Characterization of the Expression and Activity of Carboxylesterases 1 and 2 from the Beagle Dog, Cynomolgus Monkey, and Human

Williams, Eric T.; Bacon, Jeffrey W.; Bender, David M.; Lowinger, Jennifer J.; Guo, Wenkai; Ehsani, Mariam; Wang, Xiliang; Wang, He; Qian, Yue‐Wei; Ruterbories, Kenneth J.; Wrighton, Steven; Perkins, E.J.

doi:10.1124/dmd.111.041335

Cited by 61 publications

(60 citation statements)

References 16 publications

(36 reference statements)

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“…The anti-human CES1 antibody used in the present study was reported to be CES1-specific, without cross-reactivity with CES2 proteins, by probing against cynomolgus monkey, dog, and human CES1 and CES2 enzymes (Williams et al, 2011). As previously reported (Satoh and Hosokawa, 2010;Williams et al, 2011), CES1 proteins were highly expressed in the liver in the three species, and were significantly expressed in monkey intestine and kidney, dog kidney, lung, and plasma, and human lung as a single band with a molecular mass approximately at 60 kDa.…”

Section: Resultsmentioning

confidence: 99%

Different Hydrolases Involved in Bioactivation of Prodrug-Type Angiotensin Receptor Blockers: Carboxymethylenebutenolidase and Carboxylesterase 1

et al. 2013

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Olmesartan medoxomil (OM) is a prodrug-type angiotensin II type 1 receptor blocker (ARB). We recently identified carboxymethylenebutenolidase homolog (CMBL) as the responsible enzyme for OM bioactivation in humans. In the present study, we compared the bioactivating properties of OM with those of other prodrug-type ARBs, candesartan cilexetil (CC) and azilsartan medoxomil (AM), by focusing on interspecies differences and tissue specificity. In invitro experiments with pooled tissue subcellular fractions of mice, rats, monkeys, dogs, and humans, substantial OM-hydrolase activities were observed in cytosols of the liver, intestine, and kidney in all the species tested except for dog intestine, which showed negligible activity, whereas lung cytosols showed relatively low activities compared with the other tissues. AM-hydrolase activities were well correlated with the OM-hydrolase activities. In contrast, liver microsomes exhibited the highest CC-hydrolase activity among various tissue subcellular fractions in all the species tested. As a result of Western blot analysis with the tissue subcellular fractions, the band intensities stained with anti-human CMBL and carboxylesterase 1 (CES1) antibodies well reflected OM-and AM-hydrolase activities and CC-hydrolase activity, respectively, in animals and humans. Recombinant human CMBL and CES1 showed significant AM-and CC-hydrolase activities, respectively, whereas CC hydrolysis was hardly catalyzed with recombinant carboxylesterase 2 (CES2). In conclusion, OM is bioactivated mainly via intestinal and additionally hepatic CMBL not only in humans but also in mice, rats, and monkeys, while CC is bioactivated via hepatic CES1 rather than intestinal enzymes, including CES2. AM is a substrate for CMBL.

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Section: Resultsmentioning

confidence: 99%

Different Hydrolases Involved in Bioactivation of Prodrug-Type Angiotensin Receptor Blockers: Carboxymethylenebutenolidase and Carboxylesterase 1

et al. 2013

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“…The inhibition potencies of DFP and eserine to prasugrel hydrolysis by dog AADAC appeared to be slightly lower than those by human AADAC. It was reported that the IC 50 value of loperamide was 0.562 mM for purified human CES2, whereas that for purified dog CES2 was 93.6 mM (Williams et al, 2011). Thus, the differences in the inhibitory potencies of DFP and eserine are due to species differences.…”

Section: Discussionmentioning

confidence: 98%

Arylacetamide Deacetylase is Responsible for Activation of Prasugrel in Human and Dog

Kurokawa

Fukami

Yoshida

et al. 2015

Drug Metabolism and Disposition

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Prasugrel, a thienopyridine anti-platelet agent, is pharmacologically activated by hydrolysis and hydroxylation. It is efficiently hydrolyzed in the intestine after oral administration, and the enzyme responsible for the hydrolysis in humans was demonstrated to be carboxylesterase (CES)2. Prasugrel hydrolase activity is detected in dog intestines, where CES enzymes are absent; therefore, this prompted us to investigate the involvement of an enzyme(s) other than CES. Human arylacetamide deacetylase (AADAC) is highly expressed in the small intestine, catalyzing the hydrolysis of several clinical drugs containing small acyl moieties. In the present study, we investigated whether AADAC catalyzes prasugrel hydrolysis. Recombinant human AADAC was shown to catalyze prasugrel hydrolysis with a CL int value of 50.0 6 1.2 ml/min/mg protein with a similar K m value to human intestinal and liver microsomes, whereas the CL int values of human CES1 and CES2 were 4.6 6 0.1 and 6.6 6 0.3 ml/min/mg protein, respectively. Inhibition studies using various chemical inhibitors and the relative activity factor approach suggested that the contribution of AADAC to prasugrel hydrolysis in human intestine is comparable to that of CES2. In dog intestine, the expression of AADAC, but not CES1 and CES2, was confirmed by measuring the marker hydrolase activities of each human esterase. The similar K m values and inhibition profiles between recombinant dog AADAC and small intestinal microsomes suggest that AADAC is a major enzyme responsible for prasugrel hydrolysis in dog intestine. Collectively, we found that AADAC largely contributes to prasugrel hydrolysis in both human and dog intestine.

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“…4A). However, the 4-NPV assay, which measures CES activity (Williams et al, 2011), showed that total CES activity was only 2-fold lower in the skin than in the liver (Fig. 4B), confirming the potential for human skin to contribute to the metabolism of ester compounds when applied transdermally .…”

Section: Downloaded Frommentioning

confidence: 92%

“…These structural differences result in different substrate specificity. CES1 tends to hydrolyze molecules with a small alcohol moiety more efficiently, whereas CES2 is more efficient at metabolizing molecules with a larger alcohol moiety and more lipophilic molecules (Brzezinski et al, 1994;Williams et al, 2011). These differences in the substrate specificity of CES1 and CES2 could lead to differences in predicting skin absorption or metabolism.…”

Section: Downloaded Frommentioning

confidence: 99%

“…Experiments were designed to examine the effect of specific inhibitors on C2E5 hydrolysis in human skin S9 fractions. The following inhibitors were selected: Benzil (10 mM) was chosen as a pan CES inhibitor (Wadkins et al, 2005), troglitazone (10 mM) as a CES1-specific inhibitor (Fukami et al, 2010), loperamide (100 mM) as a CES2-specific inhibitor (Williams et al, 2011), and bis-para-nitrophenylphosphate (BNPP; 1 mM) as a nonspecific esterase inhibitor (Li et al, 2007). Inhibitors were incubated with human skin S9 fractions for 30 minutes at 37°C before the reaction was initiated.…”

Section: Downloaded Frommentioning

confidence: 99%

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Biotransformation Capacity of Carboxylesterase in Skin and Keratinocytes for the Penta-Ethyl Ester Prodrug of DTPA

Sadgrove

Marson

et al. 2016

Drug Metabolism and Disposition

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The penta-ethyl ester prodrug of the chelating agent diethylene triamine pentaacetic acid (DTPA), referred to as C2E5, effectively accelerated clearance of americium after transdermal delivery. Carboxylesterases (CESs) play important roles in facilitating C2E5 hydrolysis. However, whether CESs in human skin hydrolyze C2E5 remains unknown. We evaluated the gene and protein expression of CESs in distinctive human epidermal cell lines: HEKa, HEKn, HaCaT, and A431. The substrates p-nitrophenyl acetate (pNPA) and 4-nitrophenyl valerate (4-NPV) were used to access esterase and CES activity. C2E5 hydrolysis was measured by radiometric high-performance liquid chromatography after incubation of [ 14 C]C2E5 with supernatant fractions after centrifugation at 9000g (S9) prepared from skin cell lines. CESspecific inhibitors were used to access metabolism in human skin S9 fractions with analysis by liquid chromatography-tandem mass spectrometry. We identified the human carboxylesterase 1 and 2 (CES1 and CES2) bands in a Western blot. The gene expression of these enzymes was supported by a real-time polymerase chain reaction (qPCR). pNPA and 4-NPV assays demonstrated esterase and CES activity in all the cell lines that were comparable to human skin S9 fractions. The prodrug C2E5 was hydrolyzed by skin S9 fractions, resulting in a primary metabolite, C2E4. In human skin S9 fractions, inhibition of C2E5 hydrolysis was greatest with a pan-CES inhibitor (benzil). CES1 inhibition (troglitazone) was greater than CES2 (loperamide), suggesting a primary metabolic role for CES1. These results indicate that human keratinocyte cell lines are useful for the evaluation of human cutaneous metabolism and absorption of ester-based prodrugs. However, keratinocytes from skin provide a small contribution to the overall metabolism of C2E5.

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Characterization of the Expression and Activity of Carboxylesterases 1 and 2 from the Beagle Dog, Cynomolgus Monkey, and Human

Cited by 61 publications

References 16 publications

Different Hydrolases Involved in Bioactivation of Prodrug-Type Angiotensin Receptor Blockers: Carboxymethylenebutenolidase and Carboxylesterase 1

Different Hydrolases Involved in Bioactivation of Prodrug-Type Angiotensin Receptor Blockers: Carboxymethylenebutenolidase and Carboxylesterase 1

Arylacetamide Deacetylase is Responsible for Activation of Prasugrel in Human and Dog

Biotransformation Capacity of Carboxylesterase in Skin and Keratinocytes for the Penta-Ethyl Ester Prodrug of DTPA

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