Chromatographic study on the specificity of bis-p-nitrophenylphosphate in vivo Identification of labelled proteins of rat liver after intravenous injection of as carboxylesterases and amidases

Block, Wolfram; Arndt, R.

doi:10.1016/0005-2744(78)90106-7

Cited by 29 publications

(13 citation statements)

References 10 publications

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“…Recently, Quinney et al (2005) reported that loperamide, an opioid compound, competitively inhibited CES, especially human CES2 isozyme, hCE2, with a K i of 1.5 M. However, the effect of loperamide on the activity of other hydrolases is not mentioned in detail, and reduction of gastrointestinal motility by loperamide might affect drug absorption in other ways (Callreus et al, 1999). On the other hand, BNPP is well known as a potent and specific inhibitor of CES isozymes (Heymann and Krisch, 1967;Block and Arndt, 1978;Mentlein et al, 1988). We first determined that BNPP noncompetitively inhibited PNPA hydrolysis in the rat jejunal S9 with a K i value of 44.9 Ϯ 4.95 nM.…”

Section: Discussionmentioning

confidence: 99%

“…BNPP specifically combines with CES and suppresses hydrolase activity by noncompetitive inhibition without any modulation of ␣-chymotrypsin, trypsin, acetylcholinesterase, or nonspecific serum cholinesterase (Heymann and Krisch, 1967;Block and Arndt, 1978;Mentlein et al, 1988). In addition, we investigated the hydrolyzing capacity and expression level of rat intestinal CES isozymes in the jejunum and the ileum.…”

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confidence: 99%

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Intestinal First-Pass Metabolism via Carboxylesterase in Rat Jejunum and Ileum

Masaki¹,

Hashimoto²,

Imai³

2007

Drug Metab Dispos

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ABSTRACT:To determine the activity of a major intestinal esterase in the first-pass hydrolysis of O-isovaleryl-propranolol (isovaleryl-PL), a model ester compound, rat intestinal jejunum and blood vessels were perfused simultaneously after inhibition of a carboxylesterase (CES) by bis-p-nitrophenyl phosphate (BNPP). BNPP specifically inhibits approximately 90% of CES activity without influencing aminopeptidase activity or the transport of L-leucyl-p-nitroanilide and p-nitroaniline, nonester compounds. When isovaleryl-PL was perfused into the jejunal lumen after BNPP treatment, its absorption clearance (7.60 ؎ 0.74 l/min) increased approximately 3-fold compared with control, whereas its degradation clearance (32.5 ؎ 5.40 l/min) decreased to 23% of control. Therefore, CES seems to be mainly responsible for the intestinal first-pass hydrolysis of isovaleryl-PL. This finding is consistent with the results from studies of in vitro BNPP inhibition in the mucosal S9 fraction. V max values for valeryl-PL, isovaleryl-PL, and p-nitrophenyl acetate in the jejunal S9 fraction were 1.7-to 2.5-fold higher than that in the ileal S9 fraction, which agreed with the jejunum/ileum ratio (approximately 1.5-fold) of mRNA expression levels for the CES2 isozymes, AB010635 and AY034877. These findings indicated that CESs expressed in the intestine markedly contribute to first-pass hydrolysis in both jejunum and ileum.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Intestinal First-Pass Metabolism via Carboxylesterase in Rat Jejunum and Ileum

Masaki¹,

Hashimoto²,

Imai³

2007

Drug Metab Dispos

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“…Results showed that telaprevir inhibited CatA-mediated cleavage of the fluorogenic peptide substrate, (7-methoxycoumarin-4-yl)acetyl-Arg-Pro-ProGly-Phe-Ser-Ala-Phe-Lys-(2,4-dinitrophenyl)-OH, with an IC 50 value of 0.21 M, whereas telaprevir did not affect the ability of CES1 to hydrolyze 4-NPA (Table 3). BNPP, a known inhibitor of carboxylesterases (18,19), inhibited the hydrolysis of 4-NPA by CES1 (IC 50 ϭ 0.11 M) but did not inhibit the activity of CatA (Table 3). The effect of telaprevir and BNPP on the metabolism of PSI-7851 was studied by following the intracellu- (Fig.…”

Section: Metabolism Of Psi-7851 In Clone Amentioning

confidence: 99%

Mechanism of Activation of PSI-7851 and Its Diastereoisomer PSI-7977

Murakami¹,

Tolstykh²,

Bao³

et al. 2010

Journal of Biological Chemistry

265

308

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A phosphoramidate prodrug of 2-deoxy-2-␣-fluoro-␤-Cmethyluridine-5-monophosphate, PSI-7851, demonstrates potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. PSI-7851 is a mixture of two diastereoisomers, PSI-7976 and PSI-7977, with PSI-7977 being the more active inhibitor of HCV RNA replication in the HCV replicon assay. To inhibit the HCV NS5B RNA-dependent RNA polymerase, PSI-7851 must be metabolized to the active triphosphate form. The first step, hydrolysis of the carboxyl ester by human cathepsin A (CatA) and/or carboxylesterase 1 (CES1), is a stereospecific reaction. Western blot analysis showed that CatA and CES1 are both expressed in primary human hepatocytes. However, expression of CES1 is undetectable in clone A replicon cells. Studies with inhibitors of CatA and/or CES1 indicated that CatA is primarily responsible for hydrolysis of the carboxyl ester in clone A cells, although in primary human hepatocytes, both CatA and CES1 contribute to the hydrolysis. Hydrolysis of the ester is followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the spontaneous elimination of phenol and the production of an alaninyl phosphate metabolite, PSI-352707, which is common to both isomers. The removal of the amino acid moiety of PSI-352707 is catalyzed by histidine triad nucleotide-binding protein 1 (Hint1) to give the 5-monophosphate form, PSI-7411. siRNA-mediated Hint1 knockdown studies further indicate that Hint1 is, at least in part, responsible for converting PSI-352707 to PSI-7411. PSI-7411 is then consecutively phosphorylated to the diphosphate, PSI-7410, and to the active triphosphate metabolite, PSI-7409, by UMP-CMP kinase and nucleoside diphosphate kinase, respectively.Nucleoside analogs have long been the backbone therapy for the treatment of viral diseases such as HIV, HBV, and HSV infections (1-5). Recent studies have suggested that nucleoside analogs may be useful for treating hepatitis C virus (HCV) 3 infection (4, 6 -8). The most advanced anti-HCV nucleoside, RG7128, is a diisobutyrate nucleoside prodrug of ␤-D-2Ј-deoxy-2Ј-␣-fluoro-2Ј-␤-C-methylcytidine (PSI-6130) and is currently in phase IIb clinical studies. PSI-6130 demonstrated potent activity in the subgenomic HCV replicon assay (9); the incubation of radiolabeled PSI-6130 with either replicon cells or primary human hepatocytes resulted in the formation of the 5Ј-mono-, di-, and triphosphate metabolites of . The triphosphate metabolite (PSI-6130-TP) was shown to be a potent inhibitor of HCV NS5B RNA-directed RNA polymerase (RdRp) (11). However, incubation of replicon cells with the uridine analog, PSI-6206, resulted in no inhibition of HCV RNA production due to the inability of PSI-6206 to be phosphorylated by cellular nucleoside kinases to its monophosphate, 12). Biochemical studies showed that PSI-7411 was consecutively phosphorylated to its diphosphate, PSI-7410, by UMP-CMP kinase and its triphosphate, PSI-7409, by nucleoside diphosphate kinase (12). Inhibition studies using the replic...

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“…CES3 appears to show extremely low activity compared with CES1 and CES2 (Sanghani et al, 2004). The flutamide hydrolysis by human liver microsomes (HLM) is inhibited by bis-(nonylphenyl)-phenylphosphate (BNPP), a general CES inhibitor (Heymann and Krisch, 1967;Block and Arndt, 1978;Mentlein et al, 1988). Thus, it is plausible that flutamide is hydrolyzed by CES.…”

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confidence: 99%

Human Arylacetamide Deacetylase Is a Principal Enzyme in Flutamide Hydrolysis

Watanabe

Fukami²,

Nakajima³

et al. 2009

Drug Metab Dispos

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ABSTRACT:Flutamide, an antiandrogen drug, is widely used for the treatment of prostate cancer. The initial metabolic pathways of flutamide are hydroxylation and hydrolysis. It was recently reported that the hydrolyzed product, 4-nitro-3-(trifluoromethyl)phenylamine (FLU-1), is further metabolized to N-hydroxy FLU-1, an assumed hepatotoxicant. However, the esterase responsible for the flutamide hydrolysis has not been characterized. In the present study, we found that human arylacetamide deacetylase (AADAC) efficiently hydrolyzed flutamide using recombinant AADAC expressed in COS7 cells. In contrast, carboxylesterase1 (CES1) and CES2, which are responsible for the hydrolysis of many drugs, could not hydrolyze flutamide. AADAC is specifically expressed in the endoplasmic reticulum. Flutamide hydrolase activity was highly detected in human liver microsomes (K m , 794 ؎ 83 M; V max , 1.1 ؎ 0.0 nmol/min/mg protein), whereas the activity was extremely low in human liver cytosol. The flutamide hydrolase activity in human liver microsomes was strongly inhibited by bis-(nonylphenyl)-phenylphosphate, diisopropylphosphorofluoride, and physostigmine sulfate (eserine) but moderately inhibited by sodium fluoride, phenylmethylsulfonyl fluoride, and disulfiram. The same inhibition pattern was obtained with the recombinant AADAC. Moreover, human liver and jejunum microsomes showing AADAC expression could hydrolyze flutamide, but human pulmonary and renal microsomes, which do not express AADAC, showed slight activity. In human liver microsomal samples (n ‫؍‬ 50), the flutamide hydrolase activities were significantly correlated with the expression levels of AADAC protein (r ‫؍‬ 0.66, p < 0.001). In conclusion, these results clearly showed that flutamide is exclusively hydrolyzed by AADAC. AADAC would be an important enzyme responsible for flutamideinduced hepatotoxicity.Flutamide (3Ј-trifluoro-2-methyl-4Ј-nitro-2-methyl-propinoylanilide) is a nonsteroidal antiandrogen drug used for the treatment of prostate cancer. The combination of luteinizing hormone-releasing hormone agonist results in prolonged survival in prostate cancer patients (Crawford et al., 1989). However, flutamide occasionally causes severe hepatotoxicity (Thole et al., 2004). Flutamide itself is not toxic when used at the appropriate clinical dose, but bioactivation of flutamide has been considered to be the cause of flutamide-induced hepatotoxicity (Fau et al., 1994).Flutamide is mainly metabolized to 2-hydroxyflutamide by human CYP1A2. It has been suggested that 2-hydroxyflutamide is associated with the therapeutic effect of flutamide (Katchen and Buxbaum, 1975). Flutamide is also hydrolyzed to 4-nitro-3-(trifluoromethyl)phenylamine (FLU-1) by esterase (Katchen and Buxbaum, 1975;Schulz et al., 1988). FLU-1 is considered to have no therapeutic effect (Aizawa et al., 2003). Goda et al. (2006) recently reported that FLU-1 is further metabolized to N-hydroxyl FLU-1 by human CYP3A4. Many researchers have reported on the relationship between the toxicity and metabolism...

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Chromatographic study on the specificity of bis-p-nitrophenylphosphate in vivo Identification of labelled proteins of rat liver after intravenous injection of as carboxylesterases and amidases

Cited by 29 publications

References 10 publications

Intestinal First-Pass Metabolism via Carboxylesterase in Rat Jejunum and Ileum

Intestinal First-Pass Metabolism via Carboxylesterase in Rat Jejunum and Ileum

Mechanism of Activation of PSI-7851 and Its Diastereoisomer PSI-7977

Human Arylacetamide Deacetylase Is a Principal Enzyme in Flutamide Hydrolysis

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