Reductive metabolism of tiaprofenic acid by the human liver and recombinant carbonyl reducing enzymes.

Tiaprofenic acid is a widely used anti-inflammatory drug; however, the reductive metabolism of tiaprofenic acid is not yet well understood. Here, we compared the reduction of tiaprofenic acid in microsomes and cytosol from the human liver. The microsomes exhibited lower Km value toward tiaprofenic acid than the cytosol (Km = 164 ± 18 µM vs. 569 ± 74 µM, respectively), whereas the cytosol showed higher specific activity during reduction than the microsomes (Vmax = 728 ± 52 pmol mg of protein-1 min-1 vs. 285 ± 11 pmol mg of protein-1 min-1, respectively). Next, a panel of recombinant carbonyl reducing enzymes from AKR and SDR superfamilies has been studied to find the enzymes responsible for the cytosolic reduction of tiaprofenic acid. CBR1 was identified as the reductase of tiaprofenic acid with high specific activity (56,965 ± 6741 pmol mg of protein-1 min-1). Three other enzymes, AKR1A1, AKR1B10, and AKR1C4, were also able to reduce tiaprofenic acid, but with very low activity. Thus, CBR1 was shown to be a tiaprofenic acid reductase in vitro and was also suggested to be the principal tiaprofenic acid reductase in vivo.

In vitro metabolism of fenofibric acid by carbonyl reducing enzymes.

Fenofibric acid is a hypolipidemic drug that is used as an active ingredient per se or is administered in the form of fenofibrate that releases fenofibric acid after absorption. The metabolism of fenofibric acid is mediated primarily by glucuronidation. However, the other part of fenofibric acid is excreted as reduced fenofibric acid. Enzymes responsible for the formation of reduced fenofibric acid as well as their subcellular localization have remained unknown until now. We have found that the predominant site of fenofibric acid reduction is the human liver cytosol, whereas liver microsomes reduced fenofibric acid to a lower extent and exhibited a lower affinity for this drug (Km > 1000 µM). Of nine carbonyl-reducing enzymes (CREs) tested, CBR1 exhibited the greatest activity for fenofibric acid reduction (CLint = 85.975 µl/mg protein/min). CBR1 predominantly formed (-)-enantiomers of reduced fenofibric acid similar to liver cytosol and in accordance with the in vivo data. AKR1C1, AKR1C2, AKR1C3 and AKR1B1 were also identified as reductases of fenofibric acid but are expected to play only a minor role in fenofibric acid metabolism.

Carbonyl reduction of warfarin: Identification and characterization of human warfarin reductases.

Warfarin is a widely used anticoagulant and, unfortunately, is a drug that is commonly implicated in serious adverse events including fatalities. Although several factors, including the metabolism of warfarin via CYP450, have been reported to affect the safety and efficacy of warfarin therapy, the wide variance in the warfarin dosage in patients has not been completely clarified. In addition to the oxidative metabolism of warfarin mediated by CYP450, reductive metabolism is involved in warfarin biotransformation. However, the reductive metabolism of warfarin has been largely unexplored and deserves further investigation. We studied warfarin reduction by human liver fractions and found a 9-fold higher velocity of warfarin reduction in the cytosol than in microsomes (Vmax=77.2 vs. 8.7pmol/mgprotein/min, respectively). Furthermore, of nine recombinant cytosolic carbonyl reducing enzymes tested for their ability to reduce warfarin, AKR1C3 and CBR1 were identified as warfarin reductases and their kinetic parameters were determined. The internal clearance of warfarin was 3 orders of magnitude higher with AKR1C3 than with CBR1 (CLint=65.922 vs. 0.070µl/mgprotein/min, respectively). This is the first time that warfarin reducing enzymes in human liver subcellular fraction have been identified. Moreover, we have described the chiral aspects of warfarin reduction using an HPLC method that enabled the detection of individual warfarin alcohol stereoisomers. Cytosol and AKR1C3 exhibit the stereoselective metabolism of (R)-warfarin to preferentially form (SR)-warfarin alcohol as the primary in vivo metabolite of warfarin. On the other hand, microsomes and CBR1 preferentially reduce (S)-warfarin to form (RS)-warfarin alcohol and (SS)-warfarin alcohol, respectively.

The role of carbonyl reducing enzymes in oxcarbazepine in vitro metabolism in man.

Oxcarbazepine, a second generation antiepileptic drug belonging to the family of dibenz[b,f]azepines, is subjected to a rapid and extensive biotransformation. Oxcarbazepine demonstrates a low potential for drug interactions because its biotransformation is mainly mediated by the reduction pathway instead of oxidative pathways, which are very susceptible to drug interactions. The reductive metabolism of oxcarbazepine yields a 10-monohydroxy derivative (10,11-dihydro-10-hydroxy-carbazepine), which is responsible for the pharmacological activity. The identity and localization of enzymes participating in the reduction of oxcarbazepine in response to this active metabolite have remained unknown until now. Thus, we investigated the reductive metabolism of oxcarbazepine in human liver subcellular fractions and using recombinant carbonyl reducing enzymes. The reduction of oxcarbazepine was shown to occur largely in the liver cytosol rather than liver microsomes. Furthermore, the activity and stereospecificity of cytosolic carbonyl reducing enzymes toward oxcarbazepine were assessed. Of the eight tested enzymes, six reductases were identified to contribute to the reduction of oxcarbazepine. The highest activities were demonstrated by AKR1C1, AKR1C2, AKR1C3, and AKR1C4. The contribution of CBR1 and CBR3 to the reduction of oxcarbazepine was also significant, although their role in oxcarbazepine metabolism in vivo is unclear.

Carbonyl reduction pathways in drug metabolism.

The understanding of drug biotransformation is an important medical topic. The oxidative pathways that involve CYPs have been extensively studied in drug metabolism in contrast to the reductive pathways. This review focuses on drugs that have been reported to be reduced at the carbonyl group in vivo. Although the carbonyl reduction of these drugs is well known, our understanding of the carbonyl reducing enzymes (CRE) that perform these reactions is limited. We have summarized the published data in order to thoroughly describe the reductive metabolism of the selected drugs and to demonstrate the role of carbonyl reduction in the context of their overall metabolism. The number of drugs recognized as substrates for CREs has increased considerably in recent years. Moreover, the importance of carbonyl reduction in the overall metabolism of these drugs is often surprisingly high. Because only limited information is available about the CREs responsible for these reactions, additional research is needed to improve our understanding of the metabolism of drugs undergoing carbonyl reduction. Carbonyl reduction should be investigated during drug development because it can either positively or negatively influence drug efficacy.

Expression of human carbonyl reductase 3 (CBR3; SDR21C2) is inducible by pro-inflammatory stimuli.

Until today, the physiologic role of human carbonyl reductase 3 (CBR3; SDR21C2), a member of the short-chain dehydrogenase/reductase superfamily remains obscure. Since the transcriptional regulation is closely related to the function of a protein, elucidation of the regulation of CBR3 should help to understand its physiologic role. We recently identified CBR3 as a novel target gene of Nrf2, a cellular sensor of oxidative stress. In this study, we provide for the first time evidence that pro-inflammatory stimuli induce the expression of the CBR3 gene. Treatment of human cancer cells HT-29 (colon) and HepG2 (liver) with TNF-α, IL-1ß, and LPS induced CBR3 expression differentially. While TNF-α (50 ng/ml) or IL-1ß (1 and 10 ng/ml), induced CBR3 mRNA expression in HT-29 cells (up to 10-fold) and HepG2 cells (up to 20-fold), LPS activated the CBR3 gene only in HepG2 cells. Furthermore, overexpression of the NFκB subunits p65 and p50 alone or in combination elevated CBR3 mRNA levels (3.9-fold) in HT-29 cells. According to our results, CBR3 is a novel target gene of inflammatory stimuli, and elucidation of its detailed role in inflammation deserves further investigation.

Human carbonyl reductases.

Enzymatic carbonyl reduction means the formation of a hydroxy function out of a ketone or aldehyde moiety and applies for the metabolism of physiological (endogenous) or xenobiotic (exogenous) molecules. As for endogenous substrates, carbonyl reduction is often part of a reversible oxidoreductase process and involves the activation or inactivation of important signal molecules like steroids, prostaglandins, retinoids and biogenic amines. These reactions are carried out by NAD(P)(H)-dependent dehydrogenases belonging to two protein superfamilies, the aldo-keto reductases (AKR) and the short-chain dehydrogenases/reductases (SDR). With regard to exogenous substrates, carbonyl reduction of xenobiotics is generally a "one-way" detoxification reaction, since the resulting alcohol is easier to conjugate and to eliminate. Interestingly, the participating enzymes do also belong to the AKR and SDR superfamilies. Moreover, some enzymes from the two protein superfamilies exhibit pluripotency in that they are able to catalyze the oxidoreduction of endobiotics but do also function in the reductive metabolism of carbonyl group bearing xenobiotics. A special case are carbonyl reductases per se which belong to the SDR superfamily and whose substrates or physiological roles are not quite clear. Usually, carbonyl reductases have a broad and diverse substrate spectrum for xenobiotics, however, for some of them a specific physiological function has been speculated. In the human genome, three SDR genes have been identified to code for the carbonyl reductases CBR1 (SDR21C1), CBR3 (SDR21C2) and CBR4 (SDR45C1). The present review summarizes the current knowledge on these enzymes with special emphasis on their role as a defence system against toxicants, as well as their possible physiological function and medical application. In detail, we have screened the recent literature on these three enzymes with regard to endogenous and exogenous substrates, their three-dimensional structure, tissues specific expression, polymorphisms, transcriptional regulation, occurrence in pathological states, and their possible association with cancer. Combined, this review contributes to understanding the complex nature and biological roles(s) of the human carbonyl reductases CBR1, CBR3 and CBR4.

Regulation of human carbonyl reductase 3 (CBR3; SDR21C2) expression by Nrf2 in cultured cancer cells.

Carbonyl reduction is a central metabolic process that controls the level of key regulatory molecules as well as xenobiotics. Carbonyl reductase 3 (CBR3; SDR21C2), a member of the short-chain dehydrogenase/reductase (SDR) superfamily, has been poorly characterized so far, and the regulation of its expression is a complete mystery. Here, we show that CBR3 expression is regulated via Nrf2, a key regulator in response to oxidative stress. In human cancer cell lines, CBR3 mRNA was expressed differentially, ranging from very high (A549, lung) to very low (HT-29, colon; HepG2, liver) levels. CBR3 protein was highly expressed in SW-480 (colon) cells but was absent in HCT116 (colon) and HepG2 cells. CBR3 mRNA could be induced in HT-29 cells by Nrf2 agonists [sulforaphane (SUL, 7-fold) and diethyl maleate (DEM, 4-fold)] or hormone receptor ligand Z-guggulsterone (5-fold). Aryl hydrocarbon receptor agonist B[k]F failed to induce CBR3 mRNA after incubation for 8 h but elevated CBR3 levels after 24 h, most likely mediated by B[k]F metabolites that can activate Nrf2 signaling. Inhibition of Nrf2-activating upstream kinase MEK/ERK by PD98059 weakened DEM-mediated induction of CBR3 mRNA. Proteasome inhibitors MG-132 (5 µM) and bortezomib (50 nM) dramatically increased the level of CBR3 mRNA, obviously because of the increase in the level of Nrf2 protein. While siRNA-mediated knockdown of Nrf2 led to a decrease in the level of CBR3 mRNA in A549 cells (30% of control), Keap1 knockdown increased the level of CBR3 mRNA expression in HepG2 (9.3-fold) and HT-29 (2.7-fold) cells. Here, we provide for the first time evidence that human CBR3 is a new member of the Nrf2 gene battery.