The nitrosating agent in mice exposed to nitrogen dioxide: improved extraction method and localization in the skin.

We reported previously that mice exposed to atmospheric NO2 contained a nitrosating agent (NSA) that reacted with morpholine in aqueous methanol homogenates of the mice to give N-nitrosomorpholine. We have now found that N-nitrosomorpholine was also produced by reacting morpholine with ether extracts of aqueous homogenates prepared from NO2-exposed mice. After exposure to NO2 for 4 hr, mice contained NSA (5.0 nmol/g tissue, corrected to 50 ppm NO2 and assuming that 1 mol NSA yields 1 mol N-nitrosomorpholine). This is 3.6 times the concentration observed by our previous method. Some NSA (0.6 nmol/g tissue) was also detected in untreated mice. The NSA in ether extracts was nonvolatile and stable on storage at -15 degrees or for short periods in the presence of water at pH 1 to 10, but it was decomposed by a pH 1 solution of nitrite scavengers. It reacted to similar extents with three different secondary amines. Eighty-eight % of the NSA occurred in the skin, one-third of which was in the hair. The high skin concentration occurred when the bodies but not the heads of mice were exposed to NO2, indicating that the major exposure route was the skin. The NSA might consist of alpha-nitro or other activated nitrite esters derived from unsaturated lipids.

Nitrosamine formation from amines applied to the skin of mice after and before exposure to nitrogen dioxide.

Skin lipids of mice exposed to NO2 contain lipid-soluble nitrosating agent(s) (NSA) that react in vitro with amines to produce nitrosamines. To test whether this reaction occurs in skin, we exposed mice to 50 ppm NO2 for 4 h and, 20 h later, applied 25 mg morpholine or N-methylaniline to the skin, which was then analyzed for the corresponding nitrosamine. When morpholine was applied, mean N-nitrosomorpholine yield was only 0.3 nmol/mouse (not significant). When N-methylaniline was applied and mice were killed after 10-40 min, N-nitroso-N-methylaniline yield in the skin was 13-21 nmol/mouse of which 87% occurred in the hair. NSA formation when mice were exposed to 6.5 ppm NO2 was only 0.15% of that for exposure to 50 ppm NO2. NSA occurred mostly in surface lipids of the skin and its in vitro reaction to give nitrosamines was not inhibited by alpha-tocopherol. When morpholine was painted and mice were then exposed to 55 ppm NO2 for 30 min, the skins contained 19 nmol N-nitrosomorpholine/mouse, attributed to a direct reaction between NO2 and the amine. We concluded that nitrosamine formation in skin by this direct reaction may be more important than the reaction of amines with NO2-derived NSA.

Effect of diet on fecal excretion and gastrointestinal tract distribution of unmetabolized benzo(a)pyrene and 3- methylcholanthrene when these compounds are administered orally to hamsters.

3-Methylcholanthrene (3MC) administered p.o. has induced tumors of the hamster gastrointestinal tract (GIT), including the large intestine. This process may depend on the concentration of unchanged hydrocarbon in the GIT contents. Benzo(a)pyrene (BP) ingestion could be involved in human GIT carcinogenesis. Accordingly, male Syrian golden hamsters were fed diets containing BP or 3MC for 10 days. Feces collected during the last two to three days of feeding were analyzed for the unchanged hydrocarbons by KOH:methanol digestion, Florisil column and paper chromatography, and ultraviolet spectrophotometry. With a semisynthetic diet containing 5% Alphacel, 6% corn oil, and 100 microgram BP per g. fecal BP excretion was 0.45% of the dose. Variation of the corn oil content had little effect. Fecal BP excretion was increased 13 times (to 6% of the dose) when 5% wheat brain was used in place of Alphacel and 4.5 times when a commercial diet was used. This suggests that bran adsorbed or sequestered the BP. Water content of the large-intestine contents was increased when the brain diet was fed. Both these factors could affect mucosal exposure to BP. For 3MC, fecal excretion of unchanged hydrocarbon was 14 times greater than for BP under similar conditions. The GIT contents of hamsters fed BP or 3MC showed hydrocarbon concentrations in the order: stomach greater than lower large intestine greater than other sections.

Comparative mutagenicity testing of bropirimine, 3. Bropirimine does not induce cytogenetic damage in vivo in the rat but does produce micronuclei in the mouse.

Bropirimine is a biological response modifier (BRM) with potential antineoplastic and antiviral indications. Recent results have documented the negative findings in the Ames Salmonella assay, the in vitro UDS assay and the mouse lymphoma TK+/- assay as well as positive findings in the in vitro cytogenetic assay in CHO cells. Extensive mechanistic studies failed to establish the reason for positive findings in the in vitro cytogenetic assays. The data reported here cast doubt on the relevance of the in vitro cytogenetic results and suggest limited in vivo genotoxic potential. At doses as high as 150 mg/kg (i.p.) and 6.73 g/kg (p.o.), no evidence of chromosome aberration induction was observed in rat bone marrow cytogenetic assays. Consistent with these data, plasma and bone marrow tissue levels in similarly treated animals were well below those required for activity in the in vitro chromosome aberration assays. Positive results were obtained in the mouse micronucleus assay. However, the significance of these findings may be explained by markedly different pathways of metabolism in that species as compared to the rat. Hence, the findings in the mouse are of questionable relevance to human risk assessment. Exposure of humans to bropirimine, under therapeutically acceptable regimens is unlikely to constitute a genotoxic health hazard.

Absorption, distribution, metabolism, and excretion of dirlotapide in the dog.

Three once-daily oral doses of 0.2 mg/kg [(14)C]dirlotapide were administered to beagle dogs to study the absorption, distribution, metabolism, and excretion of dirlotapide. Mean (14)C recovered at 2.5 and 4.5 h after the last dose was 90%. Mean (14)C in urine, bile, and feces was <1%, 1.7%, and 56% of the dose, respectively. In tissues, 26% of the (14)C dose was present in the gastrointestinal tract, 6.0% in liver, and <1% each in kidney, gall bladder, heart, and brain. To further characterize drug disposition, a single 2.5-mg/kg oral dose of [(14)C]dirlotapide was administered to beagle dogs. More than 84% of the dose had been eliminated by 72 h in feces, with 21% of the dose present in feces as parent dirlotapide. Less than 1% of the dose was excreted in urine. In bile collected during the first 24-h postdose from three dogs, 32% and 11% of the (14)C dose was present in samples from male and female dogs, respectively. Based upon metabolite profiling of plasma, excreta, and bile samples, dirlotapide was extensively metabolized to more than 20 metabolites. Biliary/fecal excretion and the potential for enterohepatic recycling of metabolites are suggested.

A nitrosating agent from the reaction of atmospheric nitrogen dioxide (NO2) with methyl linoleate: comparison with a product from the skins of NO2-exposed mice.

We showed previously that exposure of mice to atmospheric nitrogen dioxide (NO2) leads to the formation of an ether-extractable nitrosating agent (NSA) in the skin, which produced N-nitrosomorpholine (NMOR) from morpholine in vitro but not in vivo (under our conditions). We now report that NO2 bubbled into hexane solutions of methyl linoleate (MLIN) produced a similar NSA that reacted with morpholine in dichloromethane solution to produce NMOR. The NSA yield increased sharply as MLIN concentration was raised, with a maximum 0.1% yield of NSA from MLIN. The NSA yield from MLIN was four times that from methyl oleate and seven times that from methyl stearate. The NSA derived from MLIN travelled on thin-layer chromatography more slowly than the main weight fraction; whereas TLC of NSA in the skin lipids of NO2-exposed mice and in untreated mouse skin lipids exposed in vitro to NO2 produced NSA that travelled more rapidly than the main weight fraction.

Lipidic nitrosating agents produced from atmospheric nitrogen dioxide and a nitrosamine produced in vivo from amyl nitrite.

In studies on nitrosating agent(s) formed in skin of mice exposed to nitrogen dioxide, we showed that: (i) N-nitrosomethylaniline was produced in skin of mice exposed to nitrogen dioxide and then painted with N-methylaniline; (ii) a nitrosating precursor in methyl linoleate is associated with peroxidation products; (iii) cholesterol is a major nitrosating precursor in mouse skin, probably because it produces the nitrosating agent, cholesteryl nitrite; (iv) cholesteryl nitrite enhances autoxidation of lipids in vivo and on mouse skin and, like sodium nitrite, catalyses the autoxidation of iodide; (v) N-nitrosomethylaniline was produced in mice injected intraperitoneally with methylaniline and gavaged with amyl nitrite; and (vi) nitrosating agents may occur normally in human skin lipids.

Dietary and other factors affecting nitrosomethylurea (NMU) formation in the rat stomach.

Nitrosomethylurea (NMU) formation was measured radioactively in the stomach contents of rats fed 3H-methylurea and sodium nitrite, mostly in semi-synthetic diets. When methylurea and sodium nitrite were added to various diets, the NMU concentration, averaged over 1-4 hours after the food was presented, was 4.6 micrograms/kg stomach contents for low-protein, 2.7 for control semi-synthetic, 2.4 for high-fat, 1.26 for bran, 1.24 for high-protein and 0.54 for the commercial diet. With the low-protein diet, the amount of NMU after 1 hour was 36.6 micrograms, corresponding to 5.3% conversion of methylurea. The decrease in yield as protein content increased was attributed to buffering action and competition for nitrite by the protein, as well as to the effects of the latter on consistency. It may also be correlated with the observation that human gastric cancer is associated with high-starch low-protein diets. Nitrite in diet was more effective in producing NMU than the same nitrite concentration in drinking water, except with sodium nitrite concentrations less than or equal to 0.5 g/kg vehicle, where the position was reversed. Sodium ascorbate added to a semi-synthetic diet at a level of 2.9 g/kg inhibited NMU production by 50%.

Bioactivation of the anticancer agent CPT-11 to SN-38 by human hepatic microsomal carboxylesterases and the in vitro assessment of potential drug interactions.

Human hepatic microsomes were used to investigate the carboxylesterase-mediated bioactivation of CPT-11 to the active metabolite, SN-38. SN-38 formation velocity was determined by HPLC over a concentration range of 0.25-200 microM CPT-11. Biphasic Eadie Hofstee plots were observed in seven donors, suggesting that two isoforms catalyzed the reaction. Analysis by nonlinear least squares regression gave KM estimates of 129-164 microM with a Vmax of 5.3-17 pmol/mg/min for the low affinity isoform. The high affinity isoform had KM estimates of 1.4-3.9 microM with Vmax of 1.2-2.6 pmol/mg/min. The low KM carboxylesterase may be the main contributor to SN-38 formation at clinically relevant hepatic concentrations of CPT-11. Using standard incubation conditions, the effects of potential inhibitors of carboxylesterase-mediated CPT-11 hydrolysis were evaluated at concentrations >/= 21 microM. Positive controls bis-nitrophenylphosphate (BNPP) and physostigmine decreased CPT-11 hydrolysis to 1.3-3.3% and 23% of control values, respectively. Caffeine, acetylsalicylic acid, coumarin, cisplatin, ethanol, dexamethasone, 5-fluorouracil, loperamide, and prochlorperazine had no statistically significant effect on CPT-11 hydrolysis. Small decreases were observed with metoclopramide (91% of control), acetaminophen (93% of control), probenecid (87% of control), and fluoride (91% of control). Of the compounds tested above, based on these in vitro data, only the potent inhibitors of carboxylesterase (BNPP, physostigmine) have the potential to inhibit CPT-11 bioactivation if administered concurrently. The carboxylesterase-mediated hydrolysis of alpha-naphthyl acetate (alpha-NA) was used to determine whether CPT-11 was an inhibitor of hydrolysis of high turnover substrates of carboxylesterases. Inhibition of alpha-NA hydrolysis by CPT-11 was determined relative to positive controls BNPP and NaF. Incubation with microsomes pretreated with CPT-11 (80-440 microM) decreased alpha-naphthol formation to approximately 80% of control at alpha-NA concentrations of 50-800 microM. The inhibitors BNPP (360 microM) and NaF (500 microM) inhibited alpha-naphthol formation to 9-10% of control and to 14-20% of control, respectively. Therefore, CPT-11-sensitive carboxylesterase isoforms may account for only 20% of total alpha-NA hydrolases. Thus, CPT-11 is unlikely to significantly inhibit high turnover, nonselective substrates of carboxylesterases.

Studies related to nitrosamide formation: nitrosation in solvent: water and solvent systems, nitrosomethylurea formation in the rat stomach and analysis of a fish product for ureas.

Nitrous acid (HNO2) was partly extracted from water by organic solvents, especially polar ones. Carbaryl was nitrosated in solvent: water mixtures most rapidly when nonpolar solvents, e.g. methylene chloride, carbon tetrachloride and hexane, were used. Under given conditions, carbaryl was nitrosated in methylene chloride:water mixtures 20 times faster than in water alone, mainly because of its insolubility in water. For ethylurea, hexylurea, ethyl N-ethyl-carbamate and aminopyrine, nitrosation by sodium sulfate-dried methylene chloride extracts of nitrous acid ('dried HNO2') was at least 88% complete after reaction for 5 seconds at 6 degrees C. Nitrosation of N-butylacetamide by the same extract proceeded more slowly, with a second-order rate constant 31,000 times greater than for nitrosation in water at pH 2. Butylacetamide was nitrosated in methylene chloride by equivalent concentrations of 'dried HNO2', dinitrogen trioxide and dinitrogen tetroxide at similar rates. Nitrosomethylurea (NMU) formation was measured radioactively in the stomach contents of rats fed [3H]methylurea (MU) and sodium nitrite. When both compounds were given in the food (100 mg MU and 4 g NaNO2/kg) and the rats were killed 3 hours later, the NMU yield was 0.46% of the MU. When the food also contained 11.5 g sodium ascorbate/kg, NMU production was completely inhibited. With 2-4 g/1 sodium nitrite in the drinking water and MU in the food, no NMU was detected. Ureas were determined in dried, salted bonito fish from Japan, by a method involving ion-exchange and paper chromatography. The fish sample contained 80 mg urea/kg, but no MU. When the fish was nitrosated at pH 1 and then denitrosated at pH 0, 25 mg MU/kg was detected. Mu identity was confirmed by mass spectrometry.

Pharmacokinetics, toxicokinetics, distribution, metabolism and excretion of linezolid in mouse, rat and dog.

1. Linezolid (ZYVOX), the first of a new class of antibiotics, the oxazolidinones, is approved for treatment of Gram-positive bacterial infections. 2. The aim was to determine the absorption, distribution, metabolism and excretion (ADME) of linezolid in mouse, rat and dog in support of preclinical safety studies and clinical development. 3. Conventional replicate study designs were employed in animal experiments, and biofluids were assayed by HPLC or HPLC-MS. 4. Linezolid was rapidly absorbed after p.o. dosing with an p.o. bioavailability of > 95% in rat and dog, and > 70% in mouse. Twenty-eight-day i.v./p.o. toxicokinetic studies in rat (20-200mg kg(-1) day(-1)) and dog (10-80 mg kg(-1) day(-1)) revealed neither a meaningful increase in clearance nor accumulation upon multiple dosing. 5. Linezolid had limited protein binding (<35%) and was very well distributed to most extravascular sites, with a volume of distribution at steady-state (V(ss)) approximately equal to total body water. 6. Linezolid circulated mainly as parent drug and was excreted mainly as parent drug and two inactive carboxylic acids, PNU-142586 and PNU-142300. Minor secondary metabolites were also characterized. In all species, the clearance rate was determined by metabolism. 7. Radioactivity recovery was essentially complete within 24-48 h. Renal excretion of parent drug and metabolites was a major elimination route. Parent drug underwent renal tubular reabsorption, significantly slowing parent drug excretion and allowing a slow metabolic process to become rate-limiting in overall clearance. 8. It is concluded that ADME data were relatively consistent across species and supported the rat and dog as the principal non-clinical safety species.

Pharmacokinetics, metabolism, and excretion of irinotecan (CPT-11) following I.V. infusion of [(14)C]CPT-11 in cancer patients.

This study determined the disposition of irinotecan hydrochloride trihydrate (CPT-11) after i.v. infusion of 125 mg/m(2) (100 microCi) [(14)C]CPT-11 in eight patients with solid tumors. Mean +/- S.D. recovery of radioactivity in urine and feces was 95.8 +/- 2.7% (range 92.2-100.3%, n = 7) of dose. Radioactivity in blood, plasma, urine, and feces was determined for at least 168 h after dosing. Fecal excretion accounted for 63.7 +/- 6.8 (range 54.2-74.9%, n = 7) of dose, whereas urinary excretion accounted for 32.1 +/- 6.9% (range 21.7-43.8%; n = 7) of dose. One patient with a biliary T-tube excreted 30.1% of dose in bile, 14.2% in feces, and 48.2% in urine. Quantitative radiometric HPLC revealed that CPT-11 was the major excretion product in urine, bile, and feces. Aminopentane carboxylic acid (APC) and SN-38 glucuronide (SN-38G) were the most significant metabolites in urine and bile, whereas SN-38 and NPC, a primary amine metabolite, were relatively minor excretion products. SN-38 and APC were the most significant metabolites in feces. The relatively higher amount of SN-38 in feces compared with bile is presumably due to hydrolysis of SN-38G to SN-38 by enteric bacterial beta-glucuronidases. There was close correspondence between quantitative fluorescence HPLC and mass balance findings. CPT-11 was the major circulating component in plasma (55% of the mean radiochemical area under the curve), and CPT-11, SN-38, SN-38G, and APC accounted for 93% of the mean radiochemical AUC. These results show that the parent drug and its three major metabolites account for virtually all CPT-11 disposition, with fecal excretion representing the major elimination pathway.

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.