Continuous inhibition of 20-HETE synthesis by TS-011 improves neurological and functional outcomes after transient focal cerebral ischemia in rats.

TS-011, a potent and selective inhibitor of 20-HETE synthesis, has been described as providing significant benefits in animal stroke models. However, no studies have investigated changes in brain 20-HETE levels after cerebral ischemia. Also lacking are studies of TS-011 pharmacodynamics with respect to brain 20-HETE levels that may explain the benefits of TS-011 in animal models of ischemic stroke. The present study sought to explore changes in 20-HETE levels in brain tissue, as well as in plasma, after a 90-min episode of transient focal cerebral ischemia. Pharmacodynamics of TS-011 were also examined. Then, we evaluated the long-term effects of TS-011 when administered as in this pharmacodynamics study. The major findings of the present study are as follows: (1) brain 20-HETE levels increased significantly within 7.5h after MCAO; (2) TS-011 at doses of 0.1 and 0.3mg/kg administered at regular 6-h intervals appeared to reduce brain 20-HETE levels continuously; (3) TS-011 when administered as in this pharmacodynamics study improved long-term neurological and functional outcomes. These findings strongly suggest that 20-HETE plays an important role in the development of neurological and functional deficits after focal cerebral ischemia and that TS-011 may provide benefits in patients suffering ischemic stroke.

Clinical pharmacology of oral and i.v. N-methylformamide: a pharmacologic basis for lack of clinical antineoplastic activity.

N-Methylformamide (NMF) has been an agent of considerable interest to oncologists because of its broad spectrum of preclinical antitumor activity, tumor-differentiating abilities, and radiosensitizing and chemosensitizing properties. In this report, the pharmacokinetics of NMF are described, based on data from two phase I studies exploring both iv and oral routes of administration. Mean peak NMF plasma concentrations at recommended phase II doses were 0.46 mmol/L for NMF administered orally, 600 mg/m2 three times/week X 4 weeks every 6 weeks, and 2.78 mmol/L for NMF administered as a weekly iv bolus at 2,000 mg/m2 X 3 weeks every 4 weeks. These NMF concentrations were significantly lower than the concentrations that have been demonstrated to induce antineoplastic and relevant biologic effects in preclinical studies. Plasma disappearance curves were biphasic in the majority of patients; however, 25% of the curves were best fit by a monoexponential kinetic model. Mean alpha half-life and beta half-life values (+/- SE) were 10 +/- 2 and 732 +/- 93 min, respectively. Volumes of distribution for the theoretical central compartment (Vc) and at steady-state (Vss) were 13.8 +/- 1.1 L/m2 and 18.7 +/- 1.1 L/m2, respectively. The mean plasma clearance of NMF was 19.1 +/- 2.1 mL/min per square meter, and the relative contributions to parent compound disposition by respiratory and renal routes were insignificant. No metabolites were identified. Gastrointestinal absorption of oral NMF was rapid and nearly complete; oral bioavailability was calculated to be 0.87. Pharmacodynamic associations were observed between the magnitude of the area under the plasma disappearance curves and hepatotoxicity, the dose-limiting toxic effect of iv NMF, and the symptom complex of nausea, vomiting, and malaise, which precluded dose escalation of oral NMF.

N-methylformamide: cytotoxic, radiosensitizer, or chemosensitizer.

N-methylformamide (NMF), a polar solvent, is currently being evaluated by the National Cancer Institute (NCI) as an antineoplastic agent because of its activity against colon, mammary, and lung tumor xenografts. Results from preclinical studies suggest that it has radiosensitizing, chemosensitizing, and differentiating activity. Its mechanism of action remains unknown, but may involve cellular depletion of glutathione, cell membrane changes, or modulation of proto-oncogene expression. Preclinical toxicology studies conducted in mice, rats, and beagle dogs showed reversible hepatotoxicity to be dose-limiting. Clinically, NMF is administered both orally and by intravenous (IV) injection. The bioavailability with oral administration is 90% to 95%. The highest reported plasma concentration of NMF is approximately 4 mmol/L in a patient who received a dose of 2,000 mg/m2 of IV NMF. Biphasic elimination with IV NMF is seen on both the daily for five days and weekly for 3 weeks schedule. Approximately 5% to 7% of the total administered IV dose is excreted in the urine. In phase I studies, dose-limiting toxicities included reversible hepatotoxicity, a generalized malaise syndrome, and nausea and vomiting. One partial response has been reported in the 111 patients treated on phase II trials in colorectal, head and neck, and renal carcinomas. Suggestions for the future development of this drug are presented.

Total body burden arising from a week's repeated dermal exposure to N,N-dimethylformamide.

BACKGROUND: Hazardous chemicals and their metabolites may accumulate in the body following repeated airborne exposures and skin contact. AIMS: To estimate the contribution of skin absorption to total body burden of N,N-dimethylformamide (DMF) across a working week in two groups with similar levels of respiratory exposure but dissimilar skin contact. METHODS: Twenty five workers in a synthetic leather (SL) factory, 20 in a copper laminate circuit board (CLCB) factory, and 20 age and sex matched non-DMF exposed subjects, were recruited. Environmental monitoring of DMF exposure via respiratory and dermal routes, as well as biological monitoring of pre-shift urinary N-methylformamide (U-NMF), were performed for five consecutive working days. RESULTS: Environmental and biological monitoring showed no detectable exposure in controls. The average airborne DMF concentration (geometric mean (GM) 3.98 ppm, geometric standard deviation (GSD) 1.91 ppm), was insignificantly lower for SL workers than for CLCB workers (GM 4.49, GSD 1.84 ppm). Dermal DMF exposure and U-NMF values, however, were significantly higher for SL workers. A significant pattern of linear accumulation was found across a five day work cycle for SL workers but not for CLCB workers. CONCLUSIONS: Dermal exposure to DMF over five consecutive days of occupational exposure can result in the accumulation of a significant DMF body burden. The long term exposure response under both repeated and intermittent conditions of substantial skin exposure is worthy of note.

Phenoxyphenyl sulfone N-formylhydroxylamines (retrohydroxamates) as potent, selective, orally bioavailable matrix metalloproteinase inhibitors.

A novel series of sulfone N-formylhydroxylamines (retrohydroxamates) have been investigated as matrix metalloproteinases (MMP) inhibitors. The substitution of the ether linkage of ABT-770 (5) with a sulfone group 13a led to a substantial increase in activity against MMP-9 but was accompanied by a loss of selectivity for inhibition of MMP-2 and -9 over MMP-1 and diminished oral exposure. Replacement of the biphenyl P1' substituent with a phenoxyphenyl group provided compounds that are highly selective for inhibition of MMP-2 and -9 over MMP-1. Optimization of the substituent adjacent to the retrohydroxamate center in this series led to the clinical candidate ABT-518 (6), a highly potent, selective, orally bioavailable MMP inhibitor that has been shown to significantly inhibit tumor growth in animal cancer models.

Transdermal absorption of radioprotectors using permeation-enhancing vehicles.

Radioprotectors are not currently used clinically due to concerns regarding toxicity and uncertainties regarding tumor protection. Topical radioprotection of skin might find clinical applications with protectors such as WR-2721, but laboratory studies in which protectors have been applied in water have not been promising. We have studied the absorption of 14C-WR-2721 and [14C]cysteine dissolved in skin permeation-enhancing vehicles through the skin of hairless mice and compared the absorption to that in water. Skin concentration of WR-2721 was increased most by dimethylformamide (DMF), but only propylene glycol increased absorption as far as the dermis, as measured by plasma concentration. Skin concentration of cysteine was improved by DMF, 2-pyrrolidone (2-P), and methyl-2-pyrrolidone (M-2-P); only dimethylsulfoxide (DMSO) resulted in increased plasma levels of the protector. Pretreating skin with DMSO before application of WR-2721, irrespective of the vehicle, improved its concentration within the skin. Plasma levels were improved (10 and 12 times) only with 2-P and DMF. Therefore, by choosing the appropriate vehicle, it is possible to breach the barrier of the stratum corneum and enhance the presence of the protector in all layers of the skin.

Quantitative analysis of the novel anticancer drug ABT-518, a matrix metalloproteinase inhibitor, plus the screening of six metabolites in human plasma using high-performance liquid chromatography coupled with electrospray tandem mass spectrometry.

A liquid chromatographic/tandem mass spectrometric (LC/MS/MS) assay for the quantitative analysis of the novel anticancer drug ABT-518 and the screening of six potential metabolites in human plasma has been developed and validated to support a phase I study with the drug. ABT-518 is an inhibitor of matrix metalloproteinases, which are associated with tumor growth and development of metastasis. Plasma samples were prepared for analysis using a simple solid-phase extraction method on phenyl cartridges. LC separation was performed on a Zorbax extend C18 column (150 x 2.1 mm i.d., 5 microm particle size) using a mobile phase of methanol-aqueous 10 mM ammonium hydroxide (80:20, v/v) pumped at a flow-rate of 0.2 ml min(-1). An API2000 triple-quadrupole mass spectrometer was used for specific and sensitive detection. The best chromatographic speed (total run time 8 min) and peak shapes were obtained by employing an alkaline mobile phase (pH in aqueous phase approximately 10). Furthermore, an alkaline eluent was favored in order to obtain a better overall sensitivity for the protonated analytes. The dynamic range was from 10 to 1000 ng ml(-1) from 500 microl of plasma for ABT-518 and the metabolites were detected at levels of the same order of magnitude. Inter-assay accuracies for ABT-518 at five concentration levels were between -9.24 and 6.93% and inter-assay precisions were always <10.7%. Analyte stability was not critical during either storage or processing. This method was successfully applied in a phase I clinical study of ABT-518. The active drug, ABT-518, and all of the metabolites included in the assay could be identified in plasma from dosed patients. We believe that the method described in this paper using an alkaline mobile phase in combination with a basic stable analytical column may also be generally useful for the bioanalysis of other basic drugs.

Clinical pharmacokinetics, pharmacodynamics and metabolism of the novel matrix metalloproteinase inhibitor ABT-518.

PURPOSE: To investigate the pharmacokinetics, pharmacodynamics and metabolism of the novel matrix metalloproteinase (MMP) inhibitor ABT-518. METHODS: Plasma and urine samples were obtained from six patients included in a phase I trial in which ABT-518 was given once daily via the oral route. Samples were analyzed by LC-MS/MS, ELISA and immunocapture assay. The pharmacokinetics of the parent compound and of detectable metabolites were calculated. RESULTS: After a single dose of ABT-518 peak plasma levels were reached within 4-8 h. ABT-518 had an estimated clearance (Cl/F) of approximately 3 l/h, an estimated volume of distribution (V/F) of over 70 l and a terminal half-life (T(1/2)) of 20 h. At least six different metabolites were formed. Pharmacodynamic analysis for angiogenic growth factors (bFGF and VEGF) showed plasma and urine levels in the picogram range and for total MMP-9 and MMP-2 or MMP-9 activity showed plasma and urine levels in the nanogram range. CONCLUSIONS: The MMP inhibitor ABT-518 is extensively metabolized in humans. No significant correlations between pharmacokinetics and pharmacodynamics could be established.

Cloning and expression of rat CYP2E1 in Saccharomyces cerevisiae: detection of genotoxicity of N-alkylformamides.

A cDNA coding for rat cytochrome P450 2E1 was cloned into the multicopy vector pYeDP60 and expressed in haploid RSY6 and diploid RS112 yeast strains of Saccharomyces cerevisiae under control of the GAL10-CYC1 promoter. Spectral and catalytic properties of the expressed 2E1 were examined in whole cells or microsomes of both strains. The level of CYP2E1 obtained in RS112 (200 pmol/mg microsomal protein) was the highest among CYP2E1 produced in the various expression systems. The monooxygenase activity in the microsomes of both strains, measured as aniline hydroxylase, was found comparable to that of control rat hepatic microsomes. In a reconstituted system in the presence of exogenous rat P450 reductase, their activity increased about 10-fold. When exposed to the carcinogen NDMA, a known 2E1 substrate, the recombination frequency determined in the 2E1-expressing RS112 cells was enhanced, in a dose-dependent manner, up to 20-fold. The exposure of the same cells to the hepatotoxic solvents, N-methyl- and N-ethylformamide, resulted in an induction of recombination frequency, which was not observed in the void plasmid containing RS112 cells in the presence of S9 hepatic fractions from pyrazole-induced rats, as a specific exogenous metabolic activation system. These results demonstrate that the 2E1-expressing cells metabolize the two N-alkylformamides to genotoxic intermediates and, therefore, they provide an useful tool to study the bioactivation mechanism of potential P450 2E1 substrates.

Studies on the methyl isocyanate adducts with globin.

Isocyanates such as methylisocyanate (MIC), an intermediate in the synthesis of carbamate pesticides, or diisocyanates, used in the production of plastics, are highly reactive toxic compounds that spontaneously bind to biological macromolecules. In vivo formation of stable adducts with blood protein globin offers possibilities for biomonitoring of internal exposure to various reactive species. Thus, biomonitoring of the isocyanates through determination of their specific adducts with globin is a challenge. In this study, we characterized the adducts formed in human globin upon treatment with 100-fold molar excess of MIC. The globin was subject to enzymatic hydrolysis with pronase, and the hydrolysate was analysed by high performance liquid chromatography with positive atmospheric pressure chemical ionization mass spectrometric detection (HPLC/APCI-MS). The two major MIC adducts were those with N-terminal Val and side-chain of Lys, as confirmed by comparison with the synthetic standards. About 20 other adducts were observed, and several of them were tentatively identified using their MS and MS/MS spectra. Whereas detection of the adducts with Tyr and His was expected, the adducts with Trp and Phe, and a Lys adduct containing two MIC moieties, were probably analytical artifacts resulting from the transcarbamoylation during globin hydrolysis rather than products of direct carbamoylation. The other detected products were MIC-Val-His, derived from the N-terminal dipeptide of globin beta-chain, and dipeptides consisting of MIC-Lys attached to Gly, Val, Leu, Thr, and Glu. Failure to detect the corresponding non-modified dipeptides suggests that the pronase action may be hampered by the amino acid modification. MIC is known as a metabolic intermediate of the industrial solvents N,N-dimethylformamide (DMF) and N-methylformamide (MF) in humans and rats. The HPLC/APCI-MS analysis of globin from rats injected with DMF or MF, 1000 mg/kg, revealed the presence of the MIC adducts with both Val and Lys. The level of the Val adduct in globin from the DMF-dosed rats, determined using Edman degradation and GC/MS, was ca. 40 nmol/g, which is a level common in workers occupationally exposed to DMF. This suggests that also the Lys adduct in such human globin samples can be feasible to analysis and is therefore considered for further studies as a potential biomarker of exposure to DMF.

Skin penetrating abilities and reservoir effects of neat DMF and DMF/water mixtures.

This study was set out to determine the skin permeabilities of neat N, N-dimethylformamide (DMF, denoted as DMF(100%)) and DMF/water mixtures (including 50% DMF/50% water and 10% DMF/90% water mixtures (v/v), denoted as DMF(50%) and DMF(10%), respectively) and to assess their skin reservoir effects on the systemic absorption. The penetration fluxes for DMF(10%) and DMF(50%) (=0.015 and 0.126 mg/cm(2)/h, respectively) were only approximately 1.1%and 15% in magnitude as that of DMF(100%) (=0.872+/-0.231 mg/cm(2)/h), respectively. The above results could be because the perturbation effect of the DMF content was much more significant than the rehydration effect of the water content on skin permeability. We found that 85.9%, 96.6% and 98.7% of applied doses were still remaining on the skin surface, 4.98%, 0.838% and 0.181% were still remaining in the skin layer, and 9.09%, 2.61% and 1.17% penetrated through the skin layer after the 24-h exposure for DMF(100%), DMF(50%) and DMF(10%), respectively. We found that the half-life (T(1/2)) of DMF retaining in the skin layer were 12.3, 4.07 and 1.24h for DMF(100%), DMF(50%) and DMF(10%), respectively. The estimated reservoir effect for DMF(100%) (=34.1%) was higher than that of DMF(50%) and DMF(10%) (=27.1% and 14.1%, respectively). The above results suggest that the impact associated with the internal burden of DMF could be prolonged even the external exposure of DMF is terminated, particularly for those dermal contact with DMF/water mixtures with high DMF contents.

Integration of clinical chemistry, expression, and metabolite data leads to better toxicological class separation.

A large number of databases are currently being implemented within toxicology aiming to integrate diverse biological data, such as clinical chemistry, expression, and other types of data. However, for these endeavors to be successful, tools for integration, visualization, and interpretation are needed. This paper presents a method for data integration using a hierarchical model based on either principal component analysis or partial least squares discriminant analysis of clinical chemistry, expression, and nuclear magnetic resonance data using a toxicological study as case. The study includes the three toxicants alpha-naphthyl-isothiocyanate, dimethylnitrosamine, and N-methylformamide administered to rats. Improved predictive ability of the different classes is seen, suggesting that this approach is a suitable method for data integration and visualization of biological data. Furthermore, the method allows for correlation of biological parameters between the different data types, which could lead to an improvement in biological interpretation.

Purification and functional characterization of aquaporin-8.

BACKGROUND INFORMATION: Aquaporins (AQPs) are a family of channels permeable to water and some small solutes. In mammals, 13 members (AQP0-AQP12) have been found. AQP8 is widely distributed in many tissues and organs. Previous studies in frog oocytes suggested that AQP8 was permeable to water, urea and ammonium, but no direct characterization had yet been reported. RESULTS: We expressed recombinant rAQP8, hAQP8 and mAQP8 (rat, human and mouse AQP8 respectively) in yeast, purified the proteins to homogeneity and reconstituted them into proteoliposomes. Although showing high sequence similarity, AQP8 proteins from the three species had to be purified with different detergents prior to reconstitution. In stopped-flow studies, all three AQP8 proteoliposomes showed water permeability, which was inhibited by mercuric chloride and rescued by 2-mercaptoethanol. rAQP8 and hAQP8 proteoliposomes did not transport glycerol or urea but were permeable to formamide, which was also inhibited by mercuric chloride. In the oocyte transport assay, hAQP8-injected oocytes showed significantly higher [14C]methylammonium uptake than water-injected oocytes. CONCLUSIONS: In the present study, we successfully purified rAQP8, hAQP8 and mAQP8 proteins and characterized their biochemical and biophysical properties. All three AQP8 proteins transport water. rAQP8 and hAQP8 are not permeable to urea or glycerol. Moreover, hAQP8 is permeable to ammonium analogues (formamide and methylammonium). Our results suggest that AQP8 may transport ammonium in vivo and physiologically contribute to the acid-base equilibrium.

N,N-dimethylformamide: significance of dermal absorption and adjustment method for urinary N-methylformamide concentration as a biological exposure item.

OBJECTIVES: To clarify the potential for dermal absorption of N,N-dimethylformamide (DMF) (CAS No. 68-12-2) vapor, and the appropriate adjustment method and the half-lives of urinary concentrations of N-methylformamide (NMF) as the biological exposure item of DMF. METHODS: Thirteen healthy male volunteers (mean age: 22.7 years, range: 20-27) were exposed to DMF vapor twice, via both the skin and the lung, for 4 h at concentrations below 10 ppm, the recommended occupational exposure limit set by the Japan Society for Occupational Health, the American Conference of Governmental and Industrial Hygienists, and Deutsche Forschungsgemeinschaft, under conditions of 27 degrees C and 44% humidity. Each volunteer was exposed to DMF via the skin in a whole-body type exposure chamber and outside the chamber, via the lung by a respirator connected to the chamber. Exposure levels were 6.2 +/- 1.0 ppm in dermal exposure and 7.1 +/- 1.0 ppm in inhalation exposure. Urine samples were collected at every opportunity until 72 h after exposure; and NMF, as well as volume, creatinine, and specific gravity were measured. Dermal and inhalation intakes were compared after adjusting concentrations. RESULTS AND CONCLUSIONS: DMF vapor absorptions via the skin and the lung were estimated to be 40.4 and 59.6%, respectively. Workers need to be aware of the risk of dermal absorption of DMF vapor as well as of the liquid. Though NMF concentrations adjusted by creatinine, specific gravity, and urinary volume showed good correlation with total NMF excretion and the absolute amount of NMF at each sampling time, creatinine-adjusted NMF concentration correlated better than the others. The biological half-life of urinary NMF after dermal exposure, 4.75 +/- 1.63 h, was longer than that after respiratory exposure, 2.42 +/- 0.63 h.

Occupational dimethylformamide exposure. 2. Monomethylformamide excretion in urine after occupational dimethylformamide exposure.

The relationship between the 8-h time-weighted average (TWA) intensity of exposure to N,N-dimethylformamide (DMF) vapor (with little possibility of skin contact with liquid DMF) and the subsequent excretion of N-monomethylformamide (MMF) precursor in shift-end urine samples was examined in 116 workers exposed to DMF and 92 workers exposed to DMF in combination with toluene. Urinary MMF level was examined also in 42 non-exposed subjects. The TWA vapor concentration in breathing zone air of each worker was successfully measured by means of a recently developed diffusive sampler in which water was used as an absorbent. The examination of gas chromatographic (GC) conditions for MMF determination showed that the formation of MMF was not saturated when the injection port temperature was set at 200 degrees C, reached a plateau at 250 degrees C, and showed no additional increase at 300 degrees C. There was a linear relationship between DMF in air and MMF in urine with a regression equation of y = 1.65 x + 1.69 (r = 0.723, P less than 0.01), where y is MMF (unit; mg/l, uncorrected for urine density) in urine and x is DMF (ppm) in air, when only those exposed to DMF were selected, and the injection port temperature was set at 250 degrees C. From this equation, it was possible to estimate that about 10% of the DMF absorbed will be excreted into urine as the MMF precursor. The slope of the regression line was significantly smaller among those exposed to DMF and toluene in combination as compared with those with DMF exposure only.

Metabolism and hepatotoxicity of N,N-dimethylformamide, N-hydroxymethyl-N-methylformamide, and N-methylformamide in the rat.

The metabolism and hepatotoxicity of N,N-dimethylformamide (DMF) and two of its metabolites, N-hydroxymethyl-N-methylformamide (HMMF) and N-methylformamide (NMF) were evaluated over a 4-day period in rats. DMF toxicity was dose dependent and delayed toxicity after the administration of a high DMF dose (13.7 mmol/kg) in comparison to a lower dose (4.1 mmol/kg) was observed. Treatment of rats with 13.7 mmol/kg DMF, HMMF, or NMF showed i) that DMF is more toxic than HMMF or NMF, and ii) that hepatotoxicity occurs later for DMF than for HMMF or NMF. Analysis of serum and urine samples demonstrated that DMF is first metabolized to HMMF, which is then partially converted to NMF. After HMMF administration, NMF was found both in serum and in urine. The time course of DMF and HMMF toxicity in relation to NMF formation fitted the hypothesis that the hepatotoxicity of DMF and HMMF is mediated via NMF. The degree of hepatotoxicity after HMMF and NMF treatment is similar. However, the degree of DMF hepatotoxicity is much higher than in the case of NMF or HMMF. The role of NMF as an obligatory intermediate in DMF and HMMF hepatotoxicity is discussed.

Biotransformation of aliphatic formamides: metabolites of (+-)-N-methyl-N-(1-methyl-3,3-diphenylpropyl) formamide in rats.

The in vivo biliary and urinary metabolites of (+-)-N-methyl-N-(1-methyl-3,3-diphenylpropyl) formamide (1) from male Wistar rats have been characterized by gas chromatography/mass spectrometry. In urine, non-conjugated metabolites included 1,1-diphenyl-3-butanone (4) and 3-methylamino-1,1,diphenylbutane (7). beta-Glucuronidase liberated 4, 1,1-diphenyl-3-butanol (5), 1,1-diphenyl-3-butanone oxime (6), N-hydroxymethyl-N-(1-methyl-3, 3-diphenylpropyl) formamide (3), 1-(4-hydroxyphenyl)-1-phenyl-3-butanone (11), 1-(4-hydroxyphenyl)-1-phenyl-3-butanone oxime (12), N-methyl-N-(1-methyl-3-(4-hydroxyphenyl)-3-phenylpropyl) formamide (8), 1-(4-hydroxy-3-methoxyphenyl)-1-phenyl-3-butanone (16); 1-(4-hydroxy-3-methoxyphenyl)-1-phenyl-3-butanol (17), 1-(4-hydroxy-3-methoxyphenyl)-1-phenyl-3-butanone oxime (18), N-(1-methyl-3-(4-hydroxy-3-methoxyphenyl)-3-phenylpropyl) formamide (14) and N-methyl-N-(1-methyl-3-(4-hydroxy-3-methoxyphenyl)-3-phenylpropyl) formamide (13). Most of the carbinolamide (3) decomposed in the gas chromatograph inlet to N-(1-methyl-3,3-diphenylpropyl) formamide (2) unless stabilized as a trimethylsilyl (TMS) derivative. In bile, compounds 1, 2, 3, 5, 6, 11, 12 and 16 were present as non-conjugated metabolites. beta-Glucuronidase also liberated N-(1-methyl-3-(4-hydroxyphenyl-3-phenylpropyl) formamide (9), and all of the previously listed compounds except 7. Trimethylsilylation of the conjugated bile fraction revealed the presence of an additional two compounds: N-hydroxymethyl-N-(1-methyl-3-(4-hydroxyphenyl)-3-phenylpropyl) formamide (10) and N-hydroxymethyl-N-(1-methyl-3-(4-hydroxy-3-methoxyphenyl)-3-phenylpropyl ) formamide (15). A stable carbinolamide metabolite standard was synthesized and the mass spectral fragmentations of its TMS derivative studied by tandem mass spectroscopy. This is the first report on stable carbinolamide metabolites of high-molecular-weight formamides.

Biological monitoring of workers exposed to N,N-dimethylformamide in the synthetic fibre industry.

OBJECTIVES: Monitoring of workplace air and biological monitoring of 23 workers exposed to N,N-dimethylformamide (DMF) in the polyacrylic fibre industry was carried out on 4 consecutive days. The main focus of the investigation was to study the relationship between external and internal exposure, the suitability of the metabolites of DMF for biological monitoring and their toxicokinetic behaviour in humans. METHODS: Air samples were collected using personal air samplers. The limit of detection (LOD) for DMF using an analytical method recommended by the Deutsche Forschungsgemeinschaft (DFG) was 0.1 ppm. The urinary metabolites, N-hydroxymethyl-N-methylformamide (HMMF), N-methylformamide (NMF), and N-acetyl-S-(N-methylcarbamoyl)-cysteine (AMCC), were determined in one analytical run by gas chromatography with thermionic sensitive detection (GC/TSD). The total sum of HMMF and NMF was determined in the form of NMF. The LOD was 1.0 mg/l for NMF and 0.5 mg/l for AMCC. RESULTS AND CONCLUSIONS: The external exposure to DMF vapour varied greatly depending on the workplace (median 1.74 ppm, range < 0.1-159.77 ppm). Urinary NMF concentrations were highest in post-shift samples. They also covered a wide range (< 1.0-108.7 mg/l). This variation was probably the result of different concentrations of DMF in the air at different workplaces, dermal absorption and differences in the protective measures implemented by each individual (gloves, gas masks etc.). The urinary NMF concentrations had decreased almost to zero by the beginning of the next shift. The median half-time for NMF was determined to be 5.1 h. The concentrations of AMCC in urine were determined to be in the range from < 0.5 to 204.9 mg/l. Unlike the concentrations of NMF, the AMCC concentrations did not decrease during the intervals between the shifts. For the exposure situation investigated in our study, a steady state was found between the external exposure to DMF and the levels of AMCC excreted in urine about 2 days after the beginning of exposure. AMCC is therefore excreted more slowly than NMF. The half-time for AMCC is more than 16 h. Linear regression analysis for external exposure and urinary excretion of metabolites was carried out for a sub-group of 12 workers. External exposure to 10 ppm DMF in air (the current German MAK value) corresponds to an average NMF concentration of about 27.9 mg/l in post-shift urine from the same day and an average AMCC concentration of 69.2 mg/l in pre-shift urine from the following day. NMF in urine samples therefore represents an index of daily exposure to DMF, while AMCC represents an index of the average exposure over the preceding working days. AMCC is considered to be better suited for biomonitoring purposes because (1) it has a longer half-time than NMF and (2) its formation in humans is more closely related to DMF toxicity.

Effect of processing on Celecoxib and its solvates.

Pharmaceuticals mostly exist in crystalline form and exhibit the phenomenon of differential crystal packing and configurational arrangements of molecules, called polymorphism. Pharmaceutical processing by introducing significant amount of stress alters the molecular interactions in the system engendering polymorphic transformations. The energy supplied by these processing steps tends to overcome the energy barriers between different solid-state forms, thus yielding undesirable changes in the physicochemical and material characteristics of drugs or their dosage forms. Therefore, the role of these unit processes in solid-state transformations must be cautiously studied and if required appropriate controls should be used to monitor such events. The present study was aimed at studying the effect of major energy imparting pharmaceutical unit processes, like size reduction, wet granulation, consolidation, and compression on solid-state transformation of Celecoxib, a selective cyclooxygenase-II inhibitor and its N,N-dimethyl acetamide and N,N-dimethyl formamide solvated forms. A qualitative estimation of crystal transformation in processed samples was performed using DSC, microscopy, FTIR spectroscopy, and XRPD. FTIR was also applied for the development of a quantification method to find the percentage of transformation in N,N-dimethyl acetamide solvated form during compression.

Metabolic oxidation and toxification of N-methylformamide catalyzed by the cytochrome P450 isoenzyme CYP2E1.

Alkylformamides, for example N-methylformamide, are hepatotoxic in rodents and humans. The mechanism by which N-methylformamide exerts its hepatotoxicity involves metabolic oxidation at the formyl moiety to yield a short-lived intermediate, perhaps methyl isocyanate, which reacts with glutathione to afford S-(N-methylcarbamoyl)glutathione. The hypothesis that the cytochrome P450 isozyme CYP2E1 catalyzes the metabolic toxification of N-methylformamide was tested. Hepatocytes obtained from mice that had received acetone, an inducer of CYP2E1, were incubated for up to 4 hr with N-methylformamide (5 and 10 mM). Whereas N-methylformamide caused cytotoxicity in these cells, as measured by release from the cells of lactate dehydrogenase, it was barely toxic, under these conditions, to cells from untreated mice. Coincubation of N-methylformamide with dimethylsulfoxide (10 mM), a CYP2E1 inhibitor, for 4 or 6 hr abolished the hepatocytotoxicity of N-methylformamide. Metabolism of N-methylformamide to S-(N-methylcarbamoyl) glutathione was measured in incubates with liver microsomes from rats, mice, or humans in the presence of glutathione. Pretreatment of rodents with acetone or ethanol induced the rate of metabolism of N-methylformamide and of p-nitrophenol, a known CYP2E1 substrate, but it did not increase aminopyrine N-demethylation. Metabolism of N-methylformamide and p-nitrophenol was elevated in microsomes from animals that had received acetone (1%) in their drinking water for 1 week to 230% and 200%, respectively, of control values in mouse microsomes and to 310% and 240%, respectively, of control values in rat microsomes. Pretreatment of animals with 4-methylpyrazole (200 mg/kg intraperitoneally, once daily for 3 days) increased metabolism of N-methylformamide to 410% of control values in rat liver microsomes but was without effect on murine microsomal metabolism of N-methylformamide. The metabolism of this compound was strongly inhibited by the CYP2E1 substrates or inhibitors dimethylsulfoxide (1-100 mM), p-nitrophenol (100 microM), and diethyldithiocarbamate (100 microM), which did not affect aminopyrine N-demethylation. A polyclonal antibody against rat CYP2E1 (10 mg of IgG/nmol of cytochrome P450) inhibited N-methylformamide metabolism in liver microsomes from rats and from a human by 75% and 80%, respectively. The rate of metabolism of N-methylformamide to S-(N-methylcarbamoyl) glutathione was determined in liver microsomes from six humans and correlated with extent of metabolic hydroxylation of chlorzoxazone, a CYP2E1 probe, and with amount of immunodetectable enzyme using an anti-rat CYP2E1 antibody (r = 0.81 and 0.80, respectively). The results suggest that CYP2E1 is the predominant, if not sole, cytochrome P450 isozyme responsible for the metabolic toxification of hepatotoxic N-alkylformamides.