Publications (9)20.52 Total impact
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Article: Alternate strategies to obtain mass balance without the use of radiolabeled compounds: application of quantitative fluorine (19F) nuclear magnetic resonance (NMR) spectroscopy in metabolism studies.
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ABSTRACT: Nuclear magnetic resonance (NMR) spectroscopy is playing an increasingly important role in the quantitation of small and large molecules. Recently, we demonstrated that (1)H NMR could be used to quantitate drug metabolites isolated in submilligram quantities from biological sources. It was shown that these metabolites, once quantitated by NMR, were suitable to be used as reference standards in quantitative LC/MS-based assays, hence circumventing the need for radiolabeled material or synthetic standards to obtain plasma exposure estimates in humans and preclinical species. The quantitative capabilities of high-field NMR is further demonstrated in the current study by obtaining the mass balance of fluorinated compounds using (19)F-NMR. Two fluorinated compounds which were radio-labeled with carbon-14 on metabolically stable positions were dosed in rats and urine and feces collected. The mass balance of the compounds was obtained initially by counting the radioactivity present in each sample. Subsequently, the same sets of samples were analyzed by (19)F-NMR, and the concentrations determined by this method were compared with data obtained using radioactivity counting. It was shown that the two methods produced comparable values. To demonstrate the value of this analytical technique in drug discovery, a fluorinated compound was dosed intravenously in dogs and feces and urine collected. Initial profiling of samples showed that this compound was excreted mainly unchanged in feces, and hence, an estimate of mass balance was obtained using (19)F-NMR. The data obtained by this method was confirmed by additional quantitative studies using mass spectrometry. Hence cross-validations of the quantitative (19)F-NMR method by radioactivity counting and mass spectrometric analysis were demonstrated in this study. A strategy outlining the use of fluorinated compounds in conjunction with (19)F-NMR to understand their routes of excretion or mass balance in animals is proposed. These studies demonstrate that quantitative (19)F-NMR could be used as an alternate technique to obtain an estimate of the mass balance of fluorinated compounds, especially in early drug development where attrition of the compounds is high, and cost savings could be realized through the use of such a technique rather than employing radioactive compounds. The potential application of qNMR in conducting early human ADME studies with fluorinated compounds is also discussed.Chemical Research in Toxicology 02/2012; 25(3):572-83. · 3.78 Impact Factor -
Article: Comparative pharmacokinetics and metabolism studies in lean and diet- induced obese mice: an animal efficacy model for 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitors.
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ABSTRACT: Diet-induced obese (DIO) mice have been commonly used as an animal model in the efficacy assessment for new drug candidates. Although high-fat feeding has been reported to cause profound physiological changes, including the expression of drug-metabolizing enzymes, limited studies have been reported regarding the effect of obesity/diabetes on pharmacokinetics (PK) in animals. In this study, we investigated PK profiles of three 11 -HSD-1 inhibitors in the DIO mice and compared them to the normal lean mice. After oral administration, the in vivo exposure (AUC) of all three compounds was higher in DIO mice, which was consistent with the observed lower systemic clearance (CL) in DIO mice compared to lean mice. As illustrated by Compound E, a compound metabolized predominantly by CYP3A and 2C, the metabolic profiles for Compound E were qualitatively similar between DIO and lean mice, but quantitatively lower in the DIO mice. Indeed, P-450 activities for CYP3A and 2C as well as 2D were found to be lower in liver microsomes prepared from DIO mice. The calculated hepatic clearance (CLH) from in vitro studies with liver microsomes correlated well with the observed in vivo clearance for both DIO and lean mice. The calculated oral bioavailability (F%) based on intrinsic hepatic clearance (C(LH, int)) predicted ~3 fold increase in F% for the DIO mice, which was comparable to the observed value. Collectively, these data suggest that the higher F% is most likely due to the lower first-pass effect in DIO mice. This study highlights the needs to take caution when extrapolating PK and exposure data from healthy animals to diseased animals in designing pharmacological studies.Drug metabolism letters. 01/2011; 5(1):55-63. -
Article: Reversible covalent binding of neratinib to human serum albumin in vitro.
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ABSTRACT: Neratinib (HKI-272), an irreversible inhibitor of Her 2 tyrosine kinase, is currently in development as an alternative for first and second line therapy in metastatic breast cancer patients who overexpress Her 2. Following incubation of [(14)C]neratinib in control human plasma at 37°C for 6 hours, about 60% to 70% of the radioactivity was not extractable, due to covalent binding to albumin. In this study, factors that could potentially affect the covalent binding of neratinib to plasma proteins, specifically to albumin were investigated. When [(14)C]neratinib was incubated at 10 μg/mL in human serum albumin (HSA) or control human plasma, the percent binding increased with time; the highest percentages of binding (46 and 67%, respectively) were observed at 6 hours, the longest duration of incubation examined. Binding increased with increasing temperature; the highest percentages of binding to HSA or human plasma (59 and 78%) were observed at 45°C, the highest temperature tested. The binding also increased with increasing pH of incubation; the highest percentages of binding (56 and 65%) were observed at pH 8.5, the highest pH value tested. The percentages of binding were similar (53% to 57%) when a wide range of concentrations of [(14)C]neratinib (50 ng/mL to 10 μg/mL) were incubated with human plasma at 37°C for 6 hours, indicating that the binding was independent of the substrate concentration, especially in the therapeutic range (50 to 200 ng/mL). When human plasma proteins containing covalently bound [(14)C]neratinb were suspended in a 10 fold volume of phosphate buffer at pH 4.0, 6.0, 7.4, and 8.5, and further incubated at 37°C for ~ 16 hours, about 45%, 44%, 32%, and 12% of the total radioactivity, respectively, was released as unchanged [(14)C]neratinib, indicating that the binding is reversible in nature, with more released at pH 7.4 and below. In conclusion, the covalent binding of neratinib to serum albumin is pH, time and temperature dependent, but not substrate concentration dependent, especially in the therapeutic range. Acidification and incubation of human plasma proteins that contained covalently bound [(14)C]neratinib leads to the release of the drug, indicating that the binding is reversible in nature. It is reasonable to speculate that the release of neratinib from human serum albumin provides a transport system leading to release of neratinib in the more acidic environment of the tumor.Drug metabolism letters. 12/2010; 4(4):220-7. -
Article: Characterization of HKI-272 covalent binding to human serum albumin.
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ABSTRACT: The study was initiated as an observation of incomplete extraction recovery of N-(4-(3-chloro-4-(2-pyridinylmethoxy)anilino)-3-cyano-7-ethoxy-6-quinolyl)-4-(dimethylamino)-2-butenamide (HKI-272) from human plasma. The objective of this study was to 1) identify the binding site(s) of HKI-272 to human plasma protein(s); 2) characterize the nature of the binding; and 3) evaluate the potential reversibility of the covalent binding. After incubation of [(14)C]HKI-272 with human plasma, the mixture was directly injected on liquid chromatography/mass spectrometry (LC/MS), and an intact molecular mass of HKI-272 human serum albumin (HSA) adduct was determined to be 66,999 Da, which is 556 Da (molecular mass of HKI-272) larger than the measured molecular mass of HSA (66,443 Da). For peptide mapping, the incubation mixture was separated with SDS-polyacrylamide gel electrophoresis followed by tryptic digestion combined with LC/tandem MS. A radioactive peptide fragment, LDELRDEGKASSAK [amino acid (AA) residue 182-195 of albumin], was confirmed to covalently bind to HKI-272. In addition, after HCl hydrolysis, a radioactive HKI-272-lysine adduct was identified by LC/MS. After combining the results of tryptic digestion and HCl hydrolysis, the AA residue of Lys190 of HSA was confirmed to covalently bind to HKI-272. A standard HKI-272-lysine was synthesized and characterized by NMR. The data showed that the adduct was formed via Michael addition with the epsilon-amine of lysine attacking to the beta-carbon of the amide moiety of HKI-272. Furthermore, reversibility of the covalent binding of HKI-272 to HSA was shown when a gradual release of HKI-272 was observed from protein pellet of HKI-272-treated human plasma after resuspension in phosphate buffer, pH 7.4, at 37 degrees C for 18 h.Drug metabolism and disposition: the biological fate of chemicals 07/2010; 38(7):1083-93. · 3.74 Impact Factor -
Article: Optimization of 5-vinylaryl-3-pyridinecarbonitriles as PKCtheta inhibitors.
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ABSTRACT: Analog 8, a 3-pyridinecarbonitrile with an (E)-2-[6-[(4-methylpiperazin-1-yl)methyl]pyridin-2-yl]vinyl group at C-5, had an IC(50) value of 1.1 nM for the inhibition of PKCtheta and potently blocked the production of IL-2 in both stimulated murine T cells (IC(50)=34 nM) and human whole blood (IC(50)=500 nM).Bioorganic & medicinal chemistry letters 01/2010; 20(6):1965-8. · 2.65 Impact Factor -
Article: Nuclear magnetic resonance spectroscopy as a quantitative tool to determine the concentrations of biologically produced metabolites: implications in metabolites in safety testing.
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ABSTRACT: Nuclear magnetic resonance (NMR) spectroscopy has traditionally been considered as an indispensable tool in elucidating structures of metabolites. With the advent of Fourier transform (FT) spectrometers, along with improvements in software and hardware (such as high-field magnets, cryoprobes, versatile pulse sequences, and solvent suppression techniques), NMR is increasingly being considered as a critical quantitative tool, despite its lower sensitivity as compared to mass spectrometry. A specific quantitative application of NMR is in determining the concentrations of biologically isolated metabolites, which could potentially be used as reference standards for further quantitative work by liquid chromatography/mass spectrometry. With the recent demands from regulatory agencies on quantitative information on metabolites, it is proposed that NMR will play a significant role in strategies aimed at addressing metabolite coverage in toxicological species. Traditionally, biologically isolated metabolites have not been considered as a way of generating "reference standards" for further quantitative work. However, because of the recent FDA guidance on safety testing of metabolites, one has to consider means of authenticating and quantitating biologically or nonbiologically generated metabolites. 1H NMR is being proposed as the method of choice, as it is able to be used as both a qualitative and a quantitative tool, hence allowing structure determination, purity check, and quantitative measurement of the isolated metabolite. In this publication, the application of NMR as a powerful and robust analytical technique in determining the concentrations of in vitro or in vivo isolated metabolites is discussed. Furthermore, to demonstrate the reliability and accuracy of metabolite concentrations determined by NMR, validation and cross-validation with gravimetric and mass spectrometric methods were conducted.Chemical Research in Toxicology 12/2008; 22(2):299-310. · 3.78 Impact Factor -
Article: Liquid chromatography/mass spectrometry determination of endogenous plasma acetyl and palmitoyl carnitines as potential biomarkers of beta-oxidation in mice.
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ABSTRACT: A robust bioanalytical method capable of measuring acetyl and palmitoyl carnitines was developed and validated. Application of hydrophilic interaction chromatography (HILIC) enabled retention of these highly polar and difficult to analyze compounds on a silica HPLC column. The chromatography was conducted with a high percentage of an organic component in the mobile phase, allowing high sensitivity for the pre-existing positively charged quaternary ammonium ions by electrospray ionization mass spectrometry. Successful application of the method to reliably quantify naturally occurring acyl carnitines in mouse plasma depended on the use of corresponding deuterated analogues. The specificity of the method, achieved through the use of stable isotope labeled compounds in combination with a mass spectral multiple reaction monitoring technique, permitted a non-invasive assessment of the overall change in the levels of these acyl carnitines in the plasma of intact animals administered peroxisome proliferator activated receptor (PPAR) agents. These acyl carnitines, as carriers of the corresponding long-chain fatty acids for transport into mitochondria, can be employed as potential biomarkers for significant alteration in the beta-oxidation process in an intact animal.Rapid Communications in Mass Spectrometry 10/2008; 22(21):3434-42. · 2.79 Impact Factor -
Article: Mechanism study of N-dephenylation mediated through a N-para-hydroxy metabolite.
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ABSTRACT: A P450 catalyzed N-para-hydroxy metabolite was suggested to be a prerequisite for N-dephenylation occurrence. Although two mechanisms have been proposed to describe this process as a consequence of either a chemical degradation or P450 lead epoxidation of the hydroxy metabolite, direct evidence has not been demonstrated. In this study, we started with a novel technique using a dipeptide, Lys-Phe, to trap the byproduct of N-dephenylation, a quinone-like compound, forming a peptide adduct to facilitate LC/MS characterization. N-dephenylation via chemical degradation was assessed by LC/MS characterization of the resulting (Lys-Phe)(2)-quinone from 4-hydroxyphenyl-2-naphthylamine following interaction with Lys-Phe in pH 7.4 buffer. N-dephenylation mediated by P450 catalysis proposed was investigated in N-para-hydroxy benzodioxane derivative incubated with mouse liver microsomes in the presence of Lys-Phe in 50/50 H(2)(16)O/H(2)(18)O. LC/MS demonstrated that only one of two hydroxy oxygens in the byproduct was exchanged with water and the MS signal intensity of the (16)O labeled peptide adduct was equal to that of (18)O labeled. These observations suggested us that the origin of the oxygen in the byproduct was from water only, not from O(2). Therefore, it appears that N-dephenylation occurs via a stepwise process, namely the substrate is initially metabolized to a N-para-hydroxy metabolite by P450, which was readily oxidized to a quinone imine/iminium chemically or enzymatically, then hydrolyzed resulting in N-dephenylation. However, in our studies, the proposed P450 mechanism involving epoxidation of a N-para-hydroxy metabolite was disproved.Current Drug Discovery Technologies 07/2006; 3(2):101-14. -
Article: A novel approach for predicting acyl glucuronide reactivity via Schiff base formation: development of rapidly formed peptide adducts for LC/MS/MS measurements.
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ABSTRACT: A novel technique to study the reactivity of acyl glucuronide metabolites to protein has been developed and is described herein. Considered here are acyl glucuronide metabolites, which have undergone the rearrangement of the glucuronic acid moiety at physiological temperature and pH. The investigation of the reactivity of these electrophilic metabolites was carried out by measuring the rate of reaction of rearranged AG metabolites in forming the corresponding acyl glucuronide-peptide adduct in the presence of Lys-Phe. This differs from the parallel technique used in forming AG adducts of proteins that have been previously reported. In the study described here, the Schiff base adduct, diclofenac acyl glucuronide-Lys-Phe product, was generated and structurally elucidated by liquid chromatography tandem mass spectrometry (LC/MS/MS) analysis. The product structure was proved to be a Schiff base adduct by chemical derivatization by nucleophilic addition of HCN and chemical reduction with NaCNBH(3), followed by LC/MS/MS analysis. It is proposed here that the degree of reactivity of acyl glucuronides as measured by covalent binding to protein is proportional to the amount of its peptide adduct generated with the peptide technique described. The application of this technique to the assessment of the degree of reactivity of acyl glucuronide metabolites was validated by developing a reactivity rank of seven carboxylic acid-containing drugs. Consistency was achieved between the ranking of reactivity in the peptide technique for these seven compounds and the rankings found in the literature. In addition, a correlation (R(2) = 0.95) was revealed between the formation of a peptide adduct and the rearrangement rate of the primary acyl glucuronide of seven tested compounds. A structure effect on the degree of reactivity has demonstrated the rate order: acetic acid > propionic acid > benzoic acid derivatives. A rational explanation of this order was proposed, based on the inherent electronic and steric properties of each specific aglycone. In addition, adaptation of this technique to automation in order to more rapidly assess the ranking of reactivity of acyl glucuronide covalent binding to proteins by new chemical entities is proposed.Chemical Research in Toxicology 10/2004; 17(9):1206-16. · 3.78 Impact Factor
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2010
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Pfizer Inc.
- Department of Pharmacokinetics, Dynamics and Metabolism
New York City, NY, USA
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