The relative contributions of CYP3A4 and CYP3A5 to the metabolism of vinorelbine.

Vinorelbine is a semisynthetic vinca alkaloid used in the treatment of advanced breast and non-small cell lung cancers. Vincristine, a related vinca alkaloid, is 9-fold more efficiently metabolized by CYP3A5 than by CYP3A4 in vitro. This study quantified the relative contribution of CYP3A4 and CYP3A5 to the metabolism of vinorelbine in vitro using cDNA-expressed human cytochrome P450s (P450s) and human liver microsomes (HLMs). CYP3A4 and CYP3A5 were identified as the P450s capable of oxidizing vinorelbine using a panel of human enzymes and selective P450 inhibitors in HLMs. For CYP3A4 coexpressed with cytochrome b5 (CYP3A4+b5) and CYP3A5+b5, the Michaelis-Menten constants for vinorelbine were 2.6 and 3.6 µM, respectively, but the Vmax of 1.4 pmol/min/pmol was common to both enzymes. In HLMs, the intrinsic clearance of vinorelbine metabolism was highly correlated with CYP3A4 activity, and there was no significant difference in intrinsic clearance between CYP3A5 high and low expressers. When radiolabeled vinorelbine substrate was used, there were clear qualitative differences in metabolite formation fingerprints between CYP3A4+b5 and CYP3A5+b5 as determined by NMR and mass spectrometry analysis. One major metabolite (M2), a didehydro-vinorelbine, was present in both recombinant and microsomal systems but was more abundant in CYP3A4+b5 incubations. We conclude that despite the equivalent efficiency of recombinant CYP3A4 and CYP3A5 in vinorelbine metabolism the polymorphic expression of CYP3A5, as shown by the kinetics with HLMs, may have a minimal effect on systemic clearance of vinorelbine.

A band-selective composite gradient: application to DQF-COSY.

We describe a unique band-selective method that utilizes a selective composite gradient to simultaneously achieve band selection and coherence pathway selection. This element is similar to the composite gradient known as the CLUB sandwich except the original broadband pulses have been replaced with selective pulses and the strengths of the antipolar gradients have been unbalanced. In this way, only the signals within the inversion band will continue to dephase throughout the duration of the element and satisfy the proper encoding-to-decoding gradient ratio necessary for coherence selection. Apart from the inverted polarity and asymmetry of the gradients, the band-selective CLUB sandwich is identical to the DPFGSE sequence and provides many of its desirable characteristics. We have successfully incorporated the band-selective CLUB into the DQF-COSY pulse sequence to create a band-selective experiment that offers the selectivity desired for resolution enhancement while maintaining excellent phase behavior. This is demonstrated on the congested aliphatic region of the ionophorous antibiotic Lasalocid A.

Determination by NMR of the binding constant for the molecular complex between alprostadil and alpha-cyclodextrin. Implications for a freeze-dried formulation.

A binding constant was determined for the complexation reaction between alprostadil (PGE1) and alpha-cyclodextrin (alpha-CD). This constant was used to calculate the fraction PGE1 free upon reconstitution of Caverject dual chamber syringe, indicated for the treatment of erectile dysfunction. The determination was based on the measurement of the chemical shift of the C20 methyl protons of PGE1. The observed chemical shift varies as a linear function of the amount of PGE1 bound. The binding constant was obtained from the binding isotherm, a curve of the observed chemical shift versus free ligand (alpha-CD) concentration, through the application of non-linear regression analysis. A value K11 = 966 M(-1) +/- 130 M(-1) (2s), measured at 27 degrees C, was obtained. This value is in good agreement with those reported in the literature. The percent PGE1 free was subsequently calculated for the reconstituted solution and in the corpora cavernosum after injection. The latter showed PGE1 to be delivered essentially quantitatively to the targeted site.

Novel rearrangement of a 2-aryl-3-alkyl-3H-indol-3-ol to a 1,4,5,6-tetrahydro-2,6-methano-1-benzazocin-3(2H)-one with implications for the biosynthesis of aspernomine.

[reaction: see text] Nominine (1) and aspernomine (2) are two biologically important indole diterpenoids that arise from a common digeranylindole precursor. The skeletal relationship of these two natural products was not heretofore understood. We have observed a novel rearrangement of 2-(2-bromophenyl)-3-(3-butenyl)-3H-indol-3-ol (5) to 7, which contains the uncommon 1,4,5,6-tetrahydro-2,6-methano-1-benzazocin-3(2H)-one ring system, under acidic conditions. This rearrangement suggests that aspernomine (2) may arise biosynthetically from nominine (1).

On-column nitrosation of amines observed in liquid chromatography impurity separations employing ammonium hydroxide and acetonitrile as mobile phase.

The availability of high performance liquid chromatography (HPLC) columns capable of operation at pH values up to 12 has allowed a greater selectivity space to be explored for method development in pharmaceutical analysis. Ammonium hydroxide is of particular value in the mobile phase because it is compatible with direct interfacing to electrospray mass spectrometers. This paper reports an unexpected N-nitrosation reaction that occurs with analytes containing primary and secondary amines when ammonium hydroxide is used to achieve the high pH and acetonitrile is used as the organic modifier. The nitrosation reaction has generality. It has been observed on multiple columns from different vendors and with multiple amine-containing analytes. Ammonia was established to be the source of the nitroso nitrogen. The stainless steel column frit and metal ablated from the frit have been shown to be the sites of the reactions. The process is initiated by removal of the chromium oxide protective film from the stainless steel by acetonitrile. It is hypothesized that the highly active, freshly exposed metals catalyze room temperature oxidation of ammonia to NO but that the actual nitrosating agent is likely N(2)O(3).

The disposition and metabolism of naveglitazar, a peroxisome proliferator-activated receptor alpha-gamma dual, gamma-dominant agonist in mice, rats, and monkeys.

Naveglitazar [LY519818; benzenepropanoic acid, alpha-methoxy-4-[3-(4-phenoxyphenoxy)propoxy], (alpha-S)-] is a nonthiozolidinedione peroxisome proliferator-activated receptor alpha-gamma dual, gamma-dominant agonist that has shown glucose-lowering potential in animal models and in the clinic. Studies have been conducted to characterize the disposition, metabolism, and excretion of naveglitazar in mice, rats, and monkeys after oral and/or i.v. bolus administration. After oral administration of [(14)C]naveglitazar, naveglitazar was well absorbed and moderately metabolized in all species evaluated, with total recoveries of radioactivity ranging from 90 to 96%. Naveglitazar was the most abundant peak observed in circulation at C(max), representing 68 to 81% of the total radioactivity in plasma. The most prominent metabolite observed in circulation was the R-enantiomer of naveglitazar, LY591026, which is formed via enzymatic chiral inversion. para-Hydroxy naveglitazar and the sulfate conjugate of para-hydroxy naveglitazar were also observed in circulation in most species, especially in the monkey. The metabolic pathways observed include enzymatic chiral inversion, aromatic hydroxylation, oxidative dehydrogenation, and/or various phase II conjugation pathways. Naveglitazar was highly bound to plasma proteins among the species examined (>99%), and binding was independent of concentration. Biliary excretion was recognized as the most prominent excretion pathway in bile duct-cannulated rats (79 of the 96% recovered), producing an acyl glucuronide conjugate of naveglitazar and a sulfate and glucuronide diconjugate of para-hydroxy naveglitazar, which were shown to be reversible. The primary excretory pathway observed in mice and monkeys was via the feces. In summary, naveglitazar was well absorbed, moderately metabolized, and excreted via the feces in mice, rats, and monkeys.

In vivo metabolism of [14C]ruboxistaurin in dogs, mice, and rats following oral administration and the structure determination of its metabolites by liquid chromatography/mass spectrometry and NMR spectroscopy.

Ruboxistaurin (LY333531), a potent and isoform-selective protein kinase C beta inhibitor, is currently undergoing clinical trials as a therapeutic agent for the treatment of diabetic microvascular complications. The present study describes the disposition and metabolism of [14C]ruboxistaurin following administration of an oral dose to dogs, mice, and rats. The study revealed that ruboxistaurin was highly metabolized in all species. Furthermore, the results from the bile duct-cannulated study revealed that ruboxistaurin was well absorbed in rats. The primary route of excretion of ruboxistaurin and its metabolites was through feces in all species. The major metabolite detected consistently in all matrices for all species was the N-desmethyl metabolite 1, with the exception of rat bile, in which hydroxy N-desmethyl metabolite 5 was detected as the major metabolite. Other significant metabolites detected in dog plasma were 2, 3, 5, and 6 and in mouse plasma 2, 5, and 19. The structures of the metabolites were proposed by tandem mass spectrometry with the exception of 1, 2, 3, 5, and 6, which were additionally confirmed either by direct comparison with authentic standards or by nuclear magnetic resonance spectroscopy. To assist identification by nuclear magnetic resonance spectroscopy, metabolites 3 and 5 were produced via biotransformation using recombinant human CYP2D6 and, likewise, metabolite 6 and compound 4 (regioisomer of 3 which did not correlate to metabolites found in vivo) were produced using a microbe, Mortierella zonata. The unambiguous identification of metabolites enabled the proposal of clear metabolic pathways of ruboxistaurin in dogs, mice, and rats.

Adiabatic pulses in 1H-15N direct and long-range heteronuclear correlations.

The application of adiabatic inversion pulses to the detection of (1)H-(15)N heteronuclear correlations is described. The pulse sequences studied were gHSQC, CRISIS-gHSQC, gHMBC and CRISIS-gHMBC. The poor inversion quality of rectangular 180 degrees X pulses can lead to a loss of signal at the peripheries of the spectrum. Replacing these pulses with adiabatic sweeps significantly improves sensitivity across the potentially large (15)N spectral window. Satellite spectrum profiles are shown to demonstrate the increase in sensitivity when employing adiabatic pulses on wide spectral widths. Additionally, the active pharmaceutical nizatidine was used as a model compound to demonstrate the improvements in the long-range correlation data.

Selective reduction of N-oxides to amines: application to drug metabolism.

Phase I oxidative metabolism of nitrogen-containing drug molecules to their corresponding N-oxides is a common occurrence. There are instances where liquid chromatography/tandem mass spectometry techniques are inadequate to distinguish this pathway from other oxidation processes, including C-hydroxylations and other heteroatom oxidations, such as sulfur to sulfoxide. Therefore, the purpose of the present study was to develop and optimize an efficient and practical chemical method to selectively convert N-oxides to their corresponding amines suitable for drug metabolism applications. Our results indicated that efficient conversion of N-oxides to amines could be achieved with TiCl(3) and poly(methylhydrosiloxane). Among them, we found TiCl(3) to be a facile and easy-to-use reagent, specifically applicable to drug metabolism. There are a few reports describing the use of TiCl(3) to reduce N-O bonds in drug metabolism studies, but this methodology has not been widely used. Our results indicated that TiCl(3) is nearly as efficient when the reductions were carried out in the presence of biological matrices, including plasma and urine. Finally, we have shown a number of examples where TiCl(3) can be successfully used to selectively reduce N-oxides in the presence of sulfoxides and other labile groups.