This promotor region polymorphism does not appear to play a major role in determining constitutive CYP3A4 expression.

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.

The recent guidance on "Safety Testing of Drug Metabolites" issued by the U.S. Food and Drug Administration, Center for Drug Evaluation and Research (CDER) has highlighted the importance of identifying and characterizing drug metabolites as early as possible in drug discovery and development. Furthermore, upon identifying significant circulating metabolites in human plasma, it has become important to demonstrate that these metabolites are present at an equal or greater exposure level (area under the curve, AUC) in any one of the preclinical species used in safety testing. Frequently, synthetic standards of metabolites are not available, and hence, obtaining their AUC values can be a challenge. In this report, we demonstrate how combinations of nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography/ultraviolet/mass spectrometry (LC/UV/MS), and plasma pooling methods were used to obtain reliable AUC values of metabolites present in the plasma of preclinical species from short-term safety studies. Plasma pooling methods were compared to the traditional approaches of obtaining quantitative information on the levels of circulating metabolites in preclinical species. The exposure values obtained via sample pooling were comparable to those obtained by traditional methods of analyzing samples individually. In the absence of synthetic chemical standards, calculations of AUC values of metabolites, using either sample pooling or traditional approaches, were achieved through the use of UV detectors. In cases where the UV properties of metabolites were significantly different from their parent compounds, NMR was used as a quantitative tool to obtain exposure values. NMR was found to be useful in quantitating biologically produced metabolites, which could subsequently be used as reference compounds for further quantitative studies. The limitations of UV detectors to obtain exposure estimates are discussed. A practical solution is presented that will enable us to obtain a quantitative assessment of metabolite exposure in humans and coverage in toxicology species, hence, circumventing the use of radiolabeled compounds or authentic chemically synthesized standards of metabolites.

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.

ABSTRACT:Tigecycline, a novel, first-in-class glycylcycline antibiotic, has been approved for the treatment of complicated intra-abdominal infections and complicated skin and skin structure infections. The pharmacokinetics, metabolism, and excretion of [ 14 C]tigecycline were examined in healthy male volunteers. Tigecycline has been shown to bind to bone; thus, to minimize the amount of radioactivity binding to bone and to maximize the recovery of radioactivity, tigecycline was administered intravenously (30-min infusion) as a single 100-mg dose, followed by six 50-mg doses, every 12 h, with the last dose being [ 14 C]tigecycline (50 Ci). After the final dose, the pharmacokinetics of tigecycline in serum showed a long halflife (55.8 h) and a large volume of distribution (21.0 l/kg), whereas radioactivity in serum had a shorter half-life (6.9 h) and a smaller volume of distribution (3.3 l/kg). The major route of elimination was feces, containing 59% of the radioactive dose, whereas urine contained 32%. Unchanged tigecycline was the predominant drugrelated compound in serum, urine, and feces. The major metabolic pathways identified were glucuronidation of tigecycline and amide hydrolysis followed by N-acetylation to form N-acetyl-9-aminominocycline. The glucuronide metabolites accounted for 5 to 20% of serum radioactivity, and approximately 9% of the dose was excreted as glucuronide conjugates within 48 h. Concentrations of N-acetyl-9-aminominocycline were approximately 6.5% and 11% of the tigecycline concentrations in serum and urine, respectively. Excretion of unchanged tigecycline into feces was the primary route of elimination, and the secondary elimination pathways were renal excretion of unchanged drug and metabolism to glucuronide conjugates and N-acetyl-9-aminominocycline.

1. The metabolic disposition of venlafaxine has been studied in mouse, rat, dog, rhesus monkey and man after oral doses (22, 22, 2, and 10 mg/kg, and 50 mg, respectively) of 14C-venlafaxine as the hydrochloride. 2. In all species, over 85% of the administered radioactivity was recovered in the urine within 72 h, indicating extensive absorption from the GI tract and renal excretion. 3. Venlafaxine was extensively metabolized, with only 13.0, 1.8, 7.9, 0.3 and 4.7% dose appearing as parent compound in urine of mouse, rat, dog, monkey and man, respectively. The metabolite profile varied significantly among species, but primary metabolic reactions were demethylations and the conjugation of phase I metabolites. Hydroxylation of the cyclohexyl ring also occurred in mouse, rat and monkey, and a cyclic product was formed in rat and monkey. Glucuronidation was the primary conjugation reaction, although sulphate conjugates were also detected in mouse urine. 4. While no metabolite constituted more than 20% dose in any species except man, the major urinary metabolites were: mouse, N,O-didesmethyl-venlafaxine glucuronide; rat, cis-1,4-dihydroxy-venlafaxine; dog, O-desmethyl-venlafaxine glucuronide; monkey, N,N,O-tridesmethyl-venlafaxine; and man, O-desmethyl-venlafaxine.

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Aims In order to anticipate drug-interactions of potential clinical significance the ability of the novel antidepressant, venlafaxine, to inhibit CYP2D6 dependent imipramine and desipramine 2-hydroxylation was investigated in human liver microsomes. The data obtained were compared with the selective serotonin re-uptake inhibitors, fluoxetine, sertraline, fluvoxamine and paroxetine. Venlafaxine's potential to inhibit several other major P450s was also studied (CYP3A4, CYP2D6, CYP1A2). Methods K i values for venlafaxine, paroxetine, fluoxetine, fluvoxamine and sertraline as inhibitors of imipramine and desipramine 2-hydroxylation were determined from Dixon plots of control and inhibited rate data in human hepatic microsomal incubations. The inhibitory effect of imipramine and desipramine on liver microsomal CYP2D6 dependent venlafaxine O-demethylation was determined similarly. Venlafaxine's IC 50 values for CYP3A4, CYP1A2 CYP2C9 were determined based on inhibition of probe substrate activities (testosterone 6b-hydroxylation, ethoxyresorufin O-dealkylase and tolbutamide 4-hydroxylation, respectively). Results Fluoxetine, paroxetine, and fluvoxamine were potent inhibitors of imipramine 2-hydroxylase activity (K i values of 1.6±0.8, 3.2±0.8 and 8.0±4.3 mm, respectively; mean±s.d., n=3), while sertraline was less inhibitory (K i of 24.7±8.9 mm). Fluoxetine also markedly inhibited desipramine 2-hydroxylation with a K i of 1.3±0.5 mm. Venlafaxine was less potent an inhibitor of imipramine 2-hydroxylation (K i of 41.0±9.5 mm) than the SSRIs that were studied. Imipramine and desipramine gave marked inhibition of CYP2D6 dependent venlafaxine O-demethylase activity (K i values of 3.9±1.7 and 1.7±0.9 mm, respectively). Venlafaxine did not inhibit ethoxyresorufin O-dealkylase (CYP1A2), tolbutamide 4-hydroxylase (CYP2C9) or testosterone 6b-hydroxylase (CYP3A4) activities at concentrations of up to 1 mm. Conclusions It is concluded that venlafaxine has a low potential to inhibit the metabolism of substrates for CYP2D6 such as imipramine and desipramine compared with several of the most widely used SSRIs, as well as the metabolism of substrates for several of the other major human hepatic P450s.