Biotransformation of the Novel Myeloperoxidase Inhibitor AZD4831 in Preclinical Species and Humans.

We report herein an in-depth analysis of the metabolism of the novel myeloperoxidase inhibitor AZD4831 ((R)-1-(2-(1-aminoethyl)-4-chlorobenzyl)-2-thioxo-2,3-dihydro-1H-pyrrolo[3,2-d]pyrimidin-4(5H)-one) in animals and human. Quantitative and qualitative metabolite profiling were performed on samples collected from mass balance studies in rats and humans. Exposure of circulating human metabolites with comparable levels in animal species used in safety assessment were also included. Structural characterization of 20 metabolites was performed by liquid chromatography high-resolution mass spectrometry, and quantification was performed by either 14C analysis using solid phase scintillation counting or accelerator mass spectrometry and, where available, authentication with synthesized metabolite standards. A complete mass balance study in rats is presented, while data from dogs and human are limited to metabolite profiling and characterization. The metabolism of AZD4831 is mainly comprised of reactions at the primary amine nitrogen and the thiourea sulfur, resulting in several conjugated metabolites with or without desulfurization. A carbamoyl glucuronide metabolite of AZD4831 (M7) was the most abundant plasma metabolite in both human healthy volunteers and heart failure patients after single and repeated dose administration of AZD4831, accounting for 75%-80% of the total drug-related exposure. Exposures to M7 and other human circulating metabolites were covered in rats and/or dogs, the two models most frequently used in the toxicology studies, and were also highly abundant in the mouse, the second model other than rat used in carcinogenicity studies. The carbamoyl glucuronide M7 was the main metabolite in rat bile, while a desulfurized and cyclized metabolite (M5) was abundant in rat plasma and excreta. SIGNIFICANCE STATEMENT: The biotransformation of AZD4831, a novel myeloperoxidase inhibitor inhibiting xanthine derivative bearing thiourea and primary aliphatic amine functions, is described. Twenty characterized metabolites demonstrate the involvement of carbamoylation with glucuronidation, desulfurization, and cyclization as main biotransformation reactions. The carbamoyl glucuronide was the main metabolite in human plasma, likely governed by a significant species difference in plasma protein binding for this metabolite, but this and other human plasma metabolites were covered in animals used in the toxicity studies.

Ancestral Sequence Reconstruction of a Cytochrome P450 Family Involved in Chemical Defense Reveals the Functional Evolution of a Promiscuous, Xenobiotic-Metabolizing Enzyme in Vertebrates.

The cytochrome P450 family 1 enzymes (CYP1s) are a diverse family of hemoprotein monooxygenases, which metabolize many xenobiotics including numerous environmental carcinogens. However, their historical function and evolution remain largely unstudied. Here we investigate CYP1 evolution via the reconstruction and characterization of the vertebrate CYP1 ancestors. Younger ancestors and extant forms generally demonstrated higher activity toward typical CYP1 xenobiotic and steroid substrates than older ancestors, suggesting significant diversification away from the original CYP1 function. Caffeine metabolism appears to be a recently evolved trait of the CYP1A subfamily, observed in the mammalian CYP1A lineage, and may parallel the recent evolution of caffeine synthesis in multiple separate plant species. Likewise, the aryl hydrocarbon receptor agonist, 6-formylindolo[3,2-b]carbazole (FICZ) was metabolized to a greater extent by certain younger ancestors and extant forms, suggesting that activity toward FICZ increased in specific CYP1 evolutionary branches, a process that may have occurred in parallel to the exploitation of land where UV-exposure was higher than in aquatic environments. As observed with previous reconstructions of P450 enzymes, thermostability correlated with evolutionary age; the oldest ancestor was up to 35 °C more thermostable than the extant forms, with a 10T50 (temperature at which 50% of the hemoprotein remains intact after 10 min) of 71 °C. This robustness may have facilitated evolutionary diversification of the CYP1s by buffering the destabilizing effects of mutations that conferred novel functions, a phenomenon which may also be useful in exploiting the catalytic versatility of these ancestral enzymes for commercial application as biocatalysts.

Developing an injectable co-formulation of two antidiabetic drugs: Excipient impact on peptide aggregation and pharmacokinetic properties.

Combination therapy in Type 2 Diabetes Mellitus is necessary to achieve tight glycaemic control and reduce complication risk. Current treatment plans require patients to take several drugs concomitantly leading to low therapy adherence. This study describes the development and characterisation of a stable parenteral co-formulation of a sodium glucose co-transporter 2 inhibitor (dapagliflozin) and a therapeutic lipidated peptide, using hydroxypropyl-ß-cyclodextrin as an enabling excipient. Using NMR, calorimetry, computational modelling and spectroscopic methods, we show that besides increasing the solubility of dapagliflozin, cyclodextrin prevents self-association of the peptide through interaction with the lipid chain and amino acids prone to aggregation including aromatic groups and ionisable residues. While those interactions cause a dramatic secondary structure change, no impact on potency was seen in vitro. A subcutaneous administration of the co-formulation in rat showed that both drugs reach exposure levels previously shown to be efficacious in clinical mono-therapy studies. Interestingly, a faster absorption rate was observed for the peptide formulated within the cyclodextrin vehicle with respect to the buffer vehicle, which could trigger an earlier onset of action. The cyclodextrin based co-formulation is therefore a promising approach to develop a fixed dose combination of a therapeutic peptide and a small molecule drug for increased patient adherence and better blood glucose control.

Quantification of unbound concentration of ticagrelor in plasma as a proof of mechanism biomarker of the reversal agent, MEDI2452.

Ticagrelor, a P2Y12 antagonist, is approved for prevention of thromboembolic events. MEDI2452 is a potential reversal agent for ticagrelor and ticagrelor active metabolite (TAM). The total plasma exposure of ticagrelor and TAM in patients are roughly 0.5-1 and 0.2-0.5 µmol/L, respectively. Both have similar high potency vs. P2Y12 (Ki 2 nmol/L) but are plasma protein-bound to 99.8% and only the 0.2% free fraction is able to inhibit the P2Y12 receptor. Thus, for unbound concentration measurements to be a proof of mechanism biomarker for MEDI2452 a very high sensitivity is required. Using established techniques as equilibrium dialysis and LC-MS/MS, made it possible to evaluate the efficacy of the reversal agent by measuring reduction of unbound concentration of ticagrelor in the presence of MEDI2452. With challenges such as ultra-low concentrations, small sample volumes, recovery issues and adsorption to plastic we managed to develop a highly sensitive assay for determining unbound concentration levels of ticagrelor and TAM in plasma with a quantification limit of 30 pmol/L and 45 pmol/L, respectively. With this method we were able to detect close to a 100-fold MEDI2452 mediated reduction in the unbound concentration of both ticagrelor and TAM. The assay provided proof of mechanism as MEDI2452 concentration- and dose-dependently eliminated unbound concentration of ticagrelor and reversed its antiplatelet activity in preclinical models and will support future development of MEDI2452.

Structural and functional characterization of a specific antidote for ticagrelor.

Ticagrelor is a direct-acting reversibly binding P2Y12 antagonist and is widely used as an antiplatelet therapy for the prevention of cardiovascular events in acute coronary syndrome patients. However, antiplatelet therapy can be associated with an increased risk of bleeding. Here, we present data on the identification and the in vitro and in vivo pharmacology of an antigen-binding fragment (Fab) antidote for ticagrelor. The Fab has a 20 pM affinity for ticagrelor, which is 100 times stronger than ticagrelor's affinity for its target, P2Y12. Despite ticagrelor's structural similarities to adenosine, the Fab is highly specific and does not bind to adenosine, adenosine triphosphate, adenosine 5'-diphosphate, or structurally related drugs. The antidote concentration-dependently neutralized the free fraction of ticagrelor and reversed its antiplatelet activity both in vitro in human platelet-rich plasma and in vivo in mice. Lastly, the antidote proved effective in normalizing ticagrelor-dependent bleeding in a mouse model of acute surgery. This specific antidote for ticagrelor may prove valuable as an agent for patients who require emergency procedures.