The distribution of esterases in the skin of the minipig

Skin esterases serve an important pharmacological function as they can be utilised for activation of topically applied ester prodrugs. Understanding the nature of these enzymes, with respect to their role and local activity, is essential to defining the efficacy of ester prodrugs. Minipigs are used as models to study the kinetics of absorption of topically applied drugs. Their skin has structural properties very similar to human skin. However, regional distribution differences in esterase activity from site-to-site could influence cross-species extrapolation. Investigation of the regional site variation of minipig skin esterase activity will facilitate standardization of topically applied drug studies. Furthermore, the characterization of regional skin variation, will aid in translation of minipig results to better predictions of human esterase activity. Here we report the variation in rates of hydrolysis by minipig skin taken from different regional sites, using the esterase-selective substrates: phenyl valerate (carboxylesterase), phenyl acetate (arylesterase) and p-nitrophenyl acetate (general esterase). Skin from ears and back of male minipig showed higher activity than female. Skin from minipig ears and the back showed the highest level of esterase activity and was similar to human breast skin used in vitro absorption studies. These results suggest that skin from the minipig back is an appropriate model for preclinical human skin studies, particularly breast skin. This study supports the use of the minipig, with topical application to the back, as a model for the investigation of pharmacokinetics and metabolism of ester prodrugs.     

Hydrolysis of a series of parabens by skin microsomes and cytosol from human and minipigs and in whole skin in short-term culture

Parabens are esters of 4-hydroxybenzoic acid and used as anti-microbial agents in a wide variety of toiletries, cosmetics and pharmaceuticals. It is of interest to understand the dermal absorption and hydrolysis of parabens, and to evaluate their disposition after dermal exposure and their potential to illicit localised toxicity. The use of minipig as a surrogate model for human dermal metabolism and toxicity studies, justifies the comparison of paraben metabolism in human and minipig skin. Parabens are hydrolysed by carboxylesterases to 4-hydroxybenzoic acid. The effects of the carboxylesterase inhibitors paraoxon and bis-nitrophenylphosphate provided evidence of the involvement of dermal carboxylesterases in paraben hydrolysis. Loperamide, a specific inhibitor of human carboxylesterase-2 inhibited butyl- and benzylparaben hydrolysis in human skin but not methylparaben or ethylparaben. These results show that butyl- and benzylparaben are more selective substrates for human carboxylesterase-2 in skin than the other parabens examined. Parabens applied to the surface of human or minipig skin were absorbed to a similar amount and metabolised to 4-hydroxybenzoic acid during dermal absorption. These results demonstrate that the minipig is a suitable model for man for assessing dermal absorption and hydrolysis of parabens, although the carboxylesterase profile in skin differs between human and minipig.     

Comparison of Skin Esterase Activities from Different Species

Purpose Many topically applied drugs contain esters that are hydrolyzed in the skin. Minipigs have emerged as potential models of human dermatology and, in some aspects, may be superior to commonly used rat skin. The aims of this study were to evaluate the suitability of minipig and rat skin as in vitro models of human epidermal esterase activity. Methods Naphthyl acetate and para-nitrophenyl acetate were tested as prototypical substrates of carboxylesterases from skin, plasma, and liver. Reaction products were monitored by high-performance liquid chromatography/ultraviolet analysis. Results Hydrolysis efficiency in skin was higher than plasma, but lower than liver. The esterase efficiency of rat skin microsomes (580–1100 min⁻¹ mg⁻¹) was two to three orders of magnitude higher than human (1.3–4.2 min⁻¹ mg⁻¹) and minipig microsomes (1.2–4.2 min⁻¹ mg⁻¹). Rat skin cytosol (80–100 min⁻¹ mg⁻¹) was 2- to 10-fold more efficient than human (2.4–67 min⁻¹ mg⁻¹) or minipig cytosol (18–61 min⁻¹ mg⁻¹). Most importantly, human skin fractions displayed kinetics of hydrolysis very similar to minipig skin. Conclusions These studies show minipig skin as an appropriate, potentially valuable model for human epidermal ester metabolism and support the use of minipig skin in preclinical development of topically applied compounds.     

Biochemical properties of blood esmolol esterase

The blood esterase mediating the hydrolysis of esmolol was characterized in several different species including man. In contrast to most ester-containing drugs, hydrolysis of esmolol was mediated by an esterase in the cytosol of red blood cells (RBC) in man and dogs and not in plasma or RBC membrane. Species differences in the esterase activity existed. Guinea pig and rat blood esterase activities were much greater than those in the dog followed by those in man. In addition, the esterase activity in rat and guinea pig blood was localized in plasma and not in RBC. Purified human serum cholinesterase, human RBC membrane acetylcholinesterase, human hemoglobin, human carbonic anhydrases A and B, and human and dog serum albumin were all inactive against esmolol. Esmolol esterase activity in human and dog blood was inhibited by sodium fluoride, EDTA, and p-hydroxymercuribenzoate, but not by echothiophate, eserine, and acetazolamide. In contrast, echothiophate and sodium fluoride, but not eserine, inhibited the esterase activity in rat and guinea pig plasma. Metabolic interaction studies indicated that acetylcholine, succinylcholine, procaine, and chloroprocaine did not interfere with the metabolism of esmolol by human and dog blood. Based on the results, it appeared that an arylesterase in human and dog RBC cytosol mediated the hydrolysis of esmolol while an aliphatic esterase mediated the hydrolysis of esmolol in guinea pig and rat plasma.     

Identification and characterization of hepatic carboxylesterases hydrolyzing hydrocortisone esters

The present study has provided evidence for the existence of three distinct carboxylesterases involved in the hydrolysis of steroid esters, where two enzymes are possibly responsible for the metabolism of hydrocortisone hemisuccinate (HCHS) at pH 5.5 and 8.0, and a third enzyme for the metabolism of hydrocortisone acetate (HCAC) at pH 8.0, in isolated rat liver microsomes. The activity of all three enzymes in rat liver was induced significantly by the administration of phenobarbital while no such function in enzyme activity was observed in animals receiving 3-methylcholanthrene or benzo[a] pyrene under similar experimental conditions. The increase in the activity of HCHS esterase I (HCHS-E1) active at pH 5.5, HCHS esterase II (HCHS-E2) active at pH 8.0, and HCAC esterase (HCAC-E) was approximately 7 to 8, 3- and 3-fold respectively. On the other hand, the degree of induction of nonspecific microsomal carboxylesterase acting on p-nitrophenylacetate (PNPA) was significantly less. The Km values for the hydrolysis of HCHS at pH 5.5 and 8.0 and HCAC by rat liver microsomes obtained from control rats were 2.45, 2.02 and 1.6 mM, respectively, and these Km values were not changed significantly in preparations obtained from rats treated with phenobarbital. The distinct in vitro responses displayed by hepatic microsomal steroid esterases to various inhibitors were able to distinguish three different enzymes which also differed from nonspecific carboxylesterases. The activity of HCAC-E was inhibited by NaAsO2 and AgNO3 while that of HCHS-E1 and HCHS-E2 remained unaffected. Selective inhibition of HCHS-E1 by NaF, HgCl2 and p-chloromercuribenzoate and that of HCHS-E2 by NiSO4 indicated the possible existence of different enzymes or isozymes of a carboxylesterase catalyzing HCHS hydrolysis. The effects elicited by the inhibitors on the activity of PNPA esterase were different from those observed with steroid esterases. Furthermore, the present study has also indicated species variations in the distribution of steroid esterases in the livers of rat, mouse, dog and cat.     

Metabolism of fluroxypyr, fluroxypyr methyl ester, and the herbicide fluroxypyr methylheptyl ester. II: in rat skin homogenates.

Fluroxypyr methyl ester (FPM) and the herbicide fluroxypyr methylheptyl ester (FPMH) are completely hydrolyzed during penetration through human and rat skin in vitro to the acid metabolite, fluroxypyr (FP) (). This article presents additional studies to determine the enzyme kinetics (K(m) and V(max)) of this ester hydrolysis, using crude rat whole-skin homogenate. Both FPM and FPMH were extensively metabolized in rat skin homogenates to the acid metabolite, FP. In no instance were any other metabolites detected. FPM was essentially hydrolyzed completely within 1 h. In FPMH incubations, there was still parent ester present after 24 h at all concentrations tested. The kinetics of hydrolysis of the two esters were different: V(max) was approximately 3-fold greater for FPM than FPMH (1400 and 490 micromol FP/min/g of tissue, respectively); however, K(m) values were very similar, 251 and 256 microM, respectively. Preliminary inhibitory studies suggest that FPM and FPMH are hydrolyzed by a carboxylesterase, because this reaction was inhibited by bis-p-nitrophenyl phosphate. Mercuric chloride (an inhibitor of A-esterase and arylesterase) and eserine (a cholinesterase inhibitor) had no inhibitory effect on the hydrolysis of FPM or FPMH. Taken together with the data presented by, it can be concluded that no parent ester will pass through the skin in vivo, only the metabolite, FP. Therefore, first pass metabolism will be complete before these compounds reach the systemic circulation.     

Hydrolysis of ester- and amide-type drugs by the purified isoenzymes of nonspecific carboxylesterase from rat liver

Five purified carboxylesterases from rat liver microsomes show a differing capacity for the hydrolysis of ester- and amide-type drugs. The two closely related enzymes that are responsible for the microsomal hydrolysis of palmitoyl-CoA and long chain monoacylglycerides exhibit the highest propanidid-and aspirin-cleaving rates. The predominant nonspecific esterase of microsomes is responsible for the hydrolysis of procaine, clofibrate, isoarecaidine esters, butanilicaine, octanoylamide, and possibly butyryl thiocholine. Finally, the palmitoyl carnitine-cleaving esterase splits phenacetin and acetanilide. The purified nonspecific esterase with the lowest isoelectric point is not involved in the metabolism of the drugs mentioned.     

Alteration of hepatic carboxylesterase activity by soman: inhibition in vitro and enhancement in vivo

1. Hydrolysis of the drug esters procaine, chloramphenicol succinate, and prednisolone succinate was studied. Addition of soman to guinea pig liver microsomes caused a dose-dependent inhibition of hydrolysis of all three substrates; at the highest soman concentration (1 microM), ester hydrolysis was totally abolished. 2. Ester hydrolysis was also measured in liver microsomes from guinea pigs pretreated with soman at a low dose (10% of LD50) or at a high dose (90% of LD50) either 1 h or 12 h before killing. Plasma-cholinesterase activity was decreased in all pretreated animals. Liver carboxylesterase activity, measured with the three drug substrates and by hydrolysis of 4-nitrophenyl acetate was increased by all pretreatments. 3. This enhancing effect varies with the substrate and increases with dose of soman. The 12 h pretreatment produced a greater increase in activity than did the 1 h pretreatment. 4. These studies indicate that soman is a potent inhibitor of carboxylesterase activity in vitro but increases the activity of the liver enzyme when administered in vivo.     

Involvement of human blood arylesterases and liver microsomal carboxylesterases in nafamostat hydrolysis

Metabolism of nafamostat, a clinically used serine protease inhibitor, was investigated with human blood and liver enzyme sources. All the enzyme sources examined (whole blood, erythrocytes, plasma and liver microsomes) showed nafamostat hydrolytic activity. V(max) and K(m) values for the nafamostat hydrolysis in erythrocytes were 278 nmol/min/mL blood fraction and 628 microM; those in plasma were 160 nmol/min/mL blood fraction and 8890 microM, respectively. Human liver microsomes exhibited a V(max) value of 26.9 nmol/min/mg protein and a K(m) value of 1790 microM. Hydrolytic activity of the erythrocytes and plasma was inhibited by 5, 5'-dithiobis(2-nitrobenzoic acid), an arylesterase inhibitor, in a concentration-dependent manner. In contrast, little or no suppression of these activities was seen with phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DFP), bis(p-nitrophenyl)phosphate (BNPP), BW284C51 and ethopropazine. The liver microsomal activity was markedly inhibited by PMSF, DFP and BNPP, indicating that carboxylesterase was involved in the nafamostat hydrolysis. Human carboxylesterase 2 expressed in COS-1 cells was capable of hydrolyzing nafamostat at 10 and 100 microM, whereas recombinant carboxylesterase 1 showed significant activity only at a higher substrate concentration (100 microM). The nafamostat hydrolysis in 18 human liver microsomes correlated with aspirin hydrolytic activity specific for carboxylesterase 2 (r=0.815, p<0.01) but not with imidapril hydrolysis catalyzed by carboxylesterase 1 (r=0.156, p=0.54). These results suggest that human arylesterases and carboxylesterase 2 may be predominantly responsible for the metabolism of nafamostat in the blood and liver, respectively.     

Thenoyltrifluoroacetone, a potent inhibitor of carboxylesterase activity

Thenoyltrifluoroacetone (TTFA), a conventional mitochondrial complex II inhibitor, was found to inhibit purified porcine liver carboxylesterase non-competitively with a K(i) of 0.61x10(-6)M and an IC(50) of 0.54x10(-6)M. Both rat plasma and liver mitochondrial esterases were inhibited in a concentration-dependent fashion. Results indicate that TTFA is a potent inhibitor of carboxylesterase activity, in addition to its ability to inhibit mitochondrial complex II activity. Therefore, caution is warranted in using TTFA as a mitochondrial complex inhibitor in combination with esterase substrates, such as fluorescence probes or vitamin E esters.     

Prodrugs for systemic bioreductive activation-independent delivery of phyllohydroquinone, an active form of phylloquinone (vitamin K1). 1: Preparation and in vitro evaluation

With the aim of overcoming the delivery problems (water-solubility and bioreductive activation problems) of phyllohydroquinone (PKH), an active form of phylloquinone (PK, vitamin K1), the N,N-dimethylglycine esters of phyllohydroquinone (1-mono, 1; 4-mono, 2; and 1,4-bis, 3) have been synthesized and assessed in vitro as a prodrug for the systemic bioreductive activation-independent delivery of PKH. The hydrochloride salts of the esters were found to be quite soluble in water. Hydrolysis of the esters in rat liver S9 fraction, rat plasma and phosphate buffer, pH 7.4, at 37 degrees C, was kinetically studied in the presence and absence of an esterase inhibitor. The hydrolysis was catalyzed by esterases located in rat liver and rat plasma and quantitatively yielded PKH. The enzymatic cleavage and the vitamin K-dependent carboxylation activity of the esters in the rat liver microsome preparation at pH 7.2 and 25 degrees C were studied. The regeneration of PKH from the esters was catalyzed by carboxylesterases located in the rat liver microsome, and the order was as follows: 1 > 3 > 2. The carboxylation was stimulated by selected ester 1 in the absence of dithiothreitol, an activator of the vitamin K cycle. The carboxylation activity of 1 was strongly inhibited in the presence of eserine, a carboxylesterase inhibitor. Compound 1 could also stimulate carboxylase under warfarin-poisoning conditions, where the vitamin K cycle was strongly inhibited. These results indicated that these highly water-soluble and liver-esterase hydrolyzable ester derivatives of PKH are potential candidates for parenteral prodrugs which can thus achieve the systemic bioreductive activation-independent delivery of PKH.     

Hydrolysis of pyrethroids by human and rat tissues: examination of intestinal, liver and serum carboxylesterases

Hydrolytic metabolism of pyrethroid insecticides in humans is one of the major catabolic pathways that clear these compounds from the body. Rodent models are often used to determine the disposition and clearance rates of these esterified compounds. In this study the distribution and activities of esterases that catalyze pyrethroid metabolism have been investigated in vitro using several human and rat tissues, including small intestine, liver and serum. The major esterase in human intestine is carboxylesterase 2 (hCE2). We found that the pyrethroid trans-permethrin is effectively hydrolyzed by a sample of pooled human intestinal microsomes (5 individuals), while deltamethrin and bioresmethrin are not. This result correlates well with the substrate specificity of recombinant hCE2 enzyme. In contrast, a sample of pooled rat intestinal microsomes (5 animals) hydrolyze trans-permethrin 4.5-fold slower than the sample of human intestinal microsomes. Furthermore, it is demonstrated that pooled samples of cytosol from human or rat liver are approximately 2-fold less hydrolytically active (normalized per mg protein) than the corresponding microsomal fraction toward pyrethroid substrates; however, the cytosolic fractions do have significant amounts (approximately 40%) of the total esteratic activity. Moreover, a 6-fold interindividual variation in carboxylesterase 1 protein expression in human hepatic cytosols was observed. Human serum was shown to lack pyrethroid hydrolytic activity, but rat serum has hydrolytic activity that is attributed to a single CE isozyme. We purified the serum CE enzyme to homogeneity to determine its contribution to pyrethroid metabolism in the rat. Both trans-permethrin and bioresmethrin were effectively cleaved by this serum CE, but deltamethrin, esfenvalerate, alpha-cypermethrin and cis-permethrin were slowly hydrolyzed. Lastly, two model lipase enzymes were examined for their ability to hydrolyze pyrethroids. However, no hydrolysis products could be detected. Together, these results demonstrate that extrahepatic esterolytic metabolism of specific pyrethroids may be significant. Moreover, hepatic cytosolic and microsomal hydrolytic metabolism should each be considered during the development of pharmacokinetic models that predict the disposition of pyrethroids and other esterified compounds.     

Use of ab initio calculations to predict the biological potency of carboxylesterase inhibitors

Carboxylesterases are important enzymes responsible for the hydrolysis and metabolism of numerous pharmaceuticals and xenobiotics. These enzymes are potently inhibited by trifluoromethyl ketone containing (TFK) inhibitors. We demonstrated that the ketone hydration state was affected by the surrounding chemical moieties and was related to inhibitor potency, with inhibitors that favored the gem-diol conformation exhibiting greater potency. Ab initio calculations were performed to determine the energy of hydration of the ketone, and the values were correlated with esterase inhibition data for a series of carboxylesterase inhibitors. This system was examined in three different mammalian models (human liver microsomes, murine liver microsomes, and commercial porcine liver esterase) and in an insect enzyme preparation (juvenile hormone esterase). In all cases, the extent of ketone hydration was strongly correlated with biological potency. Our results showed a very strong correlation with the extent of hydration, accounting for 94% of activity for human liver microsome esterase inhibition (p < 0.01). The atomic charge on the carbon atom of the carbonyl group in the TFK also strongly correlated with inhibitor potency, accounting for 94% of inhibition activity in human liver microsomes (p < 0.01). In addition, we provide crystallographic evidence of intramolecular hydrogen bonding in sulfur-containing inhibitors and relate these data to gem-diol formation. This study provides insight into the mechanism of carboxylesterase inhibition and raises the possibility that inhibitors that too strongly favor the gem-diol configuration have decreased potency due to low rate of ketone formation.     

A radiochemical assay method for carboxylesterase, and comparison of enzyme activity towards the substrates methyl [1-14C] butyrate and 4-nitrophenyl butyrate

A radiochemical assay for carboxylesterase based on the substrate methyl[1-14C]butyrate is described. The blank value corresponds to 1.04 micrograms (liver)-1.44 mg (plasma) of tissues with the highest and lowest activity respectively, which constitute the sensitivity of the method. The hydrolysis of methyl butyrate and 4-nitrophenyl butyrate by plasma, liver, lung, heart, diaphragm, cerebrum, kidney and duodenum of rat have been compared. The results showed that the two substrates were hydrolysed preferentially by two different groups of the enzyme. The effect of selective esterase inhibitors showed that both groups can be characterized as carboxylesterase, because bis-4-nitrophenyl phosphate inhibits the hydrolysis of both substrates, physostigmine has only a slight effect and EDTA is no inhibitor. The exception is the enzyme in the duodenum which is inhibited by all three inhibitors. The effect of phenobarbital induction and soman treatment on enzyme activity towards the two substrates were similar. Sex difference in the plasma activity towards methyl butyrate, but not 4-nitrophenyl butyrate, indicates that the group of carboxylesterase preferentially hydrolyzing 4-nitrophenyl butyrate may be the most important for the detoxification of soman.     

Differential inhibition of hepatic, pancreatic, and plasma fatty acid ethyl ester synthase by tri-o-tolylphosphate in rats

Fatty acid conjugation of alcohols, catalyzed by fatty acid ethyl ester synthase (FAEES), results in the formation of lipophilic esters. Although the activity of FAEES is reported in almost all organs, including plasma, the interrelationship among various proteins expressing FAEES activity in different organs/tissues is not well understood. Earlier, we have reported an inhibition of FAEES activity in human hepatoma cells by tri-o-tolylphosphate (TOTP; serine esterase inhibitor). The present study was undertaken to further characterize the hepatic, plasma, and pancreatic FAEES in rats after ip injection of 10, 25, 50, or 100 mg/kg TOTP in corn oil or vehicle alone. After 18 h, animals were euthanized and FAEES activity in the plasma and postnuclear fractions of hepatic and pancreatic homogenates were assayed by measuring the ester formation following incubation with [1-(14)C]oleic acid and ethanol or methanol as substrates. Significant inhibition of FAEES activity was observed in hepatic postnuclear fraction. The esterase activity also showed a pattern similar to fatty acid ethyl and methyl ester synthesizing activity. A trend similar to hepatic synthesizing and hydrolyzing activities was also found in the plasma of TOTP-treated rats. However, no inhibition of synthetic activity toward formation of fatty acid ethyl or methyl esters or p-nitrophenyl acetate hydrolyzing activity was observed in the pancreas of rats after TOTP exposure. Our results also show that the protein expressing FAEES activity in the pancreas does not cross-react with antibodies to rat adipose tissue FAEES using Western blot analysis, which recognizes approximately 60- and approximately 84-kDa proteins in the liver and plasma, respectively. Furthermore, the inhibition in liver is at the functional level of enzyme as no change was observed between control and treated animals by immunohistochemistry. We conclude that fatty acid ethyl or methyl ester synthesizing enzyme(s) in the liver and plasma, which are inhibited by TOTP, are different from that present in the pancreas.     

Enzymic hydrolysis of nicotinate esters: comparison between plasma and liver catalysis.

1. The enzymic hydrolysis of a wide series of nicotinic acid esters was investigated using human and rat plasma, and purified hog liver carboxylesterase, and compared with previously published data from rat liver microsomes. Esterase activities were always found to obey Michaelis-Menten kinetics. 2. Rat liver microsomal and plasma enzyme velocities were six orders of magnitude smaller than those of purified hog liver carboxylesterase, and three orders smaller than human plasma activities, but the Km values were of the same magnitude. 3. The binding of nicotinate esters to human plasma esterases, and purified hog liver carboxylesterase, appears to depend mainly on hydrophobic and steric factors.     

In vitro stability and metabolism of O2', O3', O5'-tri-acetyl-N6-(3-hydroxylaniline) adenosine in rat, dog and human plasma: chemical hydrolysis and role of plasma esterases

O2', O3', O5'-tri-acetyl-N(6)-(3-hydroxylaniline)adenosine (WS070117), a new structure-type lipid regulator, is being developed in pre-clinical study. In order to monitor drug kinetics it is essential to understand pre-analytical factors that may affect drug assay. In vitro stability and metabolism were investigated using high-performance liquid chromatography (HPLC) method in this study. The hydrolysis products were identified by HPLC-mass spectrometry (MS)/MS method. The esterases involved in WS070117 hydrolysis was assigned via inhibition rate assay. It was found that WS070117 was chemically unstable in alkaline solutions compared to acidic and near neutral solutions. Enzymatic hydrolysis was even more rapid. Hydrolytic rate constants differ between species, being 4.24, 5.96?×?10(-3) and 6.85?×?10(-2) min(-1) in rat, dog and human plasma at 37°C, respectively. The hydrolysis was catalyzed by plasma esterase because NaF (sodium fluoride: a general esterase inhibitor) inhibited WS070117 hydrolysis and metabolite production. Hydrolysis was fast in rat plasma and was catalysed by carboxylesterase and butyrylcholinesterase. In dog plasma, carboxylesterase, butyrylcholinesterase and paraoxonase were mainly responsible. Butyrylcholinesterase was the major esterase involved in WS070117 hydrolysis in human plasma. The WS070117 hydrolysis in plasma proceeded by gradual loss of acetyl groups. The knowledge of in vitro drug stability and metabolic pathways identified in this study will be essential for future pre-clinical and clinical pharmacokinetics studies.     

Synthesis of new carboxylesterase inhibitors and evaluation of potency and water solubility.

Carboxylesterases are essential enzymes in the hydrolysis and detoxification of numerous pharmaceuticals and pesticides. They are vital in mediating organophosphate toxicity and in activating many prodrugs such as the chemotherapeutic agent CPT-11. It is therefore important to study the catalytic mechanism responsible for carboxylesterase-induced hydrolysis, which can be accomplished through the use of potent and selective inhibitors. Trifluoromethyl ketone (TFK)-containing compounds are the most potent esterase inhibitors described to date. The inclusion of a thioether moiety beta to the carbonyl further increased TFK inhibitor potency. In this study, we have synthesized the sulfone analogues of a series of aliphatic and aromatic substituted thioether TFKs to evaluate their potency and solubility properties. This structural change shifted the keto/hydrate equilibrium from <9% hydrate to >95% hydrate, forming almost exclusively the gem-diol. These new compounds were evaluated for their inhibition of carboxylesterase activity in three different systems, rat liver microsomes, commercial porcine esterase, and juvenile hormone esterase in cabbage looper (Trichoplusia ni) hemolymph. The most potent inhibitor of rat liver carboxylesterase activity was 1,1,1-trifluoro-3-(decane-1-sulfonyl)-propan-2,2-diol, which inhibited 50% of the enzyme activity (IC(50)) at 6.3 +/- 1.3 nM and was 18-fold more potent than its thioether analogue. However, the sulfone derivatives were consistently poorer inhibitors of porcine carboxylesterase activity and juvenile hormone esterase activity, with IC(50) values ranging from low micromolar to millimolar. The compound 1,1,1-trifluoro-3-(octane-1-sulfonyl)-propan-2,2-diol was shown to have a 10-fold greater water solubility than its thioether analogue, 1,1,1-trifluoro-3-octylsulfanyl-propan-2-one (OTFP). These novel compounds provide further evidence of the differences between esterase orthologs, suggesting that additional development of esterase inhibitors may ultimately provide a battery of ortholog and/or isoform selective inhibitors analogous to those available for other complex enzyme families with overlapping substrate specificity.     

Butyrylcholinesterase, paraoxonase, and albumin esterase, but not carboxylesterase, are present in human plasma

The goal of this work was to identify the esterases in human plasma and to clarify common misconceptions. The method for identifying esterases was nondenaturing gradient gel electrophoresis stained for esterase activity. We report that human plasma contains four esterases: butyrylcholinesterase (EC 3.1.1.8), paraoxonase (EC 3.1.8.1), acetylcholinesterase (EC 3.1.1.7), and albumin. Butyrylcholinesterase (BChE), paraoxonase (PON1), and albumin are in high enough concentrations to contribute significantly to ester hydrolysis. However, only trace amounts of acetylcholinesterase (AChE) are present. Monomeric AChE is seen in wild-type as well as in silent BChE plasma. Albumin has esterase activity with alpha- and beta-naphthylacetate as well as with p-nitrophenyl acetate. Misconception #1 is that human plasma contains carboxylesterase. We demonstrate that human plasma contains no carboxylesterase (EC 3.1.1.1), in contrast to mouse, rat, rabbit, horse, cat, and tiger that have high amounts of plasma carboxylesterase. Misconception #2 is that lab animals have BChE but no AChE in their plasma. We demonstrate that mice, unlike humans, have substantial amounts of soluble AChE as well as BChE in their plasma. Plasma from AChE and BChE knockout mice allowed identification of AChE and BChE bands without the use of inhibitors. Human BChE is irreversibly inhibited by diisopropylfluorophosphate, echothiophate, and paraoxon, but mouse BChE spontaneously reactivates. Since human plasma contains no carboxylesterase, only BChE, PON1, and albumin esterases need to be considered when evaluating hydrolysis of an ester drug in human plasma.     

Stereospecific and regioselective hydrolysis of cannabinoid esters by ES46.5K, an esterase from mouse hepatic microsomes, and its differences from carboxylesterases of rabbit and porcine liver

The properties of ES46.5K, an esterase from mouse hepatic microsomes, were compared with those of carboxylesterases from rabbit and porcine liver. The inhibitory profile with a serine hydrolase inhibitor (bis-p-nitrophenylphosphate) and detergents (sodium dodecylsulfate, Emulgen 911) was different between ES46.5K and the carboxylesterases. Bis-p-nitrophenylphosphate (0.1 mM) markedly inhibited the catalytic activity of the carboxylesterases but not that of ES46.5K. Emulgen 911 (0.05-0.25%) inhibited the catalytic activity of the carboxylesterases, whereas the detergent conversely stimulated that of ES46.5K by 150%. The two carboxylesterases catalyzed the hydrolysis of acetate esters of tetrahydrocannabinol (THC) analogues with different side chain lengths (C1-C5), although ES46.5K showed marginal activity only against the acetate of Delta8-tetrahydrocannabiorcol, a methyl side chain derivative of Delta8-THC. ES46.5K hydrolyzed cannabinoid esters stereospecifically and regioselectively. The esterase hydrolyzed 8alpha-acetoxy-Delta9-tetrahydrocannabinol (8alpha-acetoxy-Delta9-THC, 5.62 nmol/min/mg protein), while the enzyme did not hydrolyze 8beta-acetoxy-Delta9-THC, 7alpha-acetoxy-, and 7beta-acetoxy-Delta8-THC at all. In contrast, the carboxylesterases from rabbit and porcine liver hydrolyzed 8beta-acetoxy-Delta9-THC efficiently but not 8alpha-acetoxy-Delta9-THC. ES46.5K hydrolyzed side chain acetoxy derivatives of Delta8-THC at the 3'- and 4'-positions, and a methyl ester of 5'-nor-Delta8-THC-4'-oic acid. The enzyme, however, could not hydrolyze methyl esters of Delta8- and Delta9-THC-11-oic acid, while both carboxylesterases hydrolyzed side chain acetoxy derivatives of Delta8-THC and three methyl esters of THC-oic acids. These differences in stereospecificity and regioselectivity between ES46.5K and carboxylesterases suggest that the configurations of important amino acids for the catalytic activities of these enzymes are different from each other.