Characterization of bropirimine O-glucuronidation in human liver microsomes.

1. The antitumour agent bropirimine undergoes significant Phase II conjugation in vivo. Incubation of [14C]bropirimine with human liver microsomes resulted in the formation of a single product peak (M1) using high-performance liquid chromatography with radiochemical detection and was tentatively assigned as bropirimine glucuronide based on sensitivity to beta-glucuronidase and by obtaining the expected mass of 442/444 amu with liquid chromatography/mass spectrometry. Following metabolite isolation, the structure of M1 was established as bropirimine O-glucuronide by 1H-nuclear magnetic spectroscopy. 2. Studies aimed at identifying the human liver UDP-glucuronosyltransferase (UGT) enzyme(s) involved in the glucuronidation of bropirimine were carried out using recombinant human UGTs and it was determined that glucuronidation of bropirimine was catalysed by UGT1A1, UGT1A3 and UGT1A9. Bropirimine O-glucuronidation followed Michaelis-Menten kinetics and the Km and Vmax (mean +/- SD; n = 3) were 1217 +/- 205 microM and 667 +/- 188 pmol min(-1) mg(-1), respectively. 3. The activity of bropirimine O-glucuronidation by human liver microsomes was inhibited by bilirubin (40%) and with mefenamic acid (80%). Although buprenorphine extensively inhibited the activity of bropirimine O-glucuronidation by UGT1A3, the inhibition profile did not parallel that observed in HLMs. 4. The results demonstrate that UGT1A9 and to a lesser extent UGT1A1 are responsible for the majority of bropirimine O-glucuronidation in man.

Pharmacokinetics, metabolism, and excretion of linezolid following an oral dose of [(14)C]linezolid to healthy human subjects.

Linezolid (Zyvox), the first of a new class of antibiotics, the oxazolidinones, is approved for treatment of Gram-positive bacterial infections, including resistant strains. The disposition of linezolid in human volunteers was determined, after a 500-mg (100-microCi) oral dose of [(14)C]linezolid. Radioactive linezolid was administered as a single dose, or at steady-state on day 4 of a 10-day, 500-mg b.i.d. regimen of unlabeled linezolid (n = 4/sex/regimen). Mean recovery of radioactivity in excreta was 93.8 +/- 1.1% (range 91.2-95.2%, n = 15), of which 83.9 +/- 3.3% (range 76.7-88.4%) was in urine and 9.9 +/- 3.4% (range 5.3-16.9%) was in feces. There was no major difference in rate or route of excretion of radioactivity by dose regimen. Linezolid was excreted primarily intact, and as two inactive, morpholine ring-oxidized metabolites, PNU-142586 and PNU-142300. Other minor metabolites were characterized by high-performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry and (19)F NMR spectroscopy. After the single radioactive dose, linezolid was the major circulating drug-related material accounting for about 78% (male) and 93% (female) of the radioactivity area under the curve (AUC). PNU-142586 (T(max) of 3-5 h) accounted for about 26% (male) and 9% (female) of the radioactivity AUC. PNU-142300 (T(max) of 2-3 h) accounted for about 7% (male) and 4% (female) of the radioactivity AUC. Overall, mean linezolid and PNU-142586 exposures at steady-state were similar across sex. In conclusion, linezolid circulates in plasma mainly as parent drug. Linezolid and two major, inactive metabolites account for the major portion of linezolid disposition, with urinary excretion representing the major elimination route. Formation of PNU-142586 was the rate-limiting step in the clearance of linezolid.

Identification of the metabolites of the HIV-1 reverse transcriptase inhibitor delavirdine in monkeys.

Delavirdine mesylate (U-90152T) is a highly specific nonnucleoside HIV-1 reverse transcriptase inhibitor currently under development for the treatment of AIDS. The metabolism of delavirdine was investigated in male and female cynomolgus monkeys after oral administration of [14C-carboxamide]delavirdine mesylate at single doses of 80 mg/kg and multiple doses of 160 to 300 mg/kg/day. Desalkyl delavirdine was the major metabolite in circulation. In urine, desalkyl delavirdine accounted for nearly half of the radioactivity, with despyridinyl delavirdine and conjugates of desalkyl delavirdine accounting for most of the remaining radioactivity. Bile was mostly composed of desalkyl delavirdine and 6'-O-glucuronide delavirdine, with parent drug, 4-O-glucuronide delavirdine, and conjugates of desalkyl delavirdine as significant components. In addition, several minor metabolites were observed in urine and bile of delavirdine treated monkeys. The metabolism of delavirdine in the monkey was extensive and involved N-desalkylation, hydroxylation at the C-4' and C-6' positions of the pyridine ring, hydroxylation at the C-4 position of the indole ring, pyridine ring-cleavage, N-glucuronidation of the indole ring, and amide bond cleavage as determined by MS and/or one-dimensional and two-dimensional NMR spectroscopies. Phase II biotransformations included glucuronidation, sulfation, and beta-N-acetylglucosaminidation. The identification of the N-linked beta-N-acetylglucosamine and 4-O-glucuronide metabolites of delavirdine represents novel biotransformation pathways.

Metabolism of the human immunodeficiency virus type 1 reverse transcriptase inhibitor delavirdine in rats.

Delavirdine mesylate (U-90152T) is a highly specific nonnucleoside reverse transcriptase inhibitor currently under development for the treatment of AIDS. The excretion, disposition, and metabolism of delavirdine were investigated in Sprague-Dawley rats after oral administration of [14C]delavirdine mesylate at single doses ranging from 10 to 250 mg/kg and multiple doses ranging from 20 to 250 mg/kg/day. Excretion studies showed that feces was the major route of elimination, delavirdine was well absorbed (>80%) after a 10 mg/kg single dose, and excretion was dose-dependent. The metabolism of delavirdine in the rat was extensive. The following metabolites were identified (% of dose in rats given 10 and 100 mg/kg, respectively): 6'-hydroxy delavirdine (7.1% and 15.6%) and its glucuronide (12.2% and 6.2%) and sulfate (5.5% and 3.2%) conjugates, despyridinyl delavirdine (12.1% and 11.7%) and its conjugate (13.0% and 11.7%), desalkyl delavirdine (16.5% and 13.4%), and its N-sulfamate, 6'- and 4'-sulfate conjugates (2.9% and 3.9%). Cleavage of the amide bond in delavirdine to give N-isopropylpyridinepiperazine and indole carboxylic acid constituted a minor pathway. Degradation of 6'-hydroxy delavirdine generated despyridinyl delavirdine and the pyridine-ring opened MET-14. The metabolic pathway of delavirdine involved N-desalkylation, pyridine ring hydroxylation, pyridine ring cleavage, and amide bond cleavage.

Diffusion in HPMC gels. I. Determination of drug and water diffusivity by pulsed-field-gradient spin-echo NMR.

PURPOSE: This work describes diffusivity measurements of drug (adinazolam mesylate) and water in a variety of solutions including polymer gels. METHODS: Pulsed-field-gradient spin-echo (PFGSE) NMR methods were employed to measure the diffusivity. RESULTS: In binary component solutions, adinazolam diffusivity is generally found to exhibit an exponential dependence on the concentration of the viscosity-inducing agent (VIA), which is glucose, lactose, maltoheptaose, hydroxypropyl methylcellulose (HPMC) or drug itself. An increasing obstruction power to drug diffusion from glucose to HPMC is observed, which can be related to the polymerization degree of the VIA. In contrast, adinazolam diffusivity in HPMC gels shows little dependence upon the polymer viscosity grades examined (K100LV, K4M, and K15M). The temperature dependence of adinazolam diffusivity in dilute VIA solutions reveals that the diffusion barrier for the drug is similar to that for self-diffusion of water. CONCLUSIONS: The retarding effect from the VIA for drug diffusion is concluded to be primarily associated with a steric obstruction mechanism. In multicomponent gels with varied concentrations of drug, lactose and HPMC, the drug diffusivity can be approximately described as an exponential function of the summation of the products of the proportionality constant (Ki) and concentration for each VIA component. In contrast, water diffusion behavior shows an universal exponential dependence upon the VIA concentration and small dependence upon the nature of the VIA. The interpretation of the diffusivity data is discussed and compared to two existing diffusion models (Yasuda and Mackie-Meares models).

The 3'-keto-diol equilibrium of trospectomycin sulfate bulk drug and freeze-dried formulation: solid-state carbon-13 cross-polarization magic angle spinning (CP/MAS) and high-resolution carbon-13 nuclear magnetic resonance (NMR) spectroscopy studies.

Understanding how moisture interacts with a drug or formulation is a critical component of product development. This study demonstrates how water affects the 3'-gem-diol<==>3'-keto equilibrium in trospectomycin sulfate bulk drug and freeze-dried formulation, as probed by solid-state carbon-13 cross-polarization magic angle spinning (CP/MAS) and high-resolution nuclear magnetic resonance (NMR) spectroscopy. Drying the bulk drug or formulation to low water levels dehydrates trospectomycin sulfate from the diol to the keto form. Carbon-13 CP/MAS NMR spectroscopy measures the keto drug concentration in solid samples directly. The bulk drug, which contains approximately 16% water, is more than 90% in the 3'-diol form. Oven drying to < 3% water converts approximately 75% of the drug to the 3'-keto form. The drug is formulated as a freeze-dried, sterile powder that can contain up to 12% water depending on the freeze-drying conditions. These studies show that the 3'-keto concentration rises uniformly (up to 75%) with decreasing residual water in the freeze-dried cake. The keto-diol equilibrium was also studied in solution by high-resolution carbon-13 NMR experiments, and it was found that raising the temperature or using dimethyl sulfoxide (DMSO) as a solvent also dehydrates the drug. For example, in aqueous solution at 25 degrees C, nearly all (> 95%) of the drug is in the 3'-diol form. After equilibration at 60 degrees C, however, the 3'-keto content increases to 7%, and in d6-DMSO solvent at 25 degrees C the drug is mostly (60%) in the 3'-keto form.

Preferred conformational state of the N-terminus section of a bovine growth hormone fragment (residues 96-133) in water is an omega loop.

The solution structure of a 38-amino-acid-residue, biologically active fragment of bovine growth hormone (bGH96-133) was investigated with a combined nuclear magnetic resonance (NMR) and computer modeling approach. With the distance geometry program DISGEO and distance constraints derived from nuclear Overhauser enhancement (NOE) experiments, it was found that residues Ser-100 to Tyr-110 circumscribe and omega-loop, a recently categorized feature of nonregular secondary protein structure.

Biosynthesis of porphyrins and corrins. 2. Isolation, purification, and NMR investigations of the porphobilinogen-deaminase covalent complex.

The procedures for the generation of enzyme-substrate complexes from labeled porphobilinogens [(2,11-13C]PBG and [2,6,11-3H]PBG) with deaminase and the methods employed for their purification are described. Use of 13C NMR failed to detect the substrate bound to the enzyme, suggesting that the line width must be inordinately large. The complex was found to disproportionate with time when stored at 25 degrees C. However, enzyme-bound uroporphyrinogen I (uro'gen I) was detected, both in the intact protein and in the oligopeptides from tryptic digestion and peptide mapping. The first detection of an enzyme-substrate complex by 3H NMR is described for [3H]PBG and deaminase. The line widths of the observed resonances were found to be extremely large and dependent upon temperature, giving chemical shifts that suggest the involvement of a sulfhydryl group as the nucleophilic enzyme group that binds the substrate. The catalytic competence of this complex was also demonstrated by displacing bound [3H]PBG with unlabeled PBG. During the resultant formation of [3H]uro'gen I, a transient low-intensity signal was detected that has been tentatively assigned to the highly reactive azafulvene species, proposed in several mechanistic schemes for porphyrin biosynthesis.

Oxidation reactions by prostaglandin cyclooxygenase-hydroperoxidase.

oxidations of organic sulfides, amines, and even enzymes catalyzed by purified and microsomal forms of prostaglandin cyclooxygenase-hydroperoxidase have been studied using O2 incorporation into arachidonic acid to monitor oxygenase and [14C]15-hydroperoxyprostaglandin E2 reduction to prostaglandin E2 to measure hydroperoxidase. The oxygenase was protected by phenol against the irreversible deactivation induced by low levels of hydroperoxides. Furthermore, the EPR signal noted during reactions with the microsomal enzyme probably reflected the adventitious oxidation of endogenous materials. As described previously for phenol and other reducing cosubstrates, methyl phenyl sulfide (MPS) increased hydroperoxidase activity at all concentrations studied, while stimulating oxygenase at low levels and inhibiting it at 5-10 mM. In stoichiometric equivalence with 15-hydroperoxyprostaglandin E2 reduction, MPS was enzymatically oxidized to its analogous sulfoxide, methylphenyl sulfoxide, acquiring an oxygen atom exclusively from the hydroperoxide and demonstrating some chiral character. In contrast, other oxidizable compounds such as N,N-dimethylphenylenediamine and aminopyrine reacted via radical intermediates. Phenylbutazone, which is oxidized using dissolved molecular oxygen, did not compete with MPS oxidation. Hence, MPS was oxidized while bound to the enzyme, whereas the amine oxidation occurred in solution via an enzyme-formed oxidant. The Soret peak noted with cyclooxygenase-hydroperoxidase was examined as a possible measure of this binding, but was also noted in denatured and deactivated enzyme, suggesting that its relevance should be reconsidered. Despite the similarities in their drug-metabolizing profiles, cyclooxygenase-hydroperoxidase is clearly distinct from cytochrome P-450. The mechanism of this hydroperoxidase is considered in the context of other more extensively studied peroxidases.

Control of porphyrin biosynthesis in Rhodopseudomonas spheroides and Propionibacterium shermanii. A direct 13C nuclear-magnetic-resonance spectroscopy study.

The facultative anaerobes Rhodopseudomonas spheroides and Propionibacterium shermanii were grown under anaerobic and aerobic conditions. The effect of light was studied with the photosynthetic R. spheroides, and the adaptation of both species to dark anaerobic life was monitored by direct observation of 5-amino[5-13C]laevulinic acid metabolism by using 13C nuclear-magnetic-resonance spectroscopy.