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.

The PET tracer [11C]MK-6884 quantifies M4 muscarinic receptor in rhesus monkeys and patients with Alzheimer's disease.

Positron emission tomography (PET) ligands play an important role in the development of therapeutics by serving as target engagement or pharmacodynamic biomarkers. Here, we describe the discovery and translation of the PET tracer [11C]MK-6884 from rhesus monkeys to patients with Alzheimer's disease (AD). [3H]MK-6884/[11C]MK-6884 binds with high binding affinity and good selectivity to an allosteric site on M4 muscarinic cholinergic receptors (M4Rs) in vitro and shows a regional distribution in the brain consistent with M4R localization in vivo. The tracer demonstrates target engagement of positive allosteric modulators of the M4R (M4 PAMs) through competitive binding interactions. [11C]MK-6884 binding is enhanced in vitro by the orthosteric M4R agonist carbachol and indirectly in vivo by the acetylcholinesterase inhibitor donepezil in rhesus monkeys and healthy volunteers, consistent with its pharmacology as a highly cooperative M4 PAM. PET imaging of [11C]MK-6884 in patients with AD identified substantial regional differences quantified as nondisplaceable binding potential (BPND) of [11C]MK-6884. These results suggest that [11C]MK-6884 is a useful target engagement biomarker for M4 PAMs but may also act as a sensitive probe of neuropathological changes in the brains of patients with AD.

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.

Discovery of [11C]MK-6884: A Positron Emission Tomography (PET) Imaging Agent for the Study of M4Muscarinic Receptor Positive Allosteric Modulators (PAMs) in Neurodegenerative Diseases.

The measurement of receptor occupancy (RO) using positron emission tomography (PET) has been instrumental in guiding discovery and development of CNS directed therapeutics. We and others have investigated muscarinic acetylcholine receptor 4 (M4) positive allosteric modulators (PAMs) for the treatment of symptoms associated with neuropsychiatric disorders. In this article, we describe the synthesis, in vitro, and in vivo characterization of a series of central pyridine-related M4 PAMs that can be conveniently radiolabeled with carbon-11 as PET tracers for the in vivo imaging of an allosteric binding site of the M4 receptor. We first demonstrated its feasibility by mapping the receptor distribution in mouse brain and confirming that a lead molecule 1 binds selectively to the receptor only in the presence of the orthosteric agonist carbachol. Through a competitive binding affinity assay and a number of physiochemical properties filters, several related compounds were identified as candidates for in vivo evaluation. These candidates were then radiolabeled with 11C and studied in vivo in rhesus monkeys. This research eventually led to the discovery of the clinical radiotracer candidate [11C]MK-6884.

Assessment of translational risk in drug research: Role of biomarker classification and mechanism-based PKPD concepts.

In 2005, Danhof and coauthors proposed a new biomarker classification in the context of the application of mechanism-based PKPD modeling. They defined the term 'biomarker' as a measure that characterizes a drug-induced response, which is on the causal path between drug administration and clinical outcome. The biomarker classification identified seven categories that provide different insights into the kinetics of drug action, such as target site distribution, target engagement, or into the impact of the drug on physiology or disease. The original biomarker classification has been further modified into a translational biomarker scheme that is used as a communication tool for drug hunting teams to guide designing translational and early clinical development plans as part of an integrated model-informed drug discovery and development strategy. It promotes a dedicated discussion on the topic of the translational relevance of biomarkers and enables efficient identification of translational gaps and opportunities. Based on the elucidated PKPD characteristics exhibited by a novel drug and the kinetics of the investigated biomarker, prospective predictions can be made for the drug response under new conditions; translating from the preclinical arena to the clinical setting, from the healthy volunteer to the patient, or from an adult to an elderly or a child. These drug response predictions provide support to decisions on appropriate next steps in the development of the drug, while keeping clear line of sight on the potential to address unmet medical need. Moreover, this framework enables a transparent translational risk assessment for drug hunting projects, and as such can underpin decisions at program and portfolio level.

Informing the Selection of Screening Hit Series with in Silico Absorption, Distribution, Metabolism, Excretion, and Toxicity Profiles.

High-throughput screening (HTS) has enabled millions of compounds to be assessed for biological activity, but challenges remain in the prioritization of hit series. While biological, absorption, distribution, metabolism, excretion, and toxicity (ADMET), purity, and structural data are routinely used to select chemical matter for further follow-up, the scarcity of historical ADMET data for screening hits limits our understanding of early hit compounds. Herein, we describe a process that utilizes a battery of in-house quantitative structure-activity relationship (QSAR) models to generate in silico ADMET profiles for hit series to enable more complete characterizations of HTS chemical matter. These profiles allow teams to quickly assess hit series for desirable ADMET properties or suspected liabilities that may require significant optimization. Accordingly, these in silico data can direct ADMET experimentation and profoundly impact the progression of hit series. Several prospective examples are presented to substantiate the value of this approach.

Optimizing DMPK Properties: Experiences from a Big Pharma DMPK Department.

BACKGROUND: The disposition of a drug is dependent on interactions between the body and the drug, its molecular properties and the physical and biological barriers presented in the body. In order for a drug to have a desired pharmacological effect it has to have the right properties to be able to reach the target site in sufficient concentration. This review details how drug metabolism and pharmacokinetics (DMPK) and physicochemical deliveries played an important role in data interpretation and compound optimization at AstraZeneca R&D in Södertälje, Sweden. METHODS: A selection of assays central in the evaluation of the DMPK properties of new chemical entities is presented, with guidance and consideration on assay outcome interpretation. Early in projects, solubility, LogD, permeability and metabolic stability were measured to support effective optimization of DMPK properties. Changes made to facilitate high throughput, efficient bioanalysis and the handling of large amounts of samples are described. Already early in drug discovery, we used an integrated approach for the prediction of the fate of drugs in human (early dose to man) based on data obtained from in vitro experiments. The early dose to man was refined with project progression, which triggered more intricate assays and experiments. At later stages, preclinical in vivo pharmacokinetic (PK) data was integrated with pharmacodynamics (PD) to allow predictions of required dose, dose intervals and exposure profile to achieve the desired effect in man. RESULTS AND CONCLUSIONS: A well-defined work flow of DMPK activities from early lead identification up to the selection of a candidate drug was developed. This resulted in a cost effective and efficient optimization of chemical series, and facilitated informed decision making throughout project progress.

Optimization of human dose prediction by using quantitative and translational pharmacology in drug discovery.

In this perspective article, we explain how quantitative and translational pharmacology, when well-implemented, is believed to lead to improved clinical candidates and drug targets that are differentiated from current treatment options. Quantitative and translational pharmacology aims to build and continuously improve the quantitative relationship between drug exposure, target engagement, efficacy, safety and its interspecies relationship at every phase of drug discovery. Drug hunters should consider and apply these concepts to develop compounds with a higher probability of interrogating the clinical biological hypothesis. We offer different approaches to set an initial effective concentration or pharmacokinetic-pharmacodynamic target in man and to predict human pharmacokinetics that determine together the predicted human dose and dose schedule. All concepts are illustrated with ample literature examples.

Revisiting the peripheral sink hypothesis: inhibiting BACE1 activity in the periphery does not alter ß-amyloid levels in the CNS.

Aggregation of amyloid beta (Aß) peptides and the subsequent neural plaque formation is a central aspect of Alzheimer's disease. Various strategies to reduce Aß load in the brain are therefore intensely pursued. It has been hypothesized that reducing Aß peptides in the periphery, that is in organs outside the brain, would be a way to diminish Aß levels and plaque load in the brain. In this report, we put this peripheral sink hypothesis to test by investigating how selective inhibition of Aß production in the periphery using a ß-secretase (BACE)1 inhibitor or reduced BACE1 gene dosage affects Aß load in the brain. Selective inhibition of peripheral BACE1 activity in wild-type mice or mice over-expressing amyloid precursor protein (APPswe transgenic mice; Tg2576) reduced Aß levels in the periphery but not in the brain, not even after chronic treatment over several months. In contrast, a BACE1 inhibitor with improved brain disposition reduced Aß levels in both brain and periphery already after acute dosing. Mice heterozygous for BACE1, displayed a 62% reduction in plasma Aß40, whereas brain Aß40 was only lowered by 11%. These data suggest that reduction of Aß in the periphery is not sufficient to reduce brain Aß levels and that BACE1 is not the rate-limiting enzyme for Aß processing in the brain. This provides evidence against the peripheral sink hypothesis and suggests that a decrease in Aß via BACE1 inhibition would need to be carried out in the brain. Aggregation of amyloid beta (Aß) peptides in the brain is a central aspect of Alzheimer's disease. In this study, we demonstrate that inhibition of Aß formation by BACE1 inhibitors needs to be carried out in the brain and that reduction of Aß in the periphery is not sufficient to reduce brain Aß levels. This information is useful for developing future Aß-targeting therapies for Alzheimer's disease.

Population pharmacokinetic and pharmacodynamic analysis of plasma Aß40 and Aß42 following single oral doses of the BACE1 inhibitor AZD3839 to healthy volunteers.

Modulating deposition of Aß-containing plaques in the brain may be beneficial in treating Alzheimer's disease. ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors have been shown to reduce Aß in plasma and CSF in healthy volunteers. In this study safety, pharmacokinetics and pharmacodynamics that is reduction of the plasma biomarkers Aß40 and Aß42 , of the BACE1 inhibitor AZD3839 were evaluated. Single oral ascending doses (1-300 mg) of AZD3839 were administered to 54 young healthy volunteers in a randomized, double-blind, placebo-controlled study. The data was analyzed using non-linear mixed effects modeling. AZD3839 reduced Aß40 and Aß42 in plasma with estimated potencies (EC50 ) of 46 and 59 nM, respectively, and a maximum effect of approximately 55%. This was in excellent agreement with the concentration-response relationships obtained in mouse and guinea pig. AZD3839 exposure displayed non-linear kinetics, described by a three-compartment model with a saturated binding compartment and an increase in bioavailability with dose. AZD3839 was safe, although, a dose-dependent QTcF prolongation was observed (mean 20 milliseconds at 300 mg). In conclusion, AZD3839 reduced plasma Aß40 and Aß42 , demonstrating clinical peripheral proof of mechanism. Pre-clinical models were predictive for the effect of AZD3839 on the human plasma biomarker in a strictly quantitative manner.

The virtue of translational PKPD modeling in drug discovery: selecting the right clinical candidate while sparing animal lives.

Translational pharmacokinetic-pharmacodynamic (PKPD) modeling has been fully implemented at AstraZeneca's drug discovery unit for central nervous system and pain indications to facilitate timely progression of the right compound to clinical studies, simultaneously assuring essential preclinical efficacy and safety knowledge. This review illustrates the impact of a translational PKPD paradigm with examples from drug discovery programs. Paradoxically, laboratory animal use decreased owing to better understanding of in vitro-in vivo relationships, optimized in vivo study designs, meta-analyses and hypothesis testing using simulations. From an ethical and effectivity perspective, we advocate that translational PKPD approaches should be implemented more broadly in drug discovery.

Biotransformation of two ß-secretase inhibitors including ring opening and contraction of a pyrimidine ring.

Recently, the discovery of the aminoisoindoles as potent and selective inhibitors of ß-secretase was reported, including the close structural analogs compound (S)-1-pyridin-4-yl-4-fluoro-1-(3-(pyrimidin-5-yl)phenyl)-1H-isoindol-3-amine [(S)-25] and (S)-1-(2-(difluoromethyl)pyridin-4-yl)-4-fluoro-1-(3-(pyrimidin-5-yl)phenyl)-1H-isoindol-3-amine hemifumarate (AZD3839), the latter being recently progressed to the clinic. The biotransformation of (S)-25 was investigated in vitro and in vivo in rat, rabbit, and human and compared with AZD3839 to further understand the metabolic fate of these compounds. In vitro, CYP3A4 was the major responsible enzyme and metabolized both compounds to a large extent in the commonly shared pyridine and pyrimidine rings. The main proposed metabolic pathways in various in vitro systems were N-oxidation of the pyridine and/or pyrimidine ring and conversion to 4-pyrimidone and pyrimidine-2,4-dione. Both compounds were extensively metabolized, and more than 90% was excreted in feces after intravenous administration of radiolabeled compound to the rat. Here, the main pathways were N-oxidation of the pyridine and/or pyrimidine ring and a ring contraction of the pyrimidine ring into an imidazole ring. Ring-contracted metabolites accounted for 25% of the total metabolism in the rat for (S)-25, whereas the contribution was much smaller for AZD3839. This metabolic pathway was not foreseen on the basis of the obtained in vitro data. In conclusion, we discovered an unusual metabolic pathway of aryl-pyrimidine-containing compounds by a ring-opening reaction followed by elimination of a carbon atom and a ring closure to form an imidazole ring.

In vivo and ex vivo inhibition of spinal nerve ligation-induced ectopic activity by sodium channel blockers correlate to in vitro inhibition of NaV1.7 and clinical efficacy: a pharmacokinetic-pharmacodynamic translational approach.

PURPOSE: In vivo and ex vivo inhibition of ectopic activity of clinically used and newly developed sodium channel (NaV) blockers were quantified in the rat spinal nerve ligation (SNL) model using a pharmacokinetic-pharmacodynamic (PKPD) approach and correlated to in vitro NaV1.7 channel inhibition and clinical effective concentrations. METHODS: In vivo, drug exposure and inhibition of ectopic activity were assessed in anaesthetized SNL rats at two dose levels. Ex vivo, compounds were applied at increasing concentrations to dorsal root ganglias isolated from SNL rats. The inhibitory potency (IC 50 ) was estimated using PKPD analysis. In vitro IC 50 was estimated using an electrophysiology-based assay using recombinant rat and human NaV1.7 expressing HEK293 cells. RESULTS: In vivo and ex vivo inhibition of ectopic activity correlated well with the in vitro inhibition on the rat NaV1.7 channel. The estimated IC 50s for inhibition of ectopic activity in the SNL model occurred at similar unbound concentrations as clinical effective concentrations in humans. CONCLUSIONS: Inhibition of ectopic activity in the SNL model could be useful in predicting clinical effective concentrations for novel sodium channel blockers. In addition, in vitro potency could be used for screening, characterization and selection of compounds, thereby reducing the need for in vivo testing.

Rat poorly predicts the combined non-absorbed and presystemically metabolized fractions in the human.

1. Intestinal loss, 1 - (Fobs/fH), is the missing fraction of the dose that is unexplained by systemic clearance. Here, we investigated whether intestinal loss in rat is predictive for human, and whether intestinal metabolism explained observed differences between rat and human. 2. For 81 marketed drugs, human and rat intestinal loss values were calculated from the literature and in-house sources. To examine the contribution of intestinal cytochrome P450-mediated metabolism to the high observed intestinal loss in the rat, metabolism was determined in rat and human intestinal microsomes for 15 compounds. 3. Oral bioavailability poorly correlated between rat and human. Twenty-two compounds in the human and 47 compounds in the rat showed an intestinal loss of more than 20%. The intestinal availability for many compounds was higher in human than in rat. Selected compounds, however, were more stable in rat than in human intestinal microsomes. 4. The rat poorly predicts the risk for intestinal loss in human; many compounds in rat had lower bioavailability than anticipated based on the hepatic clearance, but demonstrated little intestinal loss in human. This discrepancy appeared not to be caused by a higher cytochrome P450-mediated intestinal metabolism in the rat.

Presystemic metabolism of AZ'0908, a novel mPGES-1 inhibitor: an in vitro and in vivo cross-species comparison.

AZ'0908 is a novel microsomal prostaglandin E synthase-1 inhibitor intended for oral administration. Pharmacokinetic experiments in rats showed that bioavailability was much lower than anticipated and increased following pretreatment with the nonspecific cytochrome P450 (CYP) inhibitor 1-aminobenzotriazole, presumably by inhibition of intestinal metabolism. Stability experiments in rat liver and intestinal fractions revealed that the intrinsic clearance (Cl(int)) was much higher in intestinal than in liver microsomes. Caco2 experiments showed that AZ'0908 was a substrate for breast cancer resistance protein. Permeability was generally high and the efflux component was saturable predicting good absorption. The Cl(int) values in human intestinal microsome and S9 fractions were low. A correlation occurred between in vitro intestinal metabolism and in vivo intestinal loss in rats and dogs. Enzyme identification experiments showed that human CYP2J2 was involved in the oxidation of AZ'0908. In rats, the major metabolic enzyme was not identified. However, rat CYP2J2 analogs were not investigated. Intestinal metabolism appeared to be a major occurrence, explaining intestinal loss of AZ'0908 in the rats. In view of good overall permeability, low in vitro intestinal turnover, and relative low intestinal abundance of CYP2J2, we predict that intestinal metabolism of AZ'0908 in human does not exert a major issue.

Effect of solvents on the time-dependent inhibition of CYP3A4 and the biotransformation of AZD3839 in human liver microsomes and hepatocytes.

Time-dependent inhibition (TDI) of the cytochrome P450 (P450) family of enzymes is usually studied in human liver microsomes (HLM) by investigating whether the inhibitory potency is increased with increased incubation times. The presented work was initiated after a discrepancy was observed for the TDI of an important P450 enzyme, CYP3A4, during early studies of the investigational drug compound AZD3839 [(S)-1-(2-(difluoromethyl)pyridin-4-yl)-4-fluoro-1-(3-(pyrimidin-5-yl)phenyl)-1H-isoindol-3-amine hemifumarate]; TDI was detected using a regulatory method but not with an early screening method. We show here that the different solvents present in the respective studies, dimethyl sulfoxide (DMSO, screening method) versus methanol or water (regulatory method), were responsible for the different TDI results. We further demonstrate why DMSO, present at the levels of 0.2% and 0.5% in the incubations, masked the TDI effect. In addition to the TDI experiments performed in HLM, TDI studies with AZD3839 were performed in pooled human hepatocytes (Hhep) from different suppliers, using DMSO, methanol, or water. The results from these experiments show no TDI or attenuated TDI effect, depending on the supplier. Metabolite identification of the compound dissolved in DMSO, methanol, or water shows different profiles after incubations with the different systems (HLM or Hhep), which may explain the differences in the TDI outcomes. Thorough investigations of the biotransformation of AZD3839 have been performed to find the reactive pathway causing the TDI of CYP3A4, and are presented here. Our findings show that the in vitro risk profile for drug-drug interactions potential of AZD3839 is very much dependent on the chosen test system and the experimental conditions used.

Discovery of AZD3839, a potent and selective BACE1 inhibitor clinical candidate for the treatment of Alzheimer disease.

ß-Site amyloid precursor protein cleaving enzyme1 (BACE1) is one of the key enzymes involved in the processing of the amyloid precursor protein (APP) and formation of amyloid ß peptide (Aß) species. Because cerebral deposition of Aß species might be critical for the pathogenesis of Alzheimer disease, BACE1 has emerged as a key target for the treatment of this disease. Here, we report the discovery and comprehensive preclinical characterization of AZD3839, a potent and selective inhibitor of human BACE1. AZD3839 was identified using fragment-based screening and structure-based design. In a concentration-dependent manner, AZD3839 inhibited BACE1 activity in a biochemical fluorescence resonance energy transfer (FRET) assay, Aß and sAPPß release from modified and wild-type human SH-SY5Y cells and mouse N2A cells as well as from mouse and guinea pig primary cortical neurons. Selectivity against BACE2 and cathepsin D was 14 and >1000-fold, respectively. AZD3839 exhibited dose- and time-dependent lowering of plasma, brain, and cerebrospinal fluid Aß levels in mouse, guinea pig, and non-human primate. Pharmacokinetic/pharmacodynamic analyses of mouse and guinea pig data showed a good correlation between the potency of AZD3839 in primary cortical neurons and in vivo brain effects. These results suggest that AZD3839 effectively reduces the levels of Aß in brain, CSF, and plasma in several preclinical species. It might, therefore, have disease-modifying potential in the treatment of Alzheimer disease and related dementias. Based on the overall pharmacological profile and its drug like properties, AZD3839 has been progressed into Phase 1 clinical trials in man.

Phenethyl nicotinamides, a novel class of Na(V)1.7 channel blockers: structure and activity relationship.

The Na(V)1.7 ion channel is an attractive target for development of potential analgesic drugs based on strong genetic links between mutations in the gene coding for the channel protein and inheritable pain conditions. The (S)-N-chroman-3-ylcarboxamide series, exemplified by 1, was used as a starting point for development of new channel blockers, resulting in the phenethyl nicotinamide series. The structure and activity relationship for this series was established and the metabolic issues of early analogues were addressed by appropriate substitutions. Compound 33 displayed acceptable overall in vitro properties and in vivo rat PK profile.