Investigation of the chiral recognition ability of human carboxylesterase 1 using indomethacin esters

Human carboxylesterase 1 (hCES1) is an enzyme that plays an important role in hydrolysis of pharmaceuticals in the human liver. In this study, elucidation of the chiral recognition ability of hCES1 was attempted using indomethacin esters in which various chiral alcohols were introduced. Indomethacin was condensed with various chiral alcohols to synthesize indomethacin esters. The synthesized esters were hydrolyzed with a human liver microsome (HLM) solution and a human intestine microsome (HIM) solution. High hydrolytic rate and high stereoselectivity were confirmed in the hydrolysis reaction in the HLM solution but not in the HIM solution, and these indomethacin esters were thought to be hydrolyzed by hCES1. Next, these indomethacin esters were hydrolyzed in recombinant hCES1 solution and the hydrolysis rates of the esters were calculated. The stereoselectivity confirmed in HLM solution was also confirmed in the hCES1 solution. In the hydrolysis reaction of esters in which a phenyl group is bonded next to the ester, the V_max value of the (R) form was 10 times larger than that of the (S) form.     

Comparative chemical and biological hydrolytic stability of homologous esters and isosteres

Esters are one of the major functional groups present in the structures of prodrugs and bioactive compounds. Their presence is often associated with hydrolytic lability. In this paper, we describe a comparative chemical and biological stability of homologous esters and isosteres in base media as well as in rat plasma and rat liver microsomes. Our results provided evidence for the hydrolytic structure lability relationship and demonstrated that the hydrolytic stability in plasma and liver microsome might depend on carboxylesterase activity. Molecular modelling studies were performed in order to understand the experimental data. Taken together, the data could be useful to design bioactive compounds or prodrugs based on the correct choice of the ester subunit, addressing compounds with higher or lower metabolic lability.     

Comparison of the Structure and Activity of Glycosylated and Aglycosylated Human Carboxylesterase 1

Human Carboxylesterase 1 (hCES1) is the key liver microsomal enzyme responsible for detoxification and metabolism of a variety of clinical drugs. To analyse the role of the single N-linked glycan on the structure and activity of the enzyme, authentically glycosylated and aglycosylated hCES1, generated by mutating asparagine 79 to glutamine, were produced in human embryonic kidney cells. Purified enzymes were shown to be predominantly trimeric in solution by analytical ultracentrifugation. The purified aglycosylated enzyme was found to be more active than glycosylated hCES1 and analysis of enzyme kinetics revealed that both enzymes exhibit positive cooperativity. Crystal structures of hCES1 a catalytically inactive mutant (S221A) and the aglycosylated enzyme were determined in the absence of any ligand or substrate to high resolutions (1.86 Å, 1.48 Å and 2.01 Å, respectively). Superposition of all three structures showed only minor conformational differences with a root mean square deviations of around 0.5 Å over all Cα positions. Comparison of the active sites of these un-liganded enzymes with the structures of hCES1-ligand complexes showed that side-chains of the catalytic triad were pre-disposed for substrate binding. Overall the results indicate that preventing N-glycosylation of hCES1 does not significantly affect the structure or activity of the enzyme.     

Green chemistry appended synthesis,metabolic stability and pharmacokinetic assessment of medicinally important chromene dihydropyrimidinones

A green chemistry approach has been developed for the synthesis of chromene dihydropyrimidinone (CDHPM) using recyclable Fe/Al pillared clay catalyst. Pharmacokinetic parameters like aqueous solubility, lipophilicity, P-glycoprotein (P-gp) ATPase activity, permeability, plasma protein binding, red blood cell (RBC) partitioning, metabolic stability in liver microsomes and in silico computations have been studied for the most potent anticancer chromene dihydropyrimidinone hybrid 1. This compound exhibited low solubility, optimum lipophilicity, no P-gp inhibitory activity, intermediate permeability, high plasma protein binding, low RBC partitioning, acceptable metabolic stability in rat liver microsomes (RLM) as well as human liver microsomes (HLM) with transitional hepatic extraction ratio.     

In silico prediction of human carboxylesterase-1 (hCES1) metabolism combining docking analyses and MD simulations

Metabolic problems lead to numerous failures during clinical trials, and much effort is now devoted in developing in silico models predicting metabolic stability and metabolites. Such models are well known for cytochromes P450 and some transferases, whereas little has been done to predict the hydrolytic activity of human hydrolases. The present study was undertaken to develop a computational approach able to predict the hydrolysis of novel esters by human carboxylesterase hCES1. The study involves both docking analyses of known substrates to develop predictive models, and molecular dynamics (MD) simulations to reveal the in situ behavior of substrates and products, with particular attention being paid to the influence of their ionization state. The results emphasize some crucial properties of the hCES1 catalytic cavity, confirming that as a trend with several exceptions, hCES1 prefers substrates with relatively smaller and somewhat polar alkyl/aryl groups and larger hydrophobic acyl moieties. The docking results underline the usefulness of the hydrophobic interaction score proposed here, which allows a robust prediction of hCES1 catalysis, while the MD simulations show the different behavior of substrates and products in the enzyme cavity, suggesting in particular that basic substrates interact with the enzyme in their unprotonated form.     

Pharmacokinetic studies of naproxen amides of some amino acid esters with promising colorectal cancer chemopreventive activity

Naproxen (nap) is belonging to Non-steriodal anti-inflammatory drugs (NSAIDs) group of drugs that characterized by their free carboxylic group. The therapeutic activity of nap is usually accompanied by GI untoward side effects. Recently synthesized naproxen amides of some amino acid esters prodrugs to mask the free carboxylic group were reported. Those prodrugs showed a promising colorectal cancer chemopreventive activity. The current study aims to investigate the fate and hydrolysis of the prodrugs kinetically in different pH conditions, simulated gastric and intestinal fluids with pHs of 1.2, 5.5 and 7.4 in vitro at 37 °C. The effect of enzymes on the hydrolysis of prodrugs was also studied through incubation of these prodrugs at 37 °C in human plasma and rat liver homogenates. The pharmacokinetic parameters of selected prodrugs and the liberated nap were studied after oral and intraperitoneal administration in male wistar rats. The results showed the hydrolysis of naproxen amides of amino acid esters to nap through two steps first by degradation of the ester moiety to form the amide of nap with amino acid and the second was through the degradation of the amide link to liberate nap. The two reactions were followed and studied kinetically where K₁ and K₂ (rate constants of degradation) is reported. The hydrolysis of prodrugs was faster in liver homogenates than in plasma. The relative bioavailability of the liberated nap in vivo was higher in case of prodrug containing ethyl glycinate moiety than that occupied l-valine ethyl ester moiety. Each of nap. prodrugs containing ethyl glycinate and l-valine ethyl ester moieties appears promising in liberating nap, decreasing direct GI side effect and consequently their colorectal cancer chemopreventive activity.     

Capture hydrolysis signals in the microsomal stability assay: molecular mechanisms of the alkyl ester drug and prodrug metabolism

The hydrolysis of carboxylic acid esters is often catalyzed by carboxylesterases in human liver microsomes, which is also a common 'noise' in the microsomal stability assay, a widely used screening protocol in drug discovery to monitor the activity of cytochrome P450 enzymes. Herein, we captured this 'noise', the hydrolysis signal of small alkyl ester drugs and prodrugs with a unique pairwise analysis of Pfizer's microsomal clearance database. The hydrolysis mechanisms were further elucidated with density functional theory and molecular docking approaches. The results suggested that the electronic properties of ester moieties, tetrahedral intermediate formation energies, and specific drug-enzyme molecular interactions are key factors for the determination of the metabolic fate of the studied alkyl esters, but individually these factors failed to correlate with the observed rate of hydrolysis.     

In Vitro Evaluation of the Inhibitory Potential of Pharmaceutical Excipients on Human Carboxylesterase 1A and 2

Two major forms of human carboxylesterase (CES), CES1A and CES2, dominate the pharmacokinetics of most prodrugs such as imidapril and irinotecan (CPT-11). Excipients, largely used as insert vehicles in formulation, have been recently reported to affect drug enzyme activity. The influence of excipients on the activity of CES remains undefined. In this study, the inhibitory effects of 25 excipients on the activities of CES1A1 and CES2 were evaluated. Imidapril and CPT-11 were used as substrates and cultured with liver microsomes in vitro. Imidapril hydrolase activities of recombinant CES1A1 and human liver microsomes (HLM) were strongly inhibited by sodium lauryl sulphate (SLS) and polyoxyl 40 hydrogenated castor oil (RH40) [Inhibition constant (K_i) = 0.04±0.01 μg/ml and 0.20±0.09 μg/ml for CES1A1, and 0.12±0.03 μg/ml and 0.76±0.33 μg/ml, respectively, for HLM]. The enzyme hydrolase activity of recombinant CES2 was substantially inhibited by Tween 20 and polyoxyl 35 castor oil (EL35) (K_i = 0.93±0.36 μg/ml and 4.4±1.24 μg/ml, respectively). Thus, these results demonstrate that surfactants such as SLS, RH40, Tween 20 and EL35 may attenuate the CES activity; such inhibition should be taken into consideration during drug administration.     

Pharmacokinetic evaluation of medicinally important synthetic N,N′ diindolylmethane glucoside: Improved synthesis and metabolic stability

An improved route for the synthesis of N,N′-diindolyl methane (DIM) glycosides has been developed by using Fe/Al pillared clay catalyst. In-silico pharmacokinetics followed by in-vitro studies like aqueous solubility, lipophilicity, P-glycoprotein (P-gp) dependent ATPase activity, permeability, plasma protein binding, RBC partitioning, metabolic stability in different liver microsomes and its in-vitro-in-vivo extrapolation were conducted for the most potent derivative namely NGD16. The compound was found to have low solubility, optimum lipophilicity, no P-gp inhibitory activity, intermediate permeability, high plasma protein binding, low RBC partitioning, acceptable metabolic stability in rat liver microsomes (RLM) as well as human liver microsomes (HLM) with intermediate hepatic extraction ratio.     

Species differences for stereoselective hydrolysis of propranolol prodrugs in plasma and liver

Species differences and substrate specificities for the stereoselective hydrolysis of fifteen O-acyl propranolol (PL) prodrugs were investigated in pH 7.4 Tris-HCl buffer and rat and dog plasma and liver subfractions. The (R)-isomers were preferentially converted to propranolol (PL) in both rat and dog plasma with the exception of isovaleryl-PL in rat plasma, although the hydrolytic activities of prodrugs in rat plasma were 5–119-fold greater than those in dog plasma. The prodrugs with promoieties (C(=O)CH(R)CH₃) based on propionic acid showed marked preference for hydrolysis of the (R)-enantiomers in plasma from both species (R/S ratio 2.5–18.2). On the other hand, the hepatic hydrolytic activities of prodrugs were greater in dog than rat, especially in cytosolic fractions. The hydrolytic activity was predominantly located in microsomes of the liver in rat, while the cytosol also contributed to hepatic hydrolysis in dog. Hepatic microsomal hydrolysis in dog showed a preference for the (R)-isomers except acetyl- and propionyl-PL. Interestingly, in rat liver all types of prodrugs with substituents of small carbon number showed (S)-preference for hydrolysis. The hydrolyses of (R)- and (S)-isomers of straight chain acyl esters in rat liver microsomes were linearly and parabolically related with the carbon number of substituents, respectively, while these relationships were linear for both isomers in dogs. Chirality 9:661–666, 1997. © 1997 Wiley-Liss, Inc.     

Structure-activity relationship study of human liver microsomes-catalyzed hydrolysis rate of ester prodrugs of MENT by comparative molecular field analysis (CoMFA)

A series of MENT esters (3-71) was designed, prepared and tested to study the structure-activity relationship (SAR) of the hydrolysis rate with human liver microsomes of these prodrugs. Compounds were obtained covering a wide range of metabolic stability. The results are useful for the proper selection of prodrugs for different pharmaceutical formulations to deliver the potent and prostate-sparing androgen MENT. The MENT esters can especially be administered for male hormone replacement therapy and male contraception. Comparative molecular field analysis (CoMFA) was applied to a dataset of 28 esters, for which ED50 values could be obtained. The CoMFA model where the electrostatic and H-bond molecular fields were combined turned out to be most predictive. Despite the limited size of the dataset, CoMFA can help to rationalize the SAR of the ester hydrolysis rate of ester prodrugs of MENT.     

In vitro metabolism of fipronil by human and rat cytochrome P450 and its interactions with testosterone and diazepam

Fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1H-pyrazole-3-carbonitrile) is a highly active, broad spectrum insecticide from the phenyl pyrazole family, which targets the gamma-amino butyric acid (GABA) receptor. Although fipronil is presently widely used as an insecticide and acaricide, little information is available with respect to its metabolic fate and disposition in mammals. This study was designed to investigate the in vitro human metabolism of fipronil and to examine possible metabolic interactions that fipronil may have with other substrates. Fipronil was incubated with human liver microsomes (HLM) and several recombinant cytochrome P450 (CYP) isoforms obtained from BD Biosciences. HPLC was used for metabolite identification and quantification. Fipronil sulfone was the predominant metabolite via CYP oxidation. The K(m) and V(max) values for human liver microsomes are 27.2 microM and 0.11 nmol/mg proteinmin, respectively; for rat liver microsomes (RLM) the K(m) and V(max) are 19.9 microM and 0.39 nmol/mg proteinmin, respectively. CYP3A4 is the major isoform responsible for fipronil oxidation in humans while CYP2C19 is considerably less active. Other human CYP isoforms have minimal or no activity toward fipronil. Co-expression of cytochrome b(5) (b(5)) is essential for CYP3A4 to manifest high activity toward fipronil. Ketoconazole, a specific inhibitor of CYP3A4, inhibits 78% of the HLM activity toward fipronil at a concentration of 2 microM. Oxidative activity toward fipronil in 19 single-donor HLMs correlated well with their ability to oxidize testosterone. The interactions of fipronil and other CYP3A4 substrates, such as testosterone and diazepam, were also investigated. Fipronil metabolism was activated by testosterone in HLM but not in CYP3A4 Supersomes. Testosterone 6beta-hydroxylation in HLM was inhibited by fipronil. Fipronil inhibited diazepam demethylation but had little effect on diazepam hydroxylation. The results suggest that fipronil has the potential to interact with a wide range of xenobiotics or endogenous chemicals that are CYP3A4 substrates and that fipronil may be a useful substrate for the characterization of CYP3A4 in HLM.     

Chlorzoxazone esters of some non-steroidal anti-inflammatory (NSAI) carboxylic acids as mutual prodrugs: Design,synthesis, pharmacological investigations and docking studies

The discovery of the inducible isoform of cyclooxygenase enzyme (COX-2) spurred the search for anti-inflammatory agents devoid of the undesirable effects associated with classical NSAIDs. New chlorzoxazone ester prodrugs (6–8) of some acidic NSAIDs (1–3) were designed, synthesized and evaluated as mutual prodrugs with the aim of improving the therapeutic potency and retard the adverse effects of gastrointestinal origin. The structure of the synthesized mutual ester prodrugs (6–8) were confirmed by IR, ¹H NMR, mass spectroscopy (MS) and their purity was ascertained by TLC and elemental analyses. In vitro chemical stability revealed that the synthesized ester prodrugs (6–8) are chemically stable in hydrochloric acid buffer pH 1.2 as a non-enzymatic simulated gastric fluid (SGF) and in phosphate buffer pH 7.4 as non-enzymatic simulated intestinal fluid (SIF). In 80% human plasma, the mutual prodrugs were found to be susceptible to enzymatic hydrolysis at relatively faster rate (t_1/2 ≈ 37 and 34 min for prodrugs 6 and 7, respectively). Mutual ester prodrugs (6–8) were evaluated for their anti-inflammatory and muscle relaxation activities. Scanning electromicrographs of the stomach showed that the ester prodrugs induced very little irritancy in the gastric mucosa of rats after oral administration for 4 days. In addition, docking of the mutual ester prodrugs (6–8) into COX-2 active site was conducted in order to predict the affinity and orientation of these prodrugs at the enzyme active site.     

Synthesis, hydrolytic activation and cytotoxicity of etoposide prodrugs

Two 4'-propylcarbonoxy derivatives (2,3) of etoposide (1), a topoisomerase II inhibitor, were synthesized and evaluated as potential prodrugs for anticancer therapy. Their activation via hydrolysis mechanisms was determined as a function of pH in buffer solutions, in human serum and in the presence of carboxyl ester hydrolase. Cytotoxicity was determined on various tumor cell lines and compared to the parent compound. On cell lines exhibiting resistance to etoposide we observed an enhanced cytotoxicity of the prodrugs of up to three orders of magnitude.     

Inhibition of fipronil and nonane metabolism in human liver microsomes and human cytochrome P450 isoforms by chlorpyrifos

Previous studies have established that chlorpyrifos (CPS), fipronil, and nonane can all be metabolized by human liver microsomes (HLM) and a number of cytochrome P450 (CYP) isoforms. However, metabolic interactions between these three substrates have not been described. In this study the effect of either coincubation or preincubation of CPS with HLM or CYP isoforms with either fipronil or nonane as substrate was investigated. In both co- and preincubation experiments, CPS significantly inhibited the metabolism of fipronil or nonane by HLM although CPS inhibited the metabolism of fipronil more effectively than that of nonane. CPS significantly inhibited the metabolism of fipronil by CYP3A4 as well as the metabolism of nonane by CYP2B6. In both cases, preincubation with CPS caused greater inhibition than coincubation, suggesting that the inhibition is mechanism based.     

In vitro metabolism of carbofuran by human, mouse, and rat cytochrome P450 and interactions with chlorpyrifos, testosterone, and estradiol

Carbofuran is a carbamate pesticide used in agricultural practice throughout the world. Its effect as a pesticide is due to its ability to inhibit acetylcholinesterase activity. Though carbofuran has a long history of use, there is little information available with respect to its metabolic fate and disposition in mammals. The present study was designed to investigate the comparative in vitro metabolism of carbofuran from human, rat, and mouse liver microsomes (HLM, RLM, MLM, respectively), and characterize the specific enzymes involved in such metabolism, with particular reference to human metabolism. Carbofuran is metabolized by cytochrome P450 (CYP) leading to the production of one major ring oxidation metabolite, 3-hydroxycarbofuran, and two minor metabolites. The affinity of carbofuran for CYP enzymes involved in the oxidation to 3-hydroxycarbofuran is significantly less in HLM (K_m = 1.950 mM) than in RLM (K_m = 0.210 mM), or MLM (K_m = 0.550 mM). Intrinsic clearance rate calculations indicate that HLM are 14-fold less efficient in the metabolism of carbofuran to 3-hydroxycarbofuran than RLM or MLM. A screen of 15 major human CYP isoforms for metabolic ability with respect to carbofuran metabolism demonstrated that CYP3A4 is the major isoform responsible for carbofuran oxidation in humans. CYP1A2 and 2C19 are much less active while other human CYP isoforms have minimal or no activity toward carbofuran. In contrast with the human isoforms, members of the CYP2C family in rats are likely to have a primary role in carbofuran metabolism. Normalization of HLM data with the average levels of each CYP in native HLM, indicates that carbofuran metabolism is primarily mediated by CYP3A4 (percent total normalized rate (% TNR) = 77.5), although CYP1A2 and 2C19 play ancillary roles (% TNR = 9.0 and 6.0, respectively). This is substantiated by the fact that ketoconazole, a specific inhibitor of CYP3A4, is an excellent inhibitor of 3-hydroxycarbofuran formation in HLM (IC₅₀: 0.31 μM). Chlorpyrifos, an irreversible non-competitive inhibitor of CYP3A4, inhibits the formation of 3-hydroxycarbofuran in HLM (IC₅₀: 39 μM). The use of phenotyped HLM demonstrated that individuals with high levels of CYP3A4 have the greatest potential to metabolize carbofuran to its major metabolite. The variation in carbofuran metabolism among 17 single-donor HLM samples is over 5-fold and the best correlation between CYP isoform activity and carbofuran metabolism was observed with CYP3A4 (r² = 0.96). The interaction of carbofuran and the endogenous CYP3A4 substrates, testosterone and estradiol, were also investigated. Testosterone metabolism was activated by carbofuran in HLM and CYP3A4, however, less activation was observed for carbofuran metabolism by testosterone in HLM and CYP3A4. No interactions between carbofuran and estradiol metabolism were observed.     

Synthesis,molecular docking,and biological evaluation of 3-oxo-2-tolylhydrazinylidene-4,4,4-trifluorobutanoates bearing higher and natural alcohol moieties as new selective carboxylesterase inhibitors

To search for effective and selective inhibitors of carboxylesterase (CES), a series of 3-oxo-2-tolylhydrazinylidene-4,4,4-trifluorobutanoates bearing higher or natural alcohol moieties was synthesized via pre-transesterification of ethyl trifluoroacetylacetate with alcohols to isolate transesterificated oxoesters as lithium salts, which were then subjected to azo coupling with tolyldiazonium chloride. Inhibitory activity against porcine liver CES, along with two structurally related serine hydrolases, acetylcholinesterase and butyrylcholinesterase, were investigated using enzyme kinetics and molecular docking. Kinetics studies demonstrated that the tested keto-esters are reversible and selective mixed-type CES inhibitors. Analysis of X-ray crystallographic data together with our IR and NMR spectra and QM calculations indicated that the Z-isomers were the most stable. The kinetic data were well explained by the molecular docking results of the Z-isomers, which showed specific binding of the compounds in the CES catalytic active site with carbonyl oxygen atoms in the oxyanion hole and non-specific binding outside it. Some compounds were studied as inhibitors of the main human isozymes involved in biotransformation of ester-containing drugs, hCES1 and hCES2. Esters of geraniol (3d) and adamantol (3e) proved to be highly active and selective inhibitors of hCES2, inhibiting the enzyme in the nanomolar range, whereas esters of borneol (3f) and isoborneol (3g) were more active and selective against hCES1. Computational ADMET studies revealed that all test compounds had excellent intestinal absorption, medium blood-brain barrier permeability, and low hERG liability risks. Moreover, all test compounds possessed radical-scavenging properties and low acute toxicity. Overall, the results indicate that members of this novel series of esters have the potential to be good candidates as hCES1 or hCES2 inhibitors for biomedicinal applications.     

Synthesis and in vitro stability of amino acid prodrugs of 6-β-naltrexol for microneedle-enhanced transdermal delivery

A small library of amino acid ester prodrugs of 6-β-naltrexol (NTXOL, 1) was prepared in order to investigate the candidacy of these prodrugs for microneedle-enhanced transdermal delivery. Six amino acid ester prodrugs were synthesized (6a–f). 6b, 6d, and 6e were stable enough at skin pH (pH 5.0) to move forward to studies in 50% human plasma. The lead compound (6e) exhibited the most rapid bioconversion to NTXOL in human plasma (t_1/2 = 2.2 ± 0.1 h).     

Dinitroglyceryl and diazen-1-ium-1,2-diolated nitric oxide donor ester prodrugs of aspirin,indomethacin and ibuprofen: Synthesis,biological evaluation and nitric oxide release studies

A new group of hybrid nitric oxide (NO) releasing anti-inflammatory (AI) ester prodrugs (NONO-NSAIDs) wherein a 1,3-dinitrooxy-2-propyl (12a–c), or O²-acetoxymethyl-1-[2-(methyl)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (14a–c), NO-donor moiety is directly attached to the carboxylic acid group of aspirin, indomethacin or ibuprofen were synthesized. NO release from the dinitrooxypropyl, or diazen-1-ium-1,2-diolate, ester prodrugs was increased substantially upon incubation in the presence of l-cysteine (12a–c) or rat serum (14a–c). The ester prodrugs (12a–c, 14a–c), which did not inhibit the COX-1 isozyme, exhibited modest inhibitory activity against the COX-2 isozyme. The NONO-NSAIDs 12a–c and 14a–c exhibited in vivo AI activity that was similar to that exhibited by the parent drug aspirin, indomethacin or ibuprofen when the same oral dose (μmol/kg) was administered. These similarities in oral potency profiles suggest these NONO-NSAIDs act as classical prodrugs that require metabolic activation by esterase-mediated hydrolysis. Hybrid NO-donor/anti-inflammatory prodrugs of this type (NONO-NSAIDs) offer a potential drug design concept targeted toward the development of anti-inflammatory drugs with reduced adverse gastrointestinal effects.