A Randomized, Open-Label, Crossover Phase 1 Study to Evaluate the Effects of Food on the Pharmacokinetics of a Single Oral Dose of a 15-mg Tylerdipine Tablet in Healthy Chinese Male Volunteers.

Tylerdipine hydrochloride is a novel L-type and T-type dual calcium channel antagonist that has the potential effects of expanding blood vessels and lowering blood pressure. It is expected to reduce the side effect of ankle edema observed with other drugs in the same class. A randomized, open-label, crossover phase 1 study was performed to evaluate the effect of food on the bioavailability of tylerdipine. Fourteen healthy male volunteers were enrolled. The administration of tylerdipine after a high-fat meal increased the bioavailability of tylerdipine. In the fed state there was a 130% increase in the mean total systemic exposure (AUCinf ) and a 73% increase in the mean peak plasma concentration (Cmax ) compared with that in the fasting state. The geometric mean ratios (90% confidence interval) of Cmax and AUCinf were 2.54 (1.94, 3.33) and 1.75 (1.50, 2.04) for tylerdipine. The exposures of the 2 main metabolites M2 and M4 were increased by approximately 10% after a high-fat meal. The median time to peak plasma concentration of tylerdipine showed no difference between fasting and fed states.

Tissue distribution and elimination of N-methyl-N-2,4,6-tetranitroaniline (tetryl) in rats.

The elimination of tetryl was studied using ring-labeled 14C-tetryl. Tetryl was given subcutaneously to male Sprague-Dawley rats at doses of 25, 100, and 300 mg kg(-1), and urine and feces were collected 24 h post-injection. Percent urinary elimination was observed to be 10.02 +/- 2.48, 11.2 +/- 1.66, and 13.24 +/- 5.79 (mean +/- SEM) respectively. Percent fecal elimination was 15.68 +/- 6.13, 9.41 +/- 1.52, and 8.45 +/- 1.81 respectively. At 24 h post-injection, tissues from male Sprague-Dawley rats were collected from animals that received 100 mg kg(-1) 14C-tetryl. Tetryl was found to be poorly absorbed with approximately 65% of the administered dose remaining at the site of subcutaneous injection. Blood was found to be the principal depot of radioactivity, followed by muscle, liver, and kidney. Analysis of the tissue to blood radioactivity ratio revealed that the liver had the highest ratio (1.2), followed by brain (0.45), kidney (0.38), and testes (0.35). All other tissues analyzed had ratios less than 0.30. Urine of animals receiving 14C-tetryl (100 mg kg(-1)) was analyzed using HPLC coupled with UV detection (200-600 nm; 1.2 nm resolution). During HPLC analysis, 1 min fractions were collected and radioactivity measured. Two major peaks of radioactivity were identified at approximately 5 and 14 min retention times, respectively. The 14 min peak had the same retention time and UV spectrum as picric acid and 5 min peak had the same retention time and UV profile as picramic acid. The data presented demonstrates that that there is little retention of tetryl in specific tissue depots and that tetryl is eliminated in roughly equal amounts in both urine and feces. The major urinary metabolites identified picric acid and picramic acid (a known urinary metabolite observed in rabbits). From microsomal fraction studies, a major metabolite, NMPA, was identified. The formation of this metabolite was found to be dependent on at least two enzymes. One enzyme is dependent on NAD+ for NMPA formation and is likely to be NADP(H):quinone oxidoreductase. The second metabolite is NADP+ dependent and is probably related to NADPH:cytochrome-P450 reductase.

Characterization of phenotypes in Gstm1-null mice by cytosolic and in vivo metabolic studies using 1,2-dichloro-4-nitrobenzene.

Glutathione S-transferase Mu 1 (GSTM1) has been regarded as one of the key enzymes involved in phase II reactions in the liver, because of its high expression level. In this study, we generated mice with disrupted glutathione S-transferase Mu 1 gene (Gstm1-null mice) by gene targeting, and characterized the phenotypes by cytosolic and in vivo studies. The resulting Gstm1-null mice appeared to be normal and were fertile. Expression analyses for the Gstm1-null mice revealed a deletion of Gstm1 mRNA and a small decrease in glutathione S-transferase alpha 3 mRNA. In the enzymatic study, GST activities toward 1,2-dichloro-4-nitrobenzene (DCNB) and 1-chloro-2,4-dinitrobenzene (CDNB) in the liver and kidney cytosols were markedly lower in Gstm1-null mice than in the wild-type control. Gstm1-null mice had GST activities of only 6.1 to 21.0% of the wild-type control to DCNB and 26.0 to 78.6% of the wild-type control to CDNB. After a single oral administration of DCNB to Gstm1-null mice, the plasma concentration of DCNB showed larger AUC0-24 (5.1-5.3 times, versus the wild-type control) and higher Cmax (2.1-2.2 times, versus the wild-type control), with a correspondingly lower level of glutathione-related metabolite (AUC0-24, 9.4-17.9%; and Cmax, 9.7-15.6% of the wild-type control). In conclusion, Gstm1-null mice showed markedly low ability for glutathione conjugation to DCNB in the cytosol and in vivo and would be useful as a deficient model of GSTM1 for absorption, distribution, metabolism, and excretion/toxicology studies.

Low glutathione S-transferase dogs.

Liver and kidney glutathione S-transferase (GST) activities to 1,2-dichloro-4-nitrobenzene (DCNB) as a substrate (GST-D activities) were measured in 280 dogs from five different breeders, and significant individual differences in this activity were observed in both organs. Interestingly, 34 out of the 280 dogs (i.e. 12.1%) were those in which liver GST-D activities were less than 10 nmol/min per mg cytosolic protein, "low GST dogs", and the other dogs were classified as "middle" and "high" GST dogs for which the liver GST-D activities were 10-80 and >80 nmol/min per mg protein, respectively, and occurred at similar percentages (41.4% for the middle GST dog and 46.4% for the high GST dog). Furthermore, the existence of the low GST dogs was not limited to one particular breeder. There was a good correlation (r=0.910) between the liver and kidney GST-D activities, showing low activity in not only the liver but also the kidney in the low GST dogs. Although liver GST activity to 1-chloro-2,4-dinitrobenzene as a substrate (GST-C activity), catalyzed by various GST isozymes in dogs, was significantly correlated with liver GST-D activity, GST-C activity showed more than 450 nmol/min per mg protein even in the low GST dogs. There was no significant difference in cytochrome P450 content, 7-ethoxycoumarin O-deethylase activity or UDP-glucuronosyltransferase activity to p-nitrophenol as a substrate between low GST dogs and the other dogs. Finally, remarkably high plasma concentrations of DCNB were observed in the low GST dogs after single doses of DCNB at 5 or 100 mg/kg. The individual differences in GST-D activity are probably attributable to the content and/or activity of the theta class GST isozyme Yd(f)Yd(f) since it has been reported that glutathione conjugation of DCNB is specifically catalyzed by GSTYd(f)Yd(f) in dogs. In conclusion, we identified a number of low GST dogs in which the GST-D activities were not observed either in vivo or in vitro. The feasibility of using a single low dose of DCNB to phenotype dogs based on GST-D activity was confirmed. It was also suggested that low GST dogs have high susceptibility, including unexpected toxicity or abnormal exposure, to chemicals metabolized by GSTYd(f)Yd(f).

Acute nitrobenzene poisoning with severe associated methemoglobinemia: identification in whole blood by GC-FID and GC-MS.

A rare fatal case of self-poisoning with nitrobenzene following oral ingestion is reported. On presentation to the hospital, severe methemoglobinemia (70%) was observed in an 82-year-old male who had ingested 250 mL of an unknown substance in the previous 24 h. Methylene blue and exchange transfusion were the therapeutic methods applied in the treatment of the methemoglobinemia. Forty-eight hours after ingestion, a blood sample was collected in ICU and sent to our laboratory. We detected that the blood contained 3.2 microg/mL of nitrobenzene. The determination of nitrobenzene was performed using the combination of GC-FID for screening analysis and quantitation and GC-MS for confirmation of the obtained results.

Pharmacokinetic study of p-chloronitrobenzene in rat.

The pharmacokinetics of p-chloronitrobenzene (p-CNB) was evaluated in rats to propose an index for monitoring p-CNB exposure of humans exposed to it. After a single dose of 30, 100, or 333 mg/kg body weight, p-CNB was administered intraperitoneally to male Sprague-Dawley rats; blood and urine were collected periodically. p-CNB in plasma and its five major metabolites--2-chloro-5-nitrophenol, N-acetyl-S-(4-nitrophenyl)-L-cysteine,2,4-dichloroaniline,p-chloroanilin e, and 2-amino-5-chlorophenol--in urine were measured by reversed-phase HPLC methods. Pharmacokinetics was evaluated by moment analysis and compartment model analysis of the p-CNB concentration in plasma vs. time curves and of the urinary excretion rate of its metabolites vs. time curves. Urinary excretion was considered to be the most important pathway for disappearance of p-CNB, because the fraction of p-CNB metabolites excreted in urine was ca.2/3 of the dose level. N-Acetyl-S-(4-nitrophenyl)-L-cysteine was the most abundant urinary metabolite of p-CNB and comprised ca.1/2 of the total amount of the five metabolites excreted into the urine. The urinary excretion of N-acetyl-S-(4-nitrophenyl)-L-cysteine was considered to be proportional to the dose of p-CNB over a wide range of doses, because the process of metabolism of p-CNB to N-acetyl-S-(4-nitrophenyl)-L-cysteine was linear in the dose range studied. Consequently, urinary N-acetyl-S-(4-nitrophenyl)-L-cysteine was considered to be suitable as an index for monitoring p-CNB exposure.

Pharmacokinetics of a micronized, poorly water-soluble drug, HO-221, in experimental animals.

N-[[[4-(5-Bromo-2-pyrimidinyloxy)-3-chlorophenyl]amino]carbonyl] - 2-nitrobenzamide (HO-221) is presently under development as an oral anticancer agent with a novel mode of action. However, HO-221 exhibits extremely poor bioavailability after oral administration because it is only slightly soluble in water (0.055 micrograms/ml at 37 degrees C). Our previous study revealed that the micronization of HO-221 to the submicron region improved this oral bioavailability. In this study, the oral pharmacokinetics of this micronized HO-221 was investigated in rats, dogs and monkeys. After oral administration, the agent was moderately absorbed with the Tmax of 6.5-8.0, 17.3-20.0 and 12.0 h, and eliminated with the terminal half-lives of 11.9-15.0, 66.8-78.3 and 42.3 h in rats, dogs and monkeys, respectively. The bioavailability was incomplete (3.7-21.4%). In rats, the plasma concentration did not increase proportionally with increasing oral doses. In dogs, food enhanced the bioavailability 2.2-fold with a standard meal and 3.6-fold with a high fatty meal as compared with fasting conditions.

Determination of p-chloronitrobenzene in plasma by reversed-phase high-performance liquid chromatography.

A simple, accurate and precise isocratic reversed-phase high-performance liquid chromatographic method was developed and validated for the determination of p-chloronitrobenzene (p-CNB) in rat plasma. A plasma sample was deproteinized with methanol containing the internal standard (p-bromonitrobenzene). The resulting methanol eluate obtained after centrifugation was filtered and injected into a high-performance liquid chromatograph (50 microliters each). A column packed with 5 microns octadecylsilane (ODS) spherical particles was used with isocratic elution of methanol-water (45:55, v/v) at a flow-rate of 1.0 ml/min. The compounds were detected by ultraviolet absorbance at 280 nm. The retention times of p-CNB and the internal standard were 12.5 and 15.5 min, respectively, at a column oven temperature of 30 degrees C. The results were linear from 0.05 to 100 micrograms/ml (r = 0.999), and the detection limit was 0.01 microgram/ml. The relative error and the coefficient of variation on replicate assays were less than 7 and 10%, respectively, for all concentrations studied. The overall recoveries of p-CNB were between 97 and 105%. Plasma samples could be stored for up to one month at -20 degrees C.

Aniline-, phenylhydroxylamine-, nitrosobenzene-, and nitrobenzene-induced hemoglobin thiyl free radical formation in vivo and in vitro.

We have employed the ESR spin trapping technique in vivo to detect the formation of the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)/hemoglobin thiyl free radical adduct in the blood of rats following administration of either aniline, phenylhydroxylamine, nitrosobenzene, or nitrobenzene. This DMPO adduct was a six-line, strongly immobilized, radical adduct. Using rat red blood cells, both phenylhydroxylamine and nitrosobenzene were able to induce the formation of the DMPO/glutathiyl free radical adduct and the same DMPO/hemoglobin thiyl free radical adduct was detected in in vivo samples. In experiments using purified rat oxyhemoglobin, a four-line, weakly immobilized, DMPO/hemoglobin thiyl free radical adduct was detected, in addition to the six-line strongly immobilized adduct. When this study was repeated using human red blood cells, we detected only the DMPO/glutathiyl free radical adduct and, when purified human oxyhemoglobin was employed, only the four-line, weakly immobilized, DMPO/hemoglobin thiyl radical adduct could be detected. In a study using reduced glutathione, we found that phenylhydronitroxide free radicals were reduced by glutathione and that glutathione was concomitantly oxidized to its thiyl free radical. We propose that the species responsible for the oxidation of the thiols to yield the thiyl free radicals in vivo and in vitro was the phenylhydronitroxide radical produced from the reaction of phenylhydroxylamine with oxyhemoglobin.

Plasma binding of 1-butanol, phenol, nitrobenzene and pentachlorophenol in the rainbow trout and rat: a comparative study.

1. The in vitro binding of 1-butanol, phenol, nitrobenzene, and pentachlorophenol in trout plasma and rat plasma was determined. 2. Binding to rainbow trout plasma proteins agreed within 9% of that observed in rat plasma. 3. Percentage bound to rainbow trout (2-99%) or rat (10-99%) plasma proteins increased as the log octanol/water partition coefficient of the chemicals increased within the Log P 1-3 range, and was suggestive of hydrophobic interactions in binding.

Induction of hapten-specific immunological tolerance and immunity in B lymphocytes. VII. Correlation between trinitrobenzenesulfonic acid administration, serum trinitrophenyl content, and level of tolerance.

The induction of immunological tolerance with trinitrobenzenesulfonic acid (TNBS) was studied by a comparison of the concentration of trinitrophenyl (TNP) in the serum of tolerant mice (TolS) and the degree of unresponsiveness induced as the dose and time of tolerogen injection were varied. The concentration of TNP in TolS was greater with a larger dose of TNBS, as expected, and decreased with time after tolerogen injection in a biphasic manner. The rapid initial decline followed on Day 10 by a more gradual decrease in TNP concentration suggests that there were two classes of TNP conjugates produced by TNBS injection. The serum TNP concentration appeared to correlate to the in vivo response of TNBS-treated mice to thymic-dependent and thymic-independent antigenic challenge while little correlation was evident with the in vitro response.

Relationship between red blood cell uptake and methemoglobin production by nitrobenzene and dinitrobenzene in vitro.

Nitrobenzene increases methemoglobin formation when incubated with native hemoglobin but not when incubated with red blood cell suspensions. These experiments were designed to determine if transport of nitrobenzene across the red blood cell membrane is a limiting factor for methemoglobin production by red blood cell suspensions. Incubation of [14C]-m-, o- or p-dinitrobenzene, but not mononitrobenzene, with red blood cell suspensions caused a time-dependent increase in methemoglobin. All three dinitrobenzenes and mononitrobenzene crossed the red blood cell membrane and accumulated in the erythrocytes after only 1 min of incubation. Incubation of mononitrobenzene with hemolysates did not result in methemoglobin production. Incubation of red blood cells with the dinitrobenzenes or mononitrobenzene for 1 and 10 min at 4 degrees C did not influence red blood cell uptake of the nitrobenzenes, suggesting that these compounds do not enter the red blood cell by an active process. Dinitrobenzene-induced methemoglobin production was markedly inhibited at 4 degrees C, and may be a result of decreased interaction with hemoglobin and/or decreased metabolism to reactive intermediates which mediate methemoglobin production. These data indicate that red blood cell transport of nitrobenzene is not the limiting factor in methemoglobin production in vitro.

Detoxification of xenobiotics by glutathione S-transferases in erythrocytes: the transport of the conjugate of glutathione and 1-chloro-2,4-dinitrobenzene.

The incubation of human erythrocytes with 1-chloro-2,4-dinitrobenzene (CDNB) results in almost quantitative conjugation of glutathione (GSH) to form S-(2,4-dinitrophenyl) glutathione. The reaction is catalysed by erythrocyte glutathione S-transferase. During the present studies we have identified the conjugate in the incubation medium of CDNB-treated erythrocytes, indicating that the conjugate of GSH and CDNB is transported out by the erythrocytes. Quantitation of the conjugate in the incubation medium by amino acid analysis and thin layer chromatography indicates that the erythrocytes transport the conjugate at an approximate rate of 140 nmol/h/ml erythrocytes. The transport of the conjugate is inhibited by sodium fluoride. Exhaustion of ATP from the erythrocytes results in a significant decrease in the rate of transport which is restored with the regeneration of ATP by incubating the erythrocytes with adenine and inosine. This indicates that the transport of conjugate is an energy dependent process.

Penetration of 2,4,5-trinitrobenzenesulfonate into human erythrocytes. Consequences for studies on phospholipid asymmetry.

The glutathione content of human erythrocytes rapidly diminishes when cells are exposed to 2,4,6-trinitrobenzenesulfonate (20 mumol/l cells) at 37 degrees C. Even at 0 degrees C a slow decrease in glutathione content is observed. The uptake of trinitrobenzenesulfonate by the cells is retarded by inhibitors of the inorganic anion exchange system, indicating that trinitrobenzenesulfonate enters the cells by this pathway. The disappearance of glutathione most probably results from the reaction: 2 GSH + trinitrobenzenesulfonate leads to GSSG + aminodinitrobenzenesulfonate. The reaction of trinitrobenzenesulfonate with glutathione occurs prior to its covalent binding to amino groups of hemoglobin which makes this reaction a more sensitive method of detection of penetration of trinitrobenzenesulfonate into erythrocytes. Results of studies on the asymmetric distribution of phospholipids using trinitrobenzenesulfonate as the only probe should be reconsidered in the light of these new data.