I. Kinetics and metabolism of theobromine in male rats

On the basis of general pharmacological information (blood cells/plasma partition, plasma protein binding) and using HPLC as the principal analytical method, we investigated the kinetics and metabolism of theobromine (a caffeine metabolite) in male rats after a single dose and after a 2 week chronic application. Doses in both conditions varied between 1 and 100 mg/kg. In in vitro and in vivo the fraction of theobromine unbound to plasma proteins averaged 0.90 over a wide range of concentrations. No significant difference was found in the pharmacokinetic profile of the drug after acute or chronic treatment at different doses except for a reduction in the absorption rate constant as the dose increased. AUC values increased in proportion to the dose. The 2 treatment schedules were also similar as regards metabolism, at least 50% of the administered dose of theobromine being excreted unchanged, and 25% as 6-amino-5-[N-methyl-formylamino]1-methyluracil. Only at the highest doses was there a tendency for theobromine to accumulate at the expense of its major metabolite (a uracil compound).     

Kinetics of pulmonary surfactant phosphatidylcholine metabolism in the lungs of silica-treated rats

Exposure of rats to silica by intratracheal injection increased the intra- and extracellular compartments of pulmonary surfactant phospholipid. These changes were dose and time dependent, but both pools were not affected equally. Seven days after the instillation of 10 mg of silica, the intracellular pool increased 13.3-fold, from 1.49 +/- 0.30 to 19.86 +/- 0.77 mg of surfactant phospholipid per pair of lungs, and the extracellular pool increased 7.4-fold, from 1.87 +/- 0.79 to 13.79 +/- 0.72 mg of surfactant phospholipid per pair of lungs. To investigate the physiologic processes responsible for these massive accumulations of surfactant. [14C]choline was injected into the tail veins of control and silica-treated rats and the specific activity of surfactant phospholipids within the intracellular and extracellular pools was determined at various times between 0 and 26 hr after injection. All of the processes measured were increased in response to silica exposure, but not to the same extent. At 1 hr, incorporation of [14C]choline into the intracellular surfactant pool was increased 12.6-fold above controls, from 4.8 +/- 1.1 x 10(3) to 60.6 +/- 26.6 x 10(3) dpm. The flux of [14C]choline-labeled phospholipid from the intracellular to the extracellular pool was increased 7.3-fold, from 102 +/- 10 to 749 +/- 39 micrograms/hr in silica-treated animals, but its disappearance from the extracellular pool was increased only 5.0-fold, from 87 +/- 8 to 434 +/- 21 micrograms/hr. The half-life of [14C]choline-labeled phospholipids in the intracellular pool of surfactant was increased from 10.1 +/- 1.0 to 18.3 +/- 5.3 hr and that in the extracellular surfactant pool from 14.8 +/- 1.4 to 21.9 +/- 4.9 hr. Expansion of the intra- and extracellular pools of surfactant phospholipids may be explained on the basis of a metabolic imbalance in which the intracellular production of surfactant is increased above its secretion rate into the extracellular compartment, and the secretion rate is elevated above the rate at which surfactant phospholipids are cleared from the alveoli.     

The in vitro hepatic microsomal metabolism of N-benzyladamantanamine in rats

The metabolism of N-benzyladamantanamine (NBAD) was studied in vitro using rat hepatic microsomal preparations. The substrate and proposed metabolites were synthesized and characterized using spectroscopic techniques and separated using a reverse phase HPLC system. NBAD was incubated with rat microsomal preparations, extracted into DCM in the presence of NaCl and evaporated under a stream of nitrogen. The results from HPLC studies showed that NBAD produced the corresponding nitrone and hydroxylamine. This experiment also revealed that dealkylation occurred. No metabolites were observed which corresponded to authentic amide or oxaziridine. The reactions required a microsomal enzyme source and NADPH as a cofactor. The results indicate that the nitrone observed as a metabolite of NBAD is not an intermediate leading to the formation of an oxaziridine and hence an amide, under careful experimental conditions excluding light.     

Kinetics and metabolism of amphetamine in the brain of rats of different ages

The kinetics and tissue distribution of amphetamine and its metabolites p-hydroxyamphetamine (p-PH-A) and p-hydroxynorephedrine (p-OH-NE) were investigated in young adult (3-4 months) and old (20-25 months) male rats, after i.p. injection of 5 mg/kg tritium labelled D-amphetamine. The concentrations of these drugs were determined in plasma, cerebral cortex, brainstem and hypothalamus, by thin layer chromatography. 1. From 60 min up to 4 hrs after injection of amphetamine the concentration of amphetamine in plasma and brain tissue of old rats was significantly (P less than 0.05 higher than in young adult animals. In both age groups the levels of amphetamine in cerebral cortex greater than brain stem greater than hypothalamus. 2. The blood-brain barrier is permutle to p-OH-A; 10 to 20 min afer i.v. injection of 10 muCi/kg of p-OH-A (10 mCi/m mole) the ratio of brain/blood plasma was found to be 1:3. The half life of p-OH-A in blood plasma was almost identical after injection of amphetamine and p-OH-A (90 min and 98 min respectively). 3. The levels of p-OH-NE in different brain areas were significantly lower (P less than 0.05) in old animals than in young adult rats 4 hrs after application of amphetamine. This metabolite of amphetamine shows a higher concentration in the hypothalamus earlier than in other brain regions.     

Modulation of trichloroethylene in vitro metabolism by different drugs in rats

Trichloroethylene (TCE) is a widely used chemical to which humans are frequently exposed. Toxicological interactions with drugs are among factors having the potential to modulate the toxicity of TCE. The aim of this study was to identify metabolic interactions between TCE and 14 widely used drugs in rat suspended hepatocytes and characterize the strongest using microsomal assays (oxidation and/or glucuronidation). The concentrations of TCE and its metabolites, trichloroethanol (TCOH) and trichloroacetate (TCA), were measured by gas chromatography with injection headspace coupled to mass spectrometry (GC–MS). Results in hepatocyte incubations show that selected drugs can be segregated into four groups: group 1: drugs causing no significant interactions (five drugs: amoxicillin, carbamazepine, ibuprofen, mefenamic acid and ranitidine); group 2: increasing both TCE metabolites (two drugs: naproxen and salicylic acid); group 3: decreasing both TCE metabolites (five drugs: acetaminophen, gliclazide, valproic acid, cimetidine and diclofenac) and group 4: affecting only one (two drugs: erythromycin and sulphasalazine). Naproxen and salicylic acid (group 2) and acetaminophen, gliclazide and valproic acid (from group 3) presented the strongest interactions (i.e. drugs changing metabolite levels by 50% or more). For group 2 drugs, characterization in rat microsomes confirmed interaction with naproxen only, which was found to partially competitively inhibit TCOH glucuronidation (K_i = 211.6 μM). For group 3 selected drugs, confirmation was positive only for gliclazide (K_i = 58 μM for TCOH formation) and valproic acid (K_i = 1215.8 μM for TCA formation and K_i = 932.8 μM for TCOH formation). The inhibition was found to be partial non competitive for both drugs. Our results confirm the existence of interactions between TCE and a variety of widely used drugs. Further efforts are undertaken to determine if these interactions are plausible in humans and if they can impact the risk of toxicity of TCE in medicated population.     

Circadian variations of phenacetin metabolism in rats in vivo and in vitro.

The metabolism of phenacetin in vivo and in vitro at different periods of day was investigated in rats. In rats maintained on standard LD conditions the disappearance rate of phenacetin from blood and activity of phenacetin O-deethylase in liver were the highest in the morning and the lowest in the evening. Continuous illumination, adrenalectomy, phenobarbital or proadifen abolished this difference. It is postulated that these circadian changes of microsomal metabolism of phenacetin in rats liver are not fully responsible for the rhythmical changes in the antipyretic action of this drug that was observed previously. The mechanisms of this phenomenon are discussed.     

Sex-dependent pharmacokinetics and in vitro reductive metabolism of acetohexamide in Wistar-Imamichi rats.

A significant sex-related difference was observed for the pharmacokinetics of acetohexamide in Wistar-Imamichi (Wistar-IM) rats. However, there was no sex difference of the in vitro reductive metabolism of acetohexamide in the liver or kidney of these rats. Testectomy was found to decrease the plasma clearance (CLp) of acetohexamide in male rats, whereas ovariectomy had no effect on the CLp of acetohexamide in female animals, suggesting that androgens regulate the pharmacokinetics of acetohexamide. The co-administration of sulfamethazine, which is known to be metabolized by a male-specific cytochrome P450 (CYP) isoform (CYP2C11), significantly decreased the CLp of acetohexamide in male Wistar-IM rats. Based on these results, it is reasonable to assume that the sex-dependent pharmacokinetics of acetohexamide observed in Wistar-IM rats is associated with the male-specific hydroxylation catalyzed by CYP2C11.     

Kinetics of salicylate metabolism.

1 Nine patients with rheumatoid arthritis or non-inflammatory backache were given soluble aspirin (65 mg/kg body weight) daily. There was no significant difference between the plasma salicylate of those with rheumatoid arthritis and those with backache. 2 Two patients had plasma salicylate values that differed significantly from the remainder but neither these results nor the marginal differences between plasma salicylate levels of the others could be explained by individual variations in the capacity for excreting salicyluric acid or salicyl phenolic glucuronide. 3 Increasing the dose of aspirin in four patients demonstrated the reduced proportions of salicyluric acid and salicyl phenolic glucuronide excreted at high doses and the increased importance of unchanged salicylic acid as an excretory pathway. These findings are consistent with a limiting capacity for salicyluric acid and salicyl phenolic glucuronide synthesis and excretion. 4 The findings in one patient suggested that inter-subject variations in the capacity for producing salicyl phenolic glucuronide and salicyluric acid may have an effect on plasma salicylate levels at high doses of aspirin.     

Kinetics of diphenylhydantoin elimination in rats

Concentrations of diphenylhydantoin (DPH) in the blood of male Sprague-Dawley rats after intravenous injection of 10 and 40 mg/kg ¹⁴C-DPHwere determined over a sufficiently wide range to permit comparison of rates of decline at the same absolute and relative concentrations. This comparison leads to the conclusion that the elimination of DPH in the rat cannot be described by first-order or simple Michaelis-Menten kinetics but that it is qualitatively consistent with product inhibition of DPH metabolism.Supported in part by grant GM 19568 from the National Institutes of Health.On leave from the School of Pharmacy, University of Sydney, Sydney, Australia.     

Study on in vivo and in vitro metabolism of dimethylformamide in male and female rats

The study of dimethylformamide (DMF) metabolism by rat tissues in vitro indicates that formaldehyde is not a metabolic product as previously reported [1]. Furthermore, no other monocarbon derivative (CO, CH₃OH, HCOOH, CH₄) was detected when DMF was incubated with a fortified liver preparation. One metabolic product is methylhydroxymethylformamide (DMF- OH) measured as N-methylformamide (NMF) due to the breakdown of the hydroxymethyl group during gas chromatography. It was usually believed that the main metabolite excreted in urine following administration of DMF to male and female rats was N.M.F. The results of this study indicate that DMF-OH constitutes the main metabolite in vivo. A quantitatively less inmportant urinary metabolite, hydroxymethylformamide (NMFOH), is determined as formamide (F) by gase chromatography, In male and female rats, partial hepatectomy reduces markedly the in vivo biotransformation of DMF. Following administration of DMF or NMF, the total amount of metabolites (DMFOH and/or NMFOH) excerted in urine is identical in both sexes, but female rats excrete more unchanged parent compound than male rats. The rate of NMFOH excretion in urine following high doses of DMF supports the hypothesis that DMP may inhibit its own bioransformation.     

力达霉素的体外代谢

旨在研究力达霉素在血浆和肝微粒体中的体外代谢性质,指导临床合理用药。选择HPLC-MS/MS测定方法,通过测定力达霉素的活性成分,考察力达霉素在大鼠、比格犬、猕猴和人血浆及肝微粒体中的代谢稳定性以及在人肝微粒体中对细胞色素P450(cytochrome P450,CYP450)各亚型酶的抑制作用。结果表明,力达霉素在4个种属血浆中均有代谢,其代谢速率为大鼠>比格犬>人>猕猴;在4个种属肝微粒体中,只有在猕猴肝微粒体中代谢;在浓度为0.000 5～10 ng·mL-1时,对人肝微粒体中细胞色素P450各亚型酶几乎无抑制作用。可见力达霉素在人体内的代谢性质与比格犬体内较相似,而且临床上当力达霉素与通过CYP450酶代谢的药物合用时,不会导致这些药物的代谢减慢。     

Kinetics of azathioprine metabolism in fresh human blood

Azathioprine (AZA) is transformed in the whole fresh human blood in vitro to 6-mercaptopurine (6-MP). The rate of the above reaction was followed as a function of time at 25, 30 and 37 degrees C. Pseudo-first-order rate constants and thermodynamic parameters were calculated. The statistical evaluation of the parameters calculated was provided. Half-life time of 6-MP formation in blood from AZA at e.g. 37 degrees C was equal to 28.9 +/- 2.8 min.     

Effects of dimethylsulfoxide on the oxidative metabolism of certain tissues in rats in vitro.

Administration of 10 mM dimethylsulfoxide inhibits oxygen uptake in in vitro rat brain slices incubated in a Krebs-Ringer phosphate pH 7.4 solution containing 10 mM glucose. The effect is inverted when excessive potassium is added to the solution and the amount of sodium chloride is substituted by choline chloride. Using 10 mM, we observed a decrease in oxygen uptake in rat heart slices incubated in a Krebs-Ringer phosphate solution containing 131.8 mM Na+, 105.4 mM K+ and no calcium. Dimethylsulfoxide 10 mM, 1 mM and 0.1 mM does not alter the Na+/K+ ATPase activity nor the ouabain insensitive ATPase activity in in vitro preparations of rat brain, kidney and heart.     

Kinetics of benzylpenicillin metabolism in isolated rat hepatocytes

The metabolism of benzylpenicillin (PCG) in isolated rat hepatocytes was investigated. The evidence of metabolizing activity for PCG in hepatic cells was obtained as follows; the disappearance rate of PCG from the incubation medium followed Michaelis-Menten kinetics and was dependent on the cellular protein concentration, while PCG did not disappear when it was incubated with cells denaturated by heat. The rate of disappearance of PCG was reduced significantly in the presence of the structural analogue of PCG such as phenoxymethylpenicillin in the incubation medium. The major metabolite of PCG was identified, by high performance liquid chromatographic analysis, to be penicilloic acid (PA) of PCG. A kinetic model describing the intra- and extra-cellular concentrations of PCG and PA was developed. The proposed model fitted well the time course of changes in the concentration of PCG and PA. The clearance of the uptake of PCG by isolated hepatocytes was evaluated to be about 23-times greater than that of metabolism of PCG.     

Identification of new secondary metabolites of methoxyphenamine in man

Metabolites of methoxyphenamine were examined in the urine of three healthy human volunteers. The metabolites were separated by g.l.c. and identified by comparison of their chromatographic and mass-spectrometric behaviours with those of authentic synthetic compounds. 5-Hydroxy-2-methoxy-N-methylamphetamine, a metabolite previously identified by indirect methods, was conclusively identified by comparison with the now-available authentic synthetic material. In addition, three new metabolites of methoxyphenamine were identified--5-hydroxy-2-methoxyamphetamine, 2-methoxyphenylacetone and 5-hydroxy-2-methoxyphenylacetone.     

Croconazole metabolism in rats

1. Following subcutaneous administration of 1-(2-(3-chlorobenzyloxy)phenyl)vinyl)-1 H-imidazole (croconazole) to rats, four metabolites were identified by comparison of their mass and n.m.r. spectra, g.l.c., and t.l.c. with those of synthetic compounds and enzyme hydrolysis. These compounds are o-hydroxyacetophenone sulphate (M1S), 1-(1-(2-hydroxyphenacyl)vinyl)-1H-imidazole sulphate (M12S), 2-(3-chlorobenzyloxy)phenacyl alcohol (M2), and 2-(3-chlorobenzyloxy)benzoic acid (M9-1).

2. At 10?mg/kg dosing of croconazole the elimination rate of the unchanged drug from plasma was faster in male than in female rats.

3. The percentage of excretion of M12S in 24?h urine was 17% for males and 7.5% for females, and that of M1S was 6.6% for males and 6.5% for females. The percentage of excretion of M2 in 24?h bile was 14% for males and 22% for females, and that of M9-1 was 3.7% for males and 1.6% for females.     

Afobazole metabolism in rats

Metabolism of afobazole in rats has been studied using mass-spectrometry and HPLC, which revealed 17 products of afobazole biotransformation along with the parent compound. The structures of six afobazole metabolites were established and confirmed by comparison of HPLC retention times with the synthetic reference compounds and HPLC/mass spectrometry. Other metabolites were characterized by the masses of molecular ions. A significant fraction of the drug dose is biotransformed with the formation of hydroxylated benzimidazole moiety and oxidated morpholine.