plasma PCSK9; in participants on statin therapy, multiple-oral-dose regimens provided a maximum 61% geometric mean reduction (95% CI, 43-85) in low density lipoprotein cholesterol from baseline after 14 days of once-daily dosing of 20 mg MK-0616. CONCLUSIONS: This work validates the use of mRNA display technology for identification of novel oral therapeutic agents, exemplified by the identification of an oral PCSK9 inhibitor, which has the potential to be a highly effective cholesterol lowering therapy for patients in need.

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.

Generation of SARS-CoV-2 reporter replicon for high-throughput antiviral screening and testing.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and antiviral discovery are hampered by the lack of a cell-based virus replication system that can be readily adopted without biosafety level 3 (BSL-3) restrictions. Here, the construction of a noninfectious SARS-CoV-2 reporter replicon and its application in deciphering viral replication mechanisms and evaluating SARS-CoV-2 inhibitors are presented. The replicon genome is replication competent but does not produce progeny virions. Its replication can be inhibited by RdRp mutations or by known SARS-CoV-2 antiviral compounds. Using this system, a high-throughput antiviral assay has also been developed. Significant differences in potencies of several SARS-CoV-2 inhibitors in different cell lines were observed, which highlight the challenges of discovering antivirals capable of inhibiting viral replication in vivo and the importance of testing compounds in multiple cell culture models. The generation of a SARS-CoV-2 replicon provides a powerful platform to expand the global research effort to combat COVID-19.

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.

Polyethlyene Glycol 200 Can Protect Rats Against Drug-Induced Kidney Toxicity Through Inhibition of the Renal Organic Anion Transporter 3.

MK-7680, a cyclic nucleotide prodrug, caused significant kidney tubule injury in female rats when administered orally at 1000 mg/kg/day for 2 weeks using 10% Polysorbate 80 as vehicle. However, kidney injury was absent when MK-7680 was administered at the same dose regimen using 100% Polyethylene Glycol 200 (PEG 200) as the vehicle. Subsequent investigations revealed that MK-7680 triphosphate concentrations in kidney were much lower in rats treated with MK-7680 using PEG 200 compared with 10% Polysorbate 80 vehicle, whereas plasma exposures of MK-7680 prodrug were similar. In vitro studies demonstrated that PEG 200 is an inhibitor of human renal uptake transporter organic anion transporter 3 (OAT3), of which MK-7680 is a substrate. Furthermore, PEG 200 and PEG 400 were found to interfere in vitro with human renal transporters OAT3, organic cation transporter (OCT) 2, multidrug resistance-associated protein (MRP) 2 and 4, and multidrug and toxin extrusion protein (MATE) 1 and 2K, but not OAT1. These results support a conclusion that PEG 200 may prevent MK-7680-induced kidney injury by inhibiting its active uptake into proximal tubular cells by OAT3. Caution should be exercised therefore when using PEGs as vehicles for toxicity assessment for compounds that are substrates of renal transporters.

MK-7622: A First-in-Class M1 Positive Allosteric Modulator Development Candidate.

Identification of ligands that selectively activate the M1 muscarinic signaling pathway has been sought for decades to treat a range of neurological and cognitive disorders. Herein, we describe the optimization efforts focused on addressing key physicochemical and safety properties, ultimately leading to the clinical candidate MK-7622, a highly selective positive allosteric modulator of the M1 muscarinic receptor that has entered Phase II studies in patients with Alzheimer's disease.

siRNA-mediated knockdown of P450 oxidoreductase in rats: a tool to reduce metabolism by CYPs and increase exposure of high clearance compounds.

PURPOSE: To develop a tool based on siRNA-mediated knockdown of hepatic P450 oxidoreductase (POR) to decrease the CYP-mediated metabolism of small molecule drugs that suffer from rapid metabolism in vivo, with the aim of improving plasma exposure of these drugs. METHODS: siRNA against the POR gene was delivered using lipid nanoparticles (LNPs) into rats. The time course of POR mRNA knockdown, POR protein knockdown, and loss of POR enzyme activity was monitored. The rat livers were harvested to produce microsomes to determine the impact of POR knockdown on the metabolism of several probe substrates. Midazolam (a CYP3A substrate with high intrinsic clearance) was administered into LNP-treated rats to determine the impact of POR knockdown on midazolam pharmacokinetics. RESULTS: Hepatic POR mRNA and protein levels were significantly reduced by administering siRNA and the maximum POR enzyme activity reduction (~85%) occurred 2 weeks post-dose. In vitro analysis showed significant reductions in metabolism of probe substrates due to POR knockdown in liver, and in vivo POR knockdown resulted in greater than 10-fold increases in midazolam plasma concentrations following oral dosing. CONCLUSIONS: Anti-POR siRNA can be used to significantly reduce hepatic metabolism by various CYPs as well as greatly increase the bioavailability of high clearance compounds following an oral dose, thus enabling it to be used as a tool to increase drug exposure in vivo.

Discovery of naphthyl-fused 5-membered lactams as a new class of m1 positive allosteric modulators.

Selective activation of the M1 muscarinic receptor via positive allosteric modulation represents an original approach to treat the cognitive decline in patients with Alzheimer's disease. A series of naphthyl-fused 5-membered lactams were identified as a new class of M1 positive allosteric modulators and were found to possess good potency and in vivo efficacy.

Identification of a methoxynaphthalene scaffold as a core replacement in quinolizidinone amide M(1) positive allosteric modulators.

A series of methoxynaphthalene amides were prepared and evaluated as alternatives to quinolizidinone amide M1 positive allosteric modulators. A methoxy group was optimal for M1 activity and addressed key P-gp issues present in the aforementioned quinolizidinone amide series.

Identification of amides as carboxylic Acid surrogates for quinolizidinone-based m1 positive allosteric modulators.

Selective activation of the M1 muscarinic receptor via positive allosteric modulation represents an approach to treat the cognitive decline in patients with Alzheimer's disease. A series of amides were examined as a replacement for the carboxylic acid moiety in a class of quinolizidinone carboxylic acid M1 muscarinic receptor positive allosteric modulators, and leading pyran 4o and cyclohexane 5c were found to possess good potency and in vivo efficacy.

Discovery of a selective allosteric M1 receptor modulator with suitable development properties based on a quinolizidinone carboxylic acid scaffold.

One approach to ameliorate the cognitive decline in Alzheimer's disease (AD) has been to restore neuronal signaling from the basal forebrain cholinergic system via the activation of the M(1) muscarinic receptor. A number of nonselective M(1) muscarinic agonists have previously shown positive effects on cognitive behaviors in AD patients, but were limited due to cholinergic adverse events thought to be mediated by the activation of the M(2) to M(5) subtypes. One strategy to confer selectivity for M(1) is the identification of positive allosteric modulators, which would target an allosteric site on the M(1) receptor rather than the highly conserved orthosteric acetylcholine binding site. Quinoline carboxylic acids have been previously identified as highly selective M(1) positive allosteric modulators with good pharmacokinetic and in vivo properties. Herein is described the optimization of a novel quinolizidinone carboxylic acid scaffold with 4-cyanopiperidines being a key discovery in terms of enhanced activity. In particular, modulator 4i gave high plasma free fractions, enhanced central nervous system (CNS) exposure, was efficacious in a rodent in vivo model of cognition, and afforded good physicochemical properties suitable for further preclinical evaluation.

Quinolizidinone carboxylic acid selective M1 allosteric modulators: SAR in the piperidine series.

SAR study of the piperidine moiety in a series of quinolizidinone carboxylic acid M(1) positive allosteric modulators was examined. While the SAR was generally flat, compounds were identified with high CNS exposure to warrant additional in vivo evaluation.

Fused heterocyclic M1 positive allosteric modulators.

Fused aromatics such as naphthalene were identified as highly potent and CNS penetrant M(1) positive allosteric modulators during an SAR study to replace the phenyl B-ring linkage.

Heterocyclic fused pyridone carboxylic acid M(1) positive allosteric modulators.

The phenyl ring in a series of quinolone carboxylic acid M(1) positive allosteric modulators was replaced with a variety of heterocycles in order to reduce protein plasma binding and enhance CNS exposure.

Hydroxy cycloalkyl fused pyridone carboxylic acid M(1) positive allosteric modulators.

Incorporation of hydroxycycloalkyl fused pyridone carboxylic acids in lieu of quinolone carboxylic acids enhance free fraction without increased susceptibility to P-glycoprotein transport.

Quinolizidinone carboxylic acids as CNS penetrant, selective m1 allosteric muscarinic receptor modulators.

Positive allosteric modulation of the M1 muscarinic receptor represents an approach to treat the cognitive decline in patients with Alzheimer's disease. Replacement of a quinolone ring system in a quinolone carboxylic acid series of M1 modulators with a quinolizidinone bearing a basic amine linkage led to a series of compounds with higher free fraction, enhanced CNS exposure, and improved efficacy in rodent in vivo models of cognition.

Parallel synthesis of N-biaryl quinolone carboxylic acids as selective M(1) positive allosteric modulators.

An iterative analog library synthesis approach was employed in the exploration of a quinolone carboxylic acid series of selective M(1) positive allosteric modulators, and strategies for improving potency and plasma free fraction were identified.

Cyclic phosphoramidates as prodrugs of 2'-C-methylcytidine.

The currently approved treatment for hepatitis C virus infections is a combination of Ribavirin and pegylated Interferon. It leads to a sustained virologic response in approximately only half of the patients treated. For this reason there is an urgent need of new therapeutic agents. 2'-C-Methylcytidine is the first nucleoside inhibitor of the HCV NS5B polymerase that was efficacious in reducing the viral load in patients infected with HCV. The application of a monophosphate prodrug approach based on unprecedented cyclic phosphoramidates is reported. Our SAR studies led to compounds that are efficiently converted to the active triphosphate in human hepatocytes.

Comparison of drug distribution images from whole-body thin tissue sections obtained using desorption electrospray ionization tandem mass spectrometry and autoradiography.

Desorption electrospray ionization tandem mass spectrometry (DESI-MS/MS) and whole-body autoradiography (WBA) were used for chemical imaging of whole-body thin tissue sections of mice intravenously dosed with propranolol (7.5 mg/kg). DESI-MS/MS imaging utilized selected reaction monitoring detection performed on an AB/MDS SCIEX 4000 QTRAP mass spectrometer equipped with a prototype extended length particle discriminator interface. Propranolol images of the tissue sections using DESI-MS/MS were obtained at surface scan rates of 0.1, 0.5, 2, and 7 mm/s. Although signal decreased with increasing scan rate, useful whole-body images for propranolol were obtained from the tissues even at 7 mm/s, which required just 79 min of analysis time. Attempts to detect and image the distribution of the known propranolol metabolites were unsuccessful. Regions of the tissue sections showing the most radioactivity from WBA sections were excised and analyzed by high-performance liquid chromatography (HPLC) with radiochemical detection to determine relative levels of propranolol and metabolites present. Comparison of the DESI-MS/MS signal for propranolol and the radioactivity attributed to propranolol from WBA sections indicated nominal agreement between the two techniques for the amount of propranolol in the brain, lung, and liver. Data from the kidney showed an unexplained disparity between the two techniques. The results of this study show the feasibility of using DESI-MS/MS to obtain useful chemical images of a drug in whole-body thin tissue sections following drug administration at a pharmacologically relevant level. Further optimization to improve sensitivity and enable detection of the drug metabolites will be among the requirements necessary to move DESI-MS/MS chemical imaging forward as a practical tool in drug discovery.

Impact of pH on plasma protein binding in equilibrium dialysis.

Many pharmacokinetic analyses require unbound plasma concentrations, including prediction of clearance, volume of distribution, drug-drug interactions, brain uptake analysis, etc. It is most often more convenient to measure the total drug concentration in plasma rather than the unbound drug concentration. To arrive at unbound plasma concentrations, separate in vitro determinations of the plasma protein binding of a drug are usually carried out in serum or in plasma, and the plasma pharmacokinetic results are then mathematically adjusted by this fraction unbound ( f u,p). Plasma protein binding or the drug fraction unbound in plasma ( f u,p) is known to be affected by protein, drug, free fatty acid concentrations, lipoprotein partitioning, temperature, pH, and the presence or absence of other drugs/displacing agents within plasma samples. Errors in f u,p determination caused by lack of adequate pH control in newer assay formats for plasma protein binding (e.g., 96-well equilibrium thin walled polypropylene dialysis plates) will have significant drug-specific impact on these pharmacokinetic calculations. Using a diverse set of 55 drugs and a 96-well equilibrium dialysis plate format, the effect of variable pH during equilibrium dialysis experiments on measured values of f u,p was examined. Equilibrium dialysis of human plasma against Dulbecco's phosphate buffered saline at 37 degrees C under an air or 10% CO 2 atmosphere for 22 h resulted in a final pH of approximately 8.7 and 7.4, respectively. The ratio of f u,p at pH 7.4 (10% CO 2) vs pH 8.7 (air) was >or=2.0 for 40% of the 55 compounds tested. Only one of the 55 compounds tested had a ratio <0.9. Select compounds were further examined in rat and dog plasma. In addition, physicochemical properties were calculated for all compounds using ACD/Labs software or Merck in-house software and compared to plasma protein binding results. Changes in plasma protein binding due to pH increases which occurred during the equilibrium dialysis experiment were not species specific but were drug-specific, though nonpolar, cationic compounds had a higher likely hood of displaying pH-dependent binding. These studies underscore the importance of effectively controlling pH in plasma protein binding studies.