Influence of N-acetyltransferase 2 gene polymorphisms on the in vitro metabolism of the epidermal growth factor receptor inhibitor rociletinib.

AIMS: Rociletinib showed activity in T790M-positive non-small cell lung cancer patients. It undergoes amide hydrolysis to form M502, followed by N-acetylation to M544 or amide hydrolysis to M460. We identified the enzymes responsible for rociletinib metabolism, and investigated the relationship between M544 formation and N-acetyltransferase 2 (NAT2) polymorphisms. METHODS: Rociletinib and metabolites were incubated with carboxylesterase (CES)1b, CES1c, CES2, NAT1, NAT2, arylacetamide deacetylase, inhibitors, pooled human liver microsomes (HLM) and cytosols (HLC). Cytosols (n = 107) were genotyped for NAT2 polymorphisms (rs1041983 and rs1801280) and incubated with M502. Human hepatocytes from intermediate (NAT2*6/*12A) and slow (NAT2*5B/*5B) acetylators were incubated with 10 µM rociletinib and metabolites for 24 hours. Metabolites were measured by high-performance liquid chromatography. RESULTS: M502 was formed from rociletinib and M544 by CES2 and HLM; M544 and N-acetyl-M460 were formed by NAT2 and HLC; M460 was not formed by CES or arylacetamide deacetylase. M502 formation by HLM was inhibited by bis-(4-nitrophenyl)phosphate and eserine (10 µM). M544 formation in HLC was inhibited by 100 µM quercetin and was associated with NAT2 genotype (P < .0001). M460 formation in HLM was inhibited by eserine, and M460 was N-acetylated in HLC. Hepatocytes formed M502, M544 and M460. The intermediate acetylator showed higher production (range: 3.4-5.1-fold) of N-acetylated metabolites than the slow acetylator. CONCLUSIONS: Results indicate that NAT2 and CES2 are involved in rociletinib metabolism, and polymorphic NAT2 could alter drug exposure in patients. Slow NAT2 acetylators would have higher exposure to M502 and M460 and consequently, be at increased risk of experiencing hyperglycaemia and QTc prolongation.

Structure-metabolism relationships in human-AOX: Chemical insights from a large database of aza-aromatic and amide compounds.

Aldehyde oxidase (AOX) is a metabolic enzyme catalyzing the oxidation of aldehyde and aza-aromatic compounds and the hydrolysis of amides, moieties frequently shared by the majority of drugs. Despite its key role in human metabolism, to date only fragmentary information about the chemical features responsible for AOX susceptibility are reported and only "very local" structure-metabolism relationships based on a small number of similar compounds have been developed. This study reports a more comprehensive coverage of the chemical space of structures with a high risk of AOX phase I metabolism in humans. More than 270 compounds were studied to identify the site of metabolism and the metabolite(s). Both electronic [supported by density functional theory (DFT) calculations] and exposure effects were considered when rationalizing the structure-metabolism relationship.

Fast Amide Bond Cleavage Assisted by a Secondary Amino and a Carboxyl Group-A Model for yet Unknown Peptidases?

Unconstrained amides that undergo fast hydrolysis under mild conditions are valuable sources of information about how amide bonds may be activated in enzymatic transformations. We report a compound possessing an unconstrained amide bond surrounded by an amino and a carboxyl group, each mounted in close proximity on a bicyclic scaffold. Fast amide hydrolysis of this model compound was found to depend on the presence of both the amino and carboxyl functions, and to involve a proton transfer in the rate-limiting step. Possible mechanisms for the hydrolytic cleavage and their relevance to peptide bond cleavage catalyzed by natural enzymes are discussed. Experimental observations suggest that the most probable mechanisms of the model compound hydrolysis might include a twisted amide intermediate and a rate-determining proton transfer.

In vivo instability of platensimycin and platencin: Synthesis and biological evaluation of urea- and carbamate-platensimycin.

Platensimycin (PTM) and platencin (PTN), two natural products and promising drug leads that target bacterial and mammalian fatty acid synthases, are known to have unfavorable pharmacokinetic properties. It is not clear, however, what the metabolic fates of PTM and PTN are and no efforts have been reported to address this key roadblock in the development of these compounds as viable drug options. Here we describe the pharmacokinetics of PTM and PTN, and reveal rapid renal clearance as the primary metabolic liability with three additional sites of chemical liability: (i) amide hydrolysis, (ii) glucuronidation, and (iii) oxidation. We determined that hydrolysis is a viable clearance mechanism in vivo and synthesized two PTM analogues to address in vivo hydrolysis. Urea- and carbamate-PTM analogues showed no detectable hydrolysis in vivo, at the expense of antibacterial activity, with no further improvement in systemic exposure. The antibacterial sulfur-containing analogues PTM D1 and PTM ML14 showed significant decreases in renal clearance. These studies set the stage for continued generation of PTM and PTN analogues in an effort to improve their pharmacokinetics while retaining or improving their biological activities.

Mechanistic investigations of the hydrolysis of amides, oxoesters and thioesters via kinetic isotope effects and positional isotope exchange.

The hydrolysis of amides, oxoesters and thioesters is an important reaction in both organic chemistry and biochemistry. Kinetic isotope effects (KIEs) are one of the most important physical organic methods for determining the most likely transition state structure and rate-determining step of these reaction mechanisms. This method induces a very small change in reaction rates, which, in turn, results in a minimum disturbance of the natural mechanism. KIE studies were carried out on both the non-enzymatic and the enzyme-catalyzed reactions in an effort to compare both types of mechanisms. In these studies the amides and esters of formic acid were chosen because this molecular structure allowed development of methodology to determine heavy-atom solvent (nucleophile) KIEs. This type of isotope effect is difficult to measure, but is rich in mechanistic information. Results of these investigations point to transition states with varying degrees of tetrahedral character that fit a classical stepwise mechanism. This article is part of a special issue entitled: Enzyme Transition States from Theory and Experiment.

Shedding light on minipig drug metabolism - elevated amide hydrolysis in vitro.

1. In recent years, the minipig is increasingly used as a test species in non-clinical assessment of drug candidates. While there is good scientific evidence available concerning cytochrome P450-mediated metabolism in minipig, the knowledge of other metabolic pathways is more limited. 2. The aim of this study was to provide an understanding of when, why, and how drug metabolism in minipig differs from other species commonly used in non-clinical studies. In-house cross-species metabolite profile comparisons in hepatocytes and microsomes of 38 Roche development compounds were retrospectively analyzed to compare the metabolism among minipig, human, rat, dog, monkey, rabbit and mouse. 3. A significant contributor to the elevated metabolism observed for certain compounds in minipig was identified as amide hydrolysis. The hepatic amide hydrolysis activity in minipig was further investigated in subcellular liver fractions and a structure-activity relationship was established. When structural motifs according to the established SAR are excluded, coverage of major human metabolic pathways was shown to be higher in minipig than in dog, and only slightly lower than in cynomolgus monkey. 4. A strategy is presented for early identification of drug compounds which might not be suited to further investigation in minipig due to excessive hydrolytic metabolism.

Potent and selective Bruton's tyrosine kinase inhibitors: discovery of GDC-0834.

SAR studies focused on improving the pharmacokinetic (PK) properties of the previously reported potent and selective Btk inhibitor CGI-1746 (1) resulted in the clinical candidate GDC-0834 (2), which retained the potency and selectivity of CGI-1746, but with much improved PK in preclinical animal models. Structure based design efforts drove this work as modifications to 1 were investigated at both the solvent exposed region as well as 'H3 binding pocket'. However, in vitro metabolic evaluation of 2 revealed a non CYP-mediated metabolic process that was more prevalent in human than preclinical species (mouse, rat, dog, cyno), leading to a high-level of uncertainly in predicting human pharmacokinetics. Due to its promising potency, selectivity, and preclinical efficacy, a single dose IND was filed and 2 was taken in to a single dose phase I trial in healthy volunteers to quickly evaluate the human pharmacokinetics. In human, 2 was found to be highly labile at the exo-cyclic amide bond that links the tetrahydrobenzothiophene moiety to the central aniline ring, resulting in insufficient parent drug exposure. This information informed the back-up program and discovery of improved inhibitors.

Sensitive electrochemical immunosensor via amide hydrolysis by DT-diaphorase combined with five redox-cycling reactions.

Amide hydrolysis using enzyme labels, such as proteases, occurs at a slower rate than phosphoester and carboxyl ester hydrolysis. Thus, it is not very useful for obtaining high signal amplification in biosensors. However, amide hydrolysis is less sensitive to nonenzymatic spontaneous hydrolysis, allowing for lower background levels. Herein, we report that amide hydrolysis by DT-diaphorase (DT-D) occurs rapidly and that its combination with five redox-cycling reactions allows for the development of a highly sensitive electrochemical immunosensor. DT-D rapidly generates ortho-aminohydroxy-naphthalene (oAN) from its amide substrate via amide hydrolysis, which not even trypsin, a highly catalytic protease, can achieve. NADH, which is required for amide hydrolysis, advantageously acts as a reducing agent for rapid electrooxidation-based redox-cycling reactions. In the presence of oAN, DT-D, and NADH, two redox-cycling reactions rapidly occur. In the additional presence of an electron mediator, Ru(NH3)63+ [Ru(III)], three more redox-cycling reactions occur because Ru(III) reacts rapidly with oAN and DT-D. Although the O2-related redox-cycling reactions and redox reaction decrease electrochemical signals, this signal-decreasing effect is not significant in air-saturated solutions. The slow electrooxidation of NADH at an indium tin oxide electrode and sluggish reaction between NADH and Ru(III) allow for low electrochemical backgrounds. When the developed signal amplification scheme is tested for the sandwich-type electrochemical detection of parathyroid hormone (PTH), a detection limit of ∼1 pg/mL is obtained. The detection method is highly sensitive and can accurately measure PTH in serum samples.

Contributions of intestine and liver to the absorption and disposition of FZJ-003, a selective JAK1 inhibitor with structure modification of filgotinib.

FZJ-003 is a selective Janus kinase 1 (JAK1) inhibitor with structural modification of filgotinib for rheumatoid arthritis (RA) treatment. In this study, a series of in vivo and in vitro experiments were conducted to investigate the specific contribution of the intestine and liver to the disposition of FZJ-003 compared with filgotinib. Results showed that FZJ-003 exhibited over 2-fold higher systemic exposure and lower clearance than those of filgotinib, after intravenous or intragastric administration at the equivalent mole dose level to conscious rats. In anesthetized rats treated with different dosing routes, FZJ-003 exhibited higher intestinal bioavailability (Fa·Fg, 98.47 vs 34.54%) but lower hepatic bioavailability (Fh, 61.45 vs 92.07%). Permeability test in Caco-2 cells indicated that FZJ-003 was probably transported by passive diffusion (efflux ratio 1.37 < 2, indicating the approximately equivalent Papp values in two directions) with a little higher permeability (Papp,AP-to-BL, 1.42 × 10-6vs 1.01 × 10-6 cm·s-1, FZJ-003 vs filgotinib). Metabolic studies in pre-systemic incubation systems showed that FZJ-003 experienced more NADPH-dependent metabolism, especially in hepatic microsomes fractions. Unlike filgotinib, there was no obvious amide-hydrolyzed metabolite of FZJ-003 detected throughout the pre-systemic metabolic sites. Collectively, these data suggest that the higher systemic exposure of FZJ-003 than filgotinib is mainly attributed to the higher intestinal bioavailability including bypassing the amide hydrolysis and possible efflux by intestinal epithelial cells, which strongly support the structural design purpose in terms of pharmacokinetics.

Clinical Pharmacokinetics of Ombitasvir.

Ombitasvir is a potent, nonstructural protein 5A inhibitor of the hepatitis C virus (HCV) that is used in combination with other direct-acting antivirals for the treatment of chronic HCV infection. Ombitasvir is predominantly metabolized by amide hydrolysis followed by oxidative metabolism and is a substrate of P-glycoprotein. Ombitasvir displays linear pharmacokinetics with minimal accumulation and is eliminated via metabolism and biliary excretion. A negligible amount of unchanged drug is excreted in urine. Exposures are comparable across Chinese, Japanese, and non-Asian subjects. The pharmacokinetic characteristics of ombitasvir are similar in healthy subjects and HCV-infected patients, and are not appreciably altered by hepatic or renal impairment. Results from several drug interaction studies demonstrated that ombitasvir has a low potential for drug interactions.