Quantitative Analysis of mRNA and Protein Expression Levels of Aldo-Keto Reductase and Short-Chain Dehydrogenase/Reductase Isoforms in the Human Intestine.

Enzymes catalyzing the reduction reaction of xenobiotics are mainly members of the aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies. The intestine, together with the liver, is responsible for first-pass effects and is an organ that determines the bioavailability of orally administered drugs. In this study, we evaluated the mRNA and protein expression levels of 12 AKR isoforms (AKR1A1, AKR1B1, AKR1B10, AKR1B15, AKR1C1, AKR1C2, AKR1C3, AKR1C4, AKR1D1, AKR1E2, AKR7A2, and AKR7A3) and 7 SDR isoforms (CBR1, CBR3, CBR4, DCXR, DHRS4, HSD11B1, and HSD17B12) in each region of the human intestine using next-generation sequencing and data-independent acquisition proteomics. At both the mRNA and protein levels, most AKR isoforms were highly expressed in the upper regions of the intestine, namely the duodenum and jejunum, and then declined toward the rectum. Among the members in the SDR superfamily, CBR1 and DHRS4 were highly expressed in the upper regions, whereas the expression levels of the other isoforms were almost uniform in all regions. Significant positive correlations between mRNA and protein levels were observed in AKR1A1, AKR1B1, AKR1B10, AKR1C3, AKR7A2, AKR7A3, CBR1, and CBR3. The mRNA level of AKR1B10 was highest, followed by AKR7A3 and CBR1, each accounting for more than 10% of the sum of all AKR and SDR levels in the small intestine. This expression profile in the human intestine was greatly different from that in the human liver, where AKR1C isoforms are predominantly expressed. SIGNIFICANCE STATEMENT: In this study comprehensively determined the mRNA and protein expression profiles of aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase isoforms involved in xenobiotic metabolism in the human intestine and found that most of them are highly expressed in the upper region, where AKR1B10, AKR7A3, and CBR1 are predominantly expressed. Since the intestine is significantly involved in the metabolism of orally administered drugs, the information provided here is valuable for pharmacokinetic studies in drug development.

Development and Validation of a Proteomic Correlation Profiling Technique to Detect and Identify Enzymes Involved in Metabolism of Drugs of Concern.

To predict the variation of pharmacological or toxicological effect caused by pharmacokinetic variance, it is important to be able to detect previously unknown and unsuspected enzymes involved in drug metabolism. We investigated the use of proteomic correlation profiling (PCP) as a technique to identify the enzymes involved in metabolism of drugs of concern. By evaluating the metabolic activities of each enzyme (including isoforms of cytochrome P450, uridine 5' diphospho-glucuronosyltransferase, and hydrolases, plus aldehyde oxidase and carbonyl reductase) on their typical substrates using a panel of human liver samples, we were able to show the validity of PCP for this purpose. R or Rs and P values were calculated for the association between the protein abundance profile of each protein and the metabolic rate profile of each typical substrate. For the 18 enzymatic activities examined, 13 of the enzymes reported to be responsible for the reactions had correlation coefficients higher than 0.7 and were ranked first to third. For the remaining five activities, the responsible enzymes had correlation coefficients lower than 0.7 and lower rankings. The reasons for this were diverse, including confounding resulting from low protein abundance ratios, artificially high correlations of other enzymes due to limited sample numbers, the presence of inactive enzyme forms, and genetic polymorphisms. Overall, PCP was able to identify the majority of responsible drug-metabolizing enzymes across several enzyme classes (oxidoreductase, transferase, hydrolase); use of this methodology could allow more timely and accurate identification of unknown drug-metabolizing enzymes. SIGNIFICANCE STATEMENT: Proteomic correlation profiling using samples from individual human donors was proven to be a useful methodology for the identification of enzymes responsible for drug-metabolism. This methodology could accelerate the identification of unknown drug-metabolizing enzymes in the future.

Evaluation of Drug-Drug Interactions via Inhibition of Hydrolases by Orlistat, an Anti-Obesity Drug.

Drug-drug interactions (DDI) have a significant impact on drug efficacy and safety. It has been reported that orlistat, an anti-obesity drug, inhibits the hydrolysis of p-nitrophenol acetate, a common substrate of the major drug-metabolizing hydrolases, carboxylesterase (CES) 1, CES2, and arylacetamide deacetylase (AADAC), in vitro. The aim of this study was to examine whether orlistat affects the pharmacokinetics of drug(s) metabolized by hydrolases in vivo after evaluating its inhibitory potencies against CES1, CES2, and AADAC in vitro. Orlistat potently inhibited the hydrolysis of acebutolol, a specific substrate of CES2, in a non-competitive manner (inhibition constant, K i = 2.95 ± 0.16 nM), whereas it slightly inhibited the hydrolysis of temocapril and eslicarbazepine acetate, specific substrates of CES1 and AADAC, respectively (IC50 >100 nM). The in vivo DDI potential was elucidated using mice, in which orlistat showed strong inhibition against acebutolol hydrolase activities in the liver and intestinal microsomes, similar to humans. The area under the curve (AUC) of acebutolol was increased by 43%, whereas the AUC of acetolol, a hydrolyzed metabolite of acebutolol, was decreased by 47% by co-administration of orlistat. The ratio of the K i value to the maximum unbound plasma concentration of orlistat (<0.012) is lower than the risk criteria for DDI in the liver defined by the US Food and Drug Administration guideline (>0.02), whereas the ratio of the K i value to the estimated intestinal luminal concentration (3.3 × 105) is considerably higher than the risk criteria in the intestine (>10). Therefore, this suggests that orlistat causes DDI by inhibiting hydrolases in the intestine. SIGNIFICANCE STATEMENT: This study demonstrated that orlistat, an anti-obesity drug, causes drug-drug interactions in vivo by potently inhibiting carboxylesterase 2 in the intestine. This is the first evidence that inhibition of hydrolases causes drug-drug interactions.

Arylacetamide deacetylase knockout mice are sensitive to ketoconazole-induced hepatotoxicity and adrenal insufficiency.

Orally administered ketoconazole may rarely induce liver injury and adrenal insufficiency. A metabolite formed by arylacetamide deacetylase (AADAC)-mediated hydrolysis has been observed in cellulo studies, and it is relevant to ketoconazole-induced cytotoxicity. This study tried to examine the significance of AADAC in ketoconazole-induced toxicity in vivo using Aadac knockout mice. Oral administration of 150 mg/kg ketoconazole resulted in the area under the plasma concentration-time curve values of ketoconazole and N-deacetylketoconazole, a hydrolyzed metabolite of ketoconazole, in Aadac knockout mice being significantly higher and lower than those in wild-type mice, respectively. With the administration of ketoconazole (300 mg/kg/day) for 7 days, Aadac knockout mice showed higher mortality (100%) than wild-type mice (42.9%), and they also showed significantly higher plasma alanine transaminase and lower corticosterone levels, thus representing liver injury and steroidogenesis inhibition, respectively. It was suggested that a higher plasma ketoconazole concentration likely accounts for the inhibition of the synthesis of corticosterone, which has anti-inflammatory effects, in the adrenal gland in Aadac KO mice. In Aadac knockout mice, hepatic mRNA levels of immune- and inflammation-related factors were increased by the administration of 300 mg/kg ketoconazole, and the increase was restored by the replenishment of corticosterone (40 mg/kg, s.c.) along with recoveries of plasma alanine transaminase levels. In conclusion, Aadac defects exacerbate ketoconazole-induced liver injury by inhibiting glucocorticoid synthesis and enhancing the inflammatory response. This in vivo study revealed that the hydrolysis of ketoconazole by AADAC can mitigate ketoconazole-induced toxicities.

Arylacetamide deacetylase as a determinant of the hydrolysis and activation of abiraterone acetate in mice and humans.

AIM: Abiraterone acetate for metastatic castration-resistant prostate cancer is an acetylated prodrug to be hydrolyzed to abiraterone. Abiraterone acetate is known to be hydrolyzed by pancreatic cholesterol esterase secreted into the intestinal lumen. This study aimed to investigate the possibility that arylacetamide deacetylase (AADAC) expressed in enterocytes contributes to the hydrolysis of abiraterone acetate based on its substrate preference. MATERIALS AND METHODS: Abiraterone acetate hydrolase activity was measured using human intestinal (HIM) and liver microsomes (HLM) as well as recombinant AADAC. Correlation analysis between activity and AADAC expression was performed in 14 individual HIMs. The in vivo pharmacokinetics of abiraterone acetate was examined using wild-type and Aadac knockout mice administered abiraterone acetate with or without orlistat, a pancreatic cholesterol esterase inhibitor. KEY FINDINGS: Recombinant AADAC showed abiraterone acetate hydrolase activity with similar Km value to HIM and HLM. The positive correlation between activity and AADAC levels in individual HIMs supported the responsibility of AADAC for abiraterone acetate hydrolysis. The area under the plasma concentration-time curve (AUC) of abiraterone after oral administration of abiraterone acetate in Aadac knockout mice was 38% lower than that in wild-type mice. The involvement of pancreatic cholesterol esterase in abiraterone formation was revealed by the decreased AUC of abiraterone by coadministration of orlistat. Orlistat potently inhibited AADAC, implying its potential as a perpetrator of drug-drug interactions. SIGNIFICANCE: AADAC is responsible for the hydrolysis of abiraterone acetate in the intestine and liver, suggesting that concomitant use of abiraterone acetate and drugs potently inhibiting AADAC should be avoided.

Differences in Hydrolase Activities in the Liver and Small Intestine between Marmosets and Humans.

For drug development, species differences in drug-metabolism reactions present obstacles for predicting pharmacokinetics in humans. We characterized the species differences in hydrolases among humans and mice, rats, dogs, and cynomolgus monkeys. In this study, to expand the series of such studies, we attempted to characterize marmoset hydrolases. We measured hydrolase activities for 24 compounds using marmoset liver and intestinal microsomes, as well as recombinant marmoset carboxylesterase (CES) 1, CES2, and arylacetamide deacetylase (AADAC). The contributions of CES1, CES2, and AADAC to hydrolysis in marmoset liver microsomes were estimated by correcting the activities by using the ratios of hydrolase protein levels in the liver microsomes and those in recombinant systems. For six out of eight human CES1 substrates, the activities in marmoset liver microsomes were lower than those in human liver microsomes. For two human CES2 substrates and three out of seven human AADAC substrates, the activities in marmoset liver microsomes were higher than those in human liver microsomes. Notably, among the three rifamycins, only rifabutin was hydrolyzed by marmoset tissue microsomes and recombinant AADAC. The activities for all substrates in marmoset intestinal microsomes tended to be lower than those in liver microsomes, which suggests that the first-pass effects of the CES and AADAC substrates are due to hepatic hydrolysis. In most cases, the sums of the values of the contributions of CES1, CES2, and AADAC were below 100%, which indicated the involvement of other hydrolases in marmosets. In conclusion, we clarified the substrate preferences of hydrolases in marmosets. SIGNIFICANCE STATEMENT: This study confirmed that there are large differences in hydrolase activities between humans and marmosets by characterizing marmoset hydrolase activities for compounds that are substrates of human CES1, CES2, or arylacetamide deacetylase. The data obtained in this study may be useful for considering whether marmosets are appropriate for examining the pharmacokinetics and efficacies of new chemical entities in preclinical studies.

Role of Human Arylacetamide Deacetylase (AADAC) on Hydrolysis of Eslicarbazepine Acetate and Effects of AADAC Genetic Polymorphisms on Hydrolase Activity.

Human arylacetamide deacetylase (AADAC) plays a role in the detoxification or activation of drugs and is sometimes involved in the incidence of toxicity by catalyzing hydrolysis reactions. AADAC prefers compounds with relatively small acyl groups, such as acetyl groups. Eslicarbazepine acetate, an antiepileptic drug, is a prodrug rapidly hydrolyzed to eslicarbazepine. We sought to clarify whether AADAC might be responsible for the hydrolysis of eslicarbazepine acetate. Eslicarbazepine acetate was efficiently hydrolyzed by human intestinal and liver microsomes and recombinant human AADAC. The hydrolase activities in human intestinal and liver microsomes were inhibited by epigallocatechin gallate, a specific inhibitor of AADAC, by 82% and 88% of the control, respectively. The hydrolase activities in liver microsomes from 25 human livers were significantly correlated (r = 0.87, P < 0.001) with AADAC protein levels, suggesting that the enzyme AADAC is responsible for the hydrolysis of eslicarbazepine acetate. The effects of genetic polymorphisms of AADAC on eslicarbazepine acetate hydrolysis were examined by using the constructed recombinant AADAC variants with T74A, V172I, R248S, V281I, N366K, or X400Q. AADAC variants with R248S or X400Q showed lower activity than wild type (5% or 21%, respectively), whereas those with V172I showed higher activity than wild type (174%). Similar tendencies were observed in the other four substrates of AADAC; that is, p-nitrophenyl acetate, ketoconazole, phenacetin, and rifampicin. Collectively, we found that eslicarbazepine acetate is specifically and efficiently hydrolyzed by human AADAC, and several AADAC polymorphic alleles would be a factor affecting the enzyme activity and drug response. SIGNIFICANCE STATEMENT: This is the first study to clarify that arylacetamide deacetylase (AADAC) is responsible for the activation of eslicarbazepine acetate, an antiepileptic prodrug, to eslicarbazepine, an active form, in the human liver and intestines. In addition, we found that several AADAC polymorphic alleles would be a factor affecting the enzyme activity and drug response.

Methionine Sulfoxide Reductase A in Human and Mouse Tissues is Responsible for Sulindac Activation, Making a Larger Contribution than the Gut Microbiota.

Sulindac is a nonsteroidal anti-inflammatory prodrug that is converted to its pharmacologically active metabolite, sulindac sulfide, via a reduction reaction. It is widely accepted that the gut microbiota is responsible for sulindac activation; however, sulindac-induced gastrointestinal injury, which is caused by irritation of the gastrointestinal tract by its active metabolite, is uncommon. Therefore, it is surmised that sulindac is converted to its active metabolite in tissues after absorption. In this study, we sought to identify the enzyme(s) responsible for sulindac activation in tissues and to compare its/their contribution to the gut microbiota. Sulindac is enzymatically reduced in human intestinal, liver, and renal cytosols. Since sulindac is known to be reduced by methionine sulfoxide reductase (Msr) in Escherichia coli, we investigated whether the human ortholog MSRA catalyzes the sulindac reduction reaction. We found that recombinant human MSRA shows sulindac reductase activity with a similar Michaelis constant value as tissue cytosols. In addition, it was revealed that cytosolic factor(s) efficiently enhanced MSRA activity. By using the relative expression factor, the contribution of MSRA to the sulindac reductase activities in each tissue cytosol was calculated to be almost 100%. In mice, depletion of the gut microbiota by administration of antibiotics resulted in a 31% decrease in the area under the curve ratio of sulindac sulfide to sulindac, indicating that the contribution of tissue MsrA to sulindac activation is expected to be 69% in the body. In conclusion, we demonstrated that MSRA expressed in tissues is involved in sulindac activation, making a larger contribution than the gut microbiota. SIGNIFICANCE STATEMENT: Methionine sulfoxide reductase A is responsible for the activation of sulindac, a nonsteroidal anti-inflammatory prodrug, to sulindac sulfide, an active form, in human tissues. Methionine sulfoxide reductase A expressed in tissues activates sulindac with a higher contribution than gut microbiota in body.