Activation of the Antiviral Prodrug Oseltamivir Is Impaired by Two Newly Identified Carboxylesterase 1 Variants

Abstract

ABSTRACT:Oseltamivir phosphate is an ethyl ester prodrug widely used in the treatment and prevention of both Influenzavirus A and B infections. The conversion of oseltamivir to its active metabolite oseltamivir carboxylate is dependent on ester hydrolysis mediated by carboxylesterase 1 (CES1). We recently identified two functional CES1 variants p.Gly143Glu and p.Asp260fs in a research subject who displayed significant impairment in his ability to metabolize the selective CES1 substrate, methylphenidate. In vitro functional studies demonstrated that the presence of either of the two mutations can result in severe reductions in the catalytic efficiency of CES1 toward methylphenidate, which is required for hydrolysis and pharmacological deactivation. The aim of the present study was to investigate the function of these mutations on activating (hydrolyzing) oseltamivir to oseltamivir carboxylate using the cell lines expressing wild type (WT) and each mutant CES1. In vitro incubation studies demonstrated that the S9 fractions prepared from the cells transfected with WT CES1 and human liver tissues rapidly convert oseltamivir to oseltamivir carboxylate. However, the catalytic activity of the mutant hydrolases was dramatically hindered. The V max value of p.Gly143Glu was approximately 25% of that of WT enzyme, whereas the catalytic activity of p.Asp260fs was negligible. These results suggest that the therapeutic efficacy of oseltamivir could be compromised in treated patients expressing either functional CES1 mutation. Furthermore, the potential for increased adverse effects or toxicity as a result of exposure to high concentrations of the nonhydrolyzed prodrug should be considered.Oseltamivir phosphate (Tamiflu; Roche, Nutley, NJ) is widely used in the treatment and prophylaxis of both Influenzavirus A and B infections. In addition, oseltamivir may be effective in preventing or treating avian influenza or so-called "bird flu." Oseltamivir is an ester prodrug and, in general, it is readily converted to its active form oseltamivir carboxylate mediated by hepatic carboxylesterase 1 (CES1) (Fig. 1) . The active metabolite exerts its antiviral effects via the selective inhibition of neuraminidase.Carboxylesterases are members of the ␣␤ hydrolase -fold family and expressed in many tissues, especially in the liver, small intestine, and lung (Satoh and Hosokawa, 2006;Ross and Crow, 2007). The major human carboxylesterases include CES1 (UniProtKB/Swiss-Prot P23141) and carboxylesterase 2 (CES2) (UniProtKB/Swiss-Prot O00748). CES1 and CES2 are largely distinguished from one another by their substrate specificity and tissue distribution (Imai et al., 2006;Satoh and Hosokawa, 2006). CES1 more readily catalyzes substrates with a relatively large acyl group and small alcohol group such as methylphenidate, temocapril, and oseltamivir (Sun et al., 2004;Imai et al., 2005;Shi et al., 2006). In contrast, CES2 preferentially hydrolyzes compounds bearing a small acyl moiety and bulky alcohol group, which includes agents such as cocaine a...

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“…Oseltamivir phosphate is rapidly absorbed and converted to oseltamivir carboxylate by CES1 (He et al, 1999). The exception to this rapid conversion is in rare individuals who are poor converters (Zhu and Markowitz 2009). Rare genetic variants have been documented that result in low carboxylesterase activity, and this has led to concerns that individuals with variant alleles will not achieve therapeutic effects because of their inability to hydrolyze the prodrug to its active form.…”

Carboxylesterase 1 as a Determinant of Clopidogrel Metabolism and Activation

“…The Flp-In-293 cell lines stably expressing wild-type CES1 and its variants G143E and D260fs have been developed previously and applied to our ensuing CES1 pharmacogenetic studies (Zhu et al, 2008;Zhu et al, 2009b;Zhu and Markowitz, 2009). The minor allele frequency (MAF) of G143E is estimated to be 3.7%, 4.3%, 2.0%, and 0% in Caucasian, Black, Hispanic, and Asian populations, respectively, whereas D260fs appears to be a rare mutation in all racial and ethnic groups studied to date (Zhu et al, 2008).…”

“…This variability is likely to be the result of both genetic and environmental factors (Hosokawa et al, 1995;Fukami et al, 2008;Yoshimura et al, 2008;Yang et al, 2009;Zhu et al, 2009a;Shi et al, 2011;Ross et al, 2012). We have reported that the CES1 single-nucleotide polymorphisms (SNPs) G143E (rs71647871) and D260fs (rs71647872) exhibit markedly decreased enzymatic activity toward the hydrolysis of the CES1 substrates methylphenidate, oseltamivir, and trandolapril (Zhu et al, 2008;Zhu et al, 2009b;Zhu and Markowitz, 2009). Furthermore, we and others have demonstrated that some therapeutic agents can significantly inhibit CES1 activity Zhu et al, 2010;Rhoades et al, 2012).…”

Genetic Polymorphisms of theCarboxylesterase 1(CES1) Gene in a Korean Population

“…[2,6,7] In-vitro functional studies have demonstrated that the G143E and the D260fs variants have reduced catalytic function, resulting in the disruption of hydrolytic activity of CES1. [8,9] In a single-dose pharmacokinetic study, these two CES1 mutations were identified in one subject who displayed 7-fold higher total methylphenidate (i.e., combined d-and l-isomers) concentrations compared to that of other subjects (n=19). [2,9] In addition, -75T>G polymorphism was associated with the reduced appetite in attention deficit-hyperactivity disorder (ADHD) youths treated with methylphenidate [10] and the isoniazid-induced hepatotoxicity in latent tuberculosis infection patients.…”

“…[8,9] In a single-dose pharmacokinetic study, these two CES1 mutations were identified in one subject who displayed 7-fold higher total methylphenidate (i.e., combined d-and l-isomers) concentrations compared to that of other subjects (n=19). [2,9] In addition, -75T>G polymorphism was associated with the reduced appetite in attention deficit-hyperactivity disorder (ADHD) youths treated with methylphenidate [10] and the isoniazid-induced hepatotoxicity in latent tuberculosis infection patients. [11] Taken together, these findings suggest that CES1 gene variants can lead to clinically significant alterations in…”