Simultaneous Global Drug Development and Multiregional Clinical Trials (MRCT): 5 Years After Implementation of ICH E17 Guidelines.

The ICH E17 guidelines (2014-2017) on Multiregional Clinical Trials (MRCT) was a joint effort by the regulators and industry to facilitate simultaneous global drug development and registration through taking a strategic approach for clinical trials. In other words, the objective was to reduce the time it takes to bringing medications to patients around the world through minimizing unnecessary duplication of local or regional studies, which may add the regulatory burden to cost and time of bringing new therapies to patients. Under the auspices of ICH, training materials were created and provided to various stakeholders. Despite the successful promotion of the benefits of ICH E17 MRCT guidelines across the different regions, the uptake of some concepts (e.g., pooling strategy) in the ICH E17 guidelines has been slow. This paper describes various factors which could affect the conduct of MRCT at a global level, including ambiguity in definition of "region" (in MRCT), new regulatory requirements to enroll a diverse patient population, the use of decentralized clinical trials, use of data sources other than randomized clinical trials (e.g., use of Real Word Data), and the impact of the COVID-19 pandemic on the conduct of MRCT.

In vitro metabolism of a thrombin inhibitor and quantitation of metabolically generated cyanide.

During the metabolic characterization of compound I, 2-{6-cyano-3-[(2,2-difluoro-2-pyridin-2-ylethyl)amino]-2-oxopyrazin-1(2H)-yl]-N-[(3-fluoropyridin-2-yl)methyl]acetamide, evidence was obtained for extensive oxidative bioactivation of the pyrazinone ring system and some of the resulting metabolites were apparently devoid of the cyano moiety. Two assays, a spectrophotometric and a high-pressure liquid chromatography (HPLC) pre-column derivatization method, were evaluated for their ability to detect and quantify cyanide that is metabolically generated from liver microsomal incubations. When I was incubated (45 microM) in the presence of NADPH-fortified human liver microsomes for 2h, 7.5 microM of cyanide was detected using the spectrophotometric assay and 8.9 microM was measured using the HPLC methodology. Overall, the results from the two assays appeared to agree reasonably well with each other. However, the HPLC assay was the preferred method for the evaluation of cyanide formation in vitro due to its sensitivity, reliability, and ease of use.

9-hydroxyazafluorenes and their use in thrombin inhibitors.

Optimization of a previously reported thrombin inhibitor, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-trans-4-aminocyclohexylmethylamide (1), by replacing the aminocyclohexyl P1 group provided a new lead structure, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (2), with improved potency (K(i) = 0.49 nM for human thrombin, 2x APTT = 0.37 microM in human plasma) and pharmacokinetic properties (F = 39%, iv T(1/2) = 13 h in dogs). An effective strategy for reducing plasma protein binding of 2 and improving efficacy in an in vivo thrombosis model in rats was to replace the lipophilic fluorenyl group in P3 with an azafluorenyl group. Systematic investigation of all possible azafluorenyl P3 isomers and azafluorenyl-N-oxide analogues of 2 led to the identification of an optimal compound, 3-aza-9-hydroxyfluoren-9(R)-ylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (19b), with high potency (K(i) = 0.40 nM, 2x APTT = 0.18 microM), excellent pharmacokinetic properties (F = 55%, T(1/2) = 14 h in dogs), and complete efficacy in the in vivo thrombosis model in rats (inhibition of FeCl(3)-induced vessel occlusions in six of six rats receiving an intravenous infusion of 10 microg/kg/min of 19b). The stereochemistry of the azafluorenyl group in 19b was determined by X-ray crystallographic analysis of its N-oxide derivative (23b) bound in the active site of human thrombin.

Induction of CYP1A in the beagle dog by an inhibitor of kinase insert domain-containing receptor: differential effects in vitro and in vivo on mRNA and functional activity.

Compound I [3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one] is a potent inhibitor of human kinase insert domain-containing receptor (KDR kinase), which is under investigation for the treatment of cancer. Bile duct-cannulated male beagle dogs were administered 6 mg/kg compound I q.d. for 14 days. There was an approximately 2.5-fold decrease in the mean plasma area under the curve of I on days 7 and 14 (approximately 11.3 microM . h), relative to day 1 (28.2 microM . h). In the dog, compound I was eliminated by metabolism, with a major pathway being aromatic hydroxylation and subsequent sulfation to form the metabolite M3. Metabolic profiling suggested that the pathway leading to the formation of the sulfated conjugate M3 was induced upon multiple dosing of I. Studies conducted in vitro suggested that CYP1A1/2 was responsible for the formation of the hydroxylated metabolite, which is sulfated to yield M3. Additional studies confirmed induction of CYP1A protein and activity in the livers of dogs treated with I. However, studies in a dog hepatocyte model of induction showed a surprising decrease both in CYP1A mRNA and enzymatic activity in the presence of I, emphasizing the need to consider the results from a variety of in vitro and in vivo studies in deriving an understanding of the metabolic fate of a drug candidate. It is concluded that the autoinduction observed after multiple treatments with compound I occurs since compound I is both an inducer and a substrate for dog CYP1A.

Bioactivation of the 3-amino-6-chloropyrazinone ring in a thrombin inhibitor leads to novel dihydro-imidazole and imidazolidine derivatives: structures and mechanism using 13C-labels, mass spectrometry, and NMR.

Thrombin is a serine protease that plays a key role in the blood coagulation cascade. Compound I [2-[6-chloro-3-[(2,2-difluoro-2-pyridin-2-ylethyl)amino]-2-oxopyrazin-1(2H)-yl]-N-[(3-fluoropyridin-2-yl)methyl]acetamide] is a potent, selective, and orally bioavailable thrombin inhibitor that is being studied as a possible anticoagulant. Biotransformation studies in rats revealed that 84% of an i.v. dose of I was excreted in the form of two metabolites. Both metabolites were formed by metabolic activation of the pyrazinone ring in I and subsequent rearrangement leading to two novel dihydro-imidazole and imidazolidine derivatives. The structures of these metabolites and their mechanism of formation were elucidated by additional use of two 13C single labels in the pyrazinone ring of I in combination with mass spectrometry and NMR techniques. The metabolite structures described here illustrate the rich metabolic chemistry of the amino-pyrazinone heterocycle.

Pharmacokinetic optimization of 3-amino-6-chloropyrazinone acetamide thrombin inhibitors. Implementation of P3 pyridine N-oxides to deliver an orally bioavailable series containing P1 N-benzylamides.

In this manuscript we demonstrate that a modification principally directed toward the improvement of the aqueous solubility (i.e., introduction a P3 pyridine N-oxide) of the previous lead compound afforded a new series of potent orally bioavailable P1 N-benzylamide thrombin inhibitors. An expedited investigation of the P1 SAR with respect to oral bioavailability, plasma half-life, and human liver microsome stability revealed 5 as the best candidate for advanced evaluation.

Metabolism-directed optimization of 3-aminopyrazinone acetamide thrombin inhibitors. Development of an orally bioavailable series containing P1 and P3 pyridines.

Recent efforts in the field of thrombin inhibitor research have focused on the identification of compounds with good oral bioavailability and pharmacokinetics. In this manuscript we describe a metabolism-based approach to the optimization of the 3-(2-phenethylamino)-6-methylpyrazinone acetamide template (e.g., 1) which resulted in the modification of each of the three principal components (i.e., P1, P2, P3) comprising this series. As a result of these studies, several potent thrombin inhibitors (e.g., 20, 24, 25) were identified which exhibit high levels of oral bioavailability and long plasma half-lives.

Metabolic activation of a pyrazinone-containing thrombin inhibitor. Evidence for novel biotransformation involving pyrazinone ring oxidation, rearrangement, and covalent binding to proteins.

Compound I, (2-[3-[(2,2-difluoro-2(2-pyridyl)ethyl)amino]-6-methyl-2-oxohydropyrazinyl]-N-[(3-fluoro(2-pyridyl))methyl]acetamide, is a potent competitive inhibitor of thrombin that reacts stoichiometrically with the protease. Compounds of this class possess therapeutic potential as anticoagulation agents. During the metabolic characterization of compound I, evidence was obtained for extensive metabolic activation of the pyrazinone ring system. Following administration of (14)C-labeled I to rats, significant levels of irreversibly bound radioactivity to proteins were detected in rat plasma and liver. LC/MS/MS analysis of metabolites formed in rat and human liver microsomes fortified with glutathione (GSH) revealed the presence of two structurally distinct GSH adducts. It is proposed that the first of these GSH conjugates derives from a two electron oxidation of the 6-methyl-2-oxo-3-aminopyrazinone moiety to afford an electrophilic imine-methide intermediate, while the second is formed by addition of GSH to an epoxide formed by P-450-mediated oxidation of the double bond at the 5-6 position of the pyrazinone ring. The addition of GSH to the proposed epoxide facilitates opening of the pyrazinone ring and a rearrangement to afford a stable, rearranged imidazole-containing metabolite. Elucidation of the metabolic activation pathways of I provides structural guidance for the design of thrombin inhibitors with decreased potential for the generation of chemically reactive intermediates.