John Reilly

Novel zwitterionic polyphosphorylcholine monolithic column for hydrophilic interaction chromatography

A novel porous zwitterionic monolith was prepared by thermal co-polymerisation of 2-methacryloyloxyethyl phosphorylcholine (MPC) and ethylene glycol dimethacrylate (EDMA) within 100 microm I.D. capillaries. Mercury intrusion porosimetry, scanning electron microscopy (SEM), micro-HPLC (micro-HPLC), elemental analysis and zeta-potential analysis were used to evaluate the monolithic structure. No evidence of swelling or shrinking of the monolith in different polarity solvents was observed. A typical hydrophilic liquid chromatography (HILIC) mechanism was observed at high organic solvent content (acetonitrile >60%). The phosphorylcholine (PC) functionality has both a positively charged quaternary ammonium and a negatively charged phosphate group. For charged analytes, a weak electrostatic interaction was also observed by studying the influence of mobile phase pH and salt concentration on their retentions on the poly(MPC-co-EDMA) monolithic column. The optimised poly(MPC-co-EDMA) monolith showed very good selectivities for a range of polar test analytes, especially small peptides. This might be ascribed to the good biocompatibility of PC functionality. At low organic solvent content, baseline separation was also observed for a test mixture of seven alkylphenones by a reversed-phase separation mechanism.

Observed Drug-Receptor Association Rates Are Governed by Membrane Affinity: The Importance of Establishing “Micro-Pharmacokinetic/Pharmacodynamic Relationships” at the β2-Adrenoceptor

Current pharmacological models for determining affinity and kinetics of drugs for membrane receptors assume the interacting molecules are homogeneously distributed in the bulk aqueous phase. The phospholipid membrane can, however, provide a second compartment into which drugs can partition, particularly lipophilic/basic compounds. In this study we measured the phospholipid affinity and receptor binding kinetics of several clinically relevant β2-adrenoceptor agonists and antagonists and demonstrated that the degree of phospholipid interaction directly affects the observed kinetic association rate (kon) and dissociation constant (Kd), but not the dissociation rate (koff) from the target, by concentrating drug in the local environment around the receptor. When the local drug concentration was accounted for, the kon was comparable across the cohort and the corrected Kd was directly related to the koff. In conclusion, we propose a new approach to determining the pharmacology of drugs for membrane targets that accounts for differences in local drug concentration brought about by direct affinity for phospholipids, establishing “micro-pharmacokinetic/pharmacodynamic relationships” for drugs.

Discovery of a 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one (MK-2461) inhibitor of c-Met kinase for the treatment of cancer.

c-Met is a transmembrane tyrosine kinase that mediates activation of several signaling pathways implicated in aggressive cancer phenotypes. In recent years, research into this area has highlighted c-Met as an attractive cancer drug target, triggering a number of approaches to disrupt aberrant c-Met signaling. Screening efforts identified a unique class of 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one kinase inhibitors, exemplified by 1. Subsequent SAR studies led to the development of 81 (MK-2461), a potent inhibitor of c-Met that was efficacious in preclinical animal models of tumor suppression. In addition, biochemical studies and X-ray analysis have revealed that this unique class of kinase inhibitors binds preferentially to the activated (phosphorylated) form of the kinase. This report details the development of 81 and provides a description of its unique biochemical properties.

Observed drug-receptor association rates are governed by membrane affinity: The importance of establishing "micro PK/PD relationships" at the β2-adrenoceptor

Current pharmacological models for determining affinity and kinetics of drugs for membrane receptors assume the interacting molecules are homogeneously distributed in the bulk aqueous phase. The phospholipid membrane can, however, provide a second compartment into which drugs can partition, particularly lipophilic/basic compounds. In this study we measured the phospholipid affinity and receptor binding kinetics of several clinically relevant β2-adrenoceptor agonists and antagonists and demonstrated that the degree of phospholipid interaction directly affects the observed kinetic association rate (kon) and dissociation constant (Kd), but not the dissociation rate (koff) from the target, by concentrating drug in the local environment around the receptor. When the local drug concentration was accounted for, the kon was comparable across the cohort and the corrected Kd was directly related to the koff. In conclusion, we propose a new approach to determining the pharmacology of drugs for membrane targets that accounts for differences in local drug concentration brought about by direct affinity for phospholipids, establishing “micro-pharmacokinetic/pharmacodynamic relationships” for drugs.

HandaPhos: A General Ligand Enabling Sustainable ppm Levels of Palladium‐catalyzed Cross‐Couplings in Water at Room Temperature

The new monophosphine ligand HandaPhos has been identified such that when complexed in a 1:1 ratio with Pd(OAc)2, enables Pd-catalyzed cross-couplings to be run using ≤1000 ppm of this pre-catalyst. Applications to Suzuki-Miyaura reactions involving highly funtionalized reaction partners are demonstrated, all run using environmentally benign nanoreactors in water at ambient temperatures. Comparisons with existing state-of-the-art ligands and catalysts are discussed herein.

7-Azaindole-3-acetic acid derivatives: potent and selective CRTh2 receptor antagonists.

High throughput screening identified a 7-azaindole-3-acetic acid scaffold as a novel CRTh2 receptor antagonist chemotype, which could be optimised to furnish a highly selective compound with good functional potency for inhibition of human eosinophil shape change in whole blood and oral bioavailability in the rat.

A rapid vesicle electrokinetic chromatography method for the in vitro prediction of non-specific binding for potential PET ligands

High non-specific binding (NSB) is one of the most common reasons for candidate failure in potential positron emission tomography (PET) radiotracer development. It is of interest to develop high throughput in vitro methods for predicting non-specific binding prior to radiolabeling, which would help guide radiotracer candidate selection and assist decision making in new radiotracer discovery. We evaluated several electrokinetic chromatographic (EKC) systems to help identify PET ligands with low non-specific binding characteristics by mimicking the ligand-brain tissue interaction. The measured retention factors of tracers in clinical use or terminated candidates within AOT vesicle EKC systems were compared with literature in vitro or in vivo NSB data. We conclude that there is a statistical correlation between the chromatographic retention parameters of tested drugs and their NSB. The AOT vesicle EKC method can provide NSB in vitro trend analysis for a large number of drug candidates early in the novel radiotracer discovery process with minimal resources.

Fevipiprant (QAW039), a Slowly Dissociating CRTh2 Antagonist with the Potential for Improved Clinical Efficacy

Here we describe the pharmacologic properties of a series of clinically relevant chemoattractant receptor-homologous molecules expressed on T-helper type 2 (CRTh2) receptor antagonists, including fevipiprant (NVP-QAW039 or QAW039), which is currently in development for the treatment of allergic diseases. [3H]-QAW039 displayed high affinity for the human CRTh2 receptor (1.14 ± 0.44 nM) expressed in Chinese hamster ovary cells, the binding being reversible and competitive with the native agonist prostaglandin D2 (PGD2). The binding kinetics of QAW039 determined directly using [3H]-QAW039 revealed mean kinetic on (kon) and off (koff) values for QAW039 of 4.5 × 107 M-1min−1 and 0.048 minute−1, respectively. Importantly, the koff of QAW039 (half-life = 14.4 minutes) was >7-fold slower than the slowest reference compound tested, AZD-1981. In functional studies, QAW039 behaved as an insurmountable antagonist of PGD2-stimulated [35S]-GTPγS activation, and its effects were not fully reversed by increasing concentrations of PGD2 after an initial 15-minute incubation period. This behavior is consistent with its relatively slow dissociation from the human CRTh2 receptor. In contrast for the other ligands tested this time-dependent effect on maximal stimulation was fully reversed by the 15-minute time point, whereas QAW039’s effects persisted for >180 minutes. All CRTh2 antagonists tested inhibited PGD2-stimulated human eosinophil shape change, but importantly QAW039 retained its potency in the whole-blood shape-change assay relative to the isolated shape change assay, potentially reflective of its relatively slower off rate from the CRTh2 receptor. QAW039 was also a potent inhibitor of PGD2-induced cytokine release in human Th2 cells. Slow CRTh2 antagonist dissociation could provide increased receptor coverage in the face of pathologic PGD2 concentrations, which may be clinically relevant.

Chromatography approaches for early screening of the phospholipidosis-inducing potential of pharmaceuticals

Drug-induced phospholipidosis (PLD) is an excessive accumulation of polar phospholipids within cells as a result of medical treatment. Even though a direct link between in vitro drug-induced PLD and toxicity in humans has not yet been firmly established, the development of PLD during preclinical testing in animals is a recognized problem in the pharmaceutical industry and can delay or abort the development process. Therefore, it is of interest to investigate the potential PLD risk of candidates at an early stage of the drug discovery process. In this work, a high throughput physicochemical approach, which is based on measuring the retention factors of the test compound within several different separation systems, was developed for screening phospholipidosis-inducing potential (PLIP) of pharmaceuticals. The measured retention factors of 36 drugs were compared with literature data on PLIP risk from three different sources. It is clearly shown that there is a statistical correlation between the chromatographic retention parameters of tested drugs and their PLIP risk. In conclusion, the retention factor (logk(AOT)) observed on a docusate sodium salt (AOT) surfactant vesicle electrokinetic chromatography (EKC) system and the logarithm of the volume of distribution (logV(d)) calculated from immobilized artificial membrane chromatography at pH 7.4 (CHI IAM(7.4)) and HSA binding value (% HSA) can provide primary profile prediction for a large number of drug candidates early in the drug discovery process with minimal resources. The observations are that the higher the value of both logk(AOT) and logV(d), the higher the PLIP risk, and we would recommend this dual approach.

The discovery of potent, orally bioavailable pyrimidine-5-carbonitrile-6-alkyl CXCR2 receptor antagonists.

A hit-to-lead optimisation programme was carried out on the Novartis archive screening hit, pyrimidine 2-((2,6-dichlorobenzyl)thio)-5-isocyano-6-phenylpyrimidin-4-ol 4, resulting in the discovery of CXCR2 receptor antagonist 2-((2,3-difluorobenzyl)thio)-6-(2-(hydroxymethyl)cyclopropyl)-5-isocyanopyrimidin-4-ol 24. The SAR was investigated by systematic variation of the aromatic group at c-6, the linker between c-2 and the halogenated ring, and the c-5 nitrile moiety.