体外小鼠、豚鼠、犬及人血液对梭曼的解毒作用

目的　研究不同种属动物及人血液对梭曼的解毒能力与血液中几种梭曼解毒酶活性的关系。方法　测定血液中梭曼残留浓度及解毒酶活性。结果　小鼠、豚鼠、犬、人血浆对梭曼的解毒能力要明显高于其自身红细胞的解毒能力 ,血浆对梭曼的解毒能力依小鼠、豚鼠、犬、人的顺序由高到低依次排列。啮齿类动物血液中羧酸酯酶 (CaE)活性位点数目多且与梭曼结合快 ,因此在梭曼解毒中可发挥重要作用。犬、人血液中乙酰胆碱酯酶 (AChE)在梭曼解毒中占据了重要的解毒地位 ,犬、人血液中CaE基本没有参与梭曼解毒。结论　血液解毒能力的种属差异与种属间解毒酶结合位点数量的多少及梭曼与酶的结合速率密切相关     

In vitro degradation of the four isomers of soman in human serum

Starting from racemic soman (1,2,2-trimethylpropyl methylphosphonofluoridate), the degradation of its four stereoisomers in human serum (25 degrees, pH 8.8), has been studied at the nM level. Phosphylation of serum proteins is eliminated by preincubation of the serum with soman. A capillary gas chromatographic method with nitrogen-phosphorous detection allows the separation of the diastereoisomers. The total hydrolysis (enzymatic and non-enzymatic) rate constants of the isomers can then be resolved indirectly on the basis of the important rate difference between P(+) and P(-) isomers. The enzymatic hydrolysis rate constants are obtained by subtracting, for each isomer, the spontaneous (non-enzymatic) rate constant from the total hydrolysis rate constant. The non-enzymatic part of the hydrolysis is obtained from experiments in serum ultrafiltrate (30,000 NMWL). Enzymatic hydrolysis of C(+) P(+) soman occurs so rapidly that only a lower limit of the rate constant can be given. The other enzymatic rate constants are 0.016 min-1 for C(+)P(-), 0.74 min-1 for C(-)P(+) and 0.028 min-1 for C(-)P(-).     

In vitro percutaneous absorption in mouse skin: influence of skin appendages

Skin appendages are often envisaged as channels that bypass the stratum corneum barrier and are generally thought to facilitate the dermal absorption of topical agents. However, the significance of this transappendageal pathway in percutaneous absorption remains to be assessed experimentally. With the use of a skin organ culture penetration chamber system, the influence of skin appendages on the in vitro permeation of topically applied benzo[a]pyrene and testosterone (5 micrograms/2 cm2) was examined in skin preparations from both haired and hairless mice. Haired mice examined included the C57BL6, C3H, DBA2, Balbc, and Sencar strains and the hairless mice were the HRS and SKH. In all mouse strains examined, the overall permeation of testosterone (greater than 65% of applied dose) 16 hr following in vitro topical application was greater than that of benzo[a]pyrene (less than 10%). No strain differences were observed with respect to the percutaneous permeation of testosterone; however, percutaneous permeation of benzo[a]pyrene in the haired mice (7-10% of applied dose) was higher than that in the hairless mice (2%). In an in-house derived mouse strain which showed three phenotypic variants due to hair densities, the permeability to both compounds was highest in the skin of the haired phenotype (testosterone 67%, benzo[a]pyrene 7%), lowest in the hairless phenotype (35 and 1%, respectively) and intermediate in the fuzzy-haired animal (57 and 3%, respectively). Examination by fluorescence microscopy of cryosections of skin, prepared 1 hr after topical benzo[a]pyrene, showed areas of intense fluorescence deep within the nonfluorescing dermis of skin from the haired phenotype. These fluorescent areas were correlated with follicular ducts and sebaceous glands. In contrast, skin from the hairless phenotype showed no evidence of fluorescence in the dermis and intermediate was the fluorescence observed in the skin from the fuzzy-haired animal. These observations showed that transappendageal penetration could contribute significantly to the overall skin absorption of topical agents. They also suggest that regional distribution of skin appendages could influence the percutaneous fate of topically applied chemicals.     

In vitro formation of selegiline-N-oxide as a metabolite of selegiline in human, hamster, mouse, rat, guinea-pig, rabbit and dog.

It is well established in the litrature, that selegiline is metabolised to its N-dealkylated metabolites, N-desmethylselegiline, methamphetamine and amphetamine. However, most studies on selegiline metabolism did not characterize the species differences in the formation of the metabolites. Therefore, in this study, we investigated the in vitro metabolism of selegiline in liver microsomes of different species. In addition, to the previously well-characterized metabolites, selegiline-N-oxide (selegiline-NO) was found to be formed as a metabolite of selegiline in rat liver microsomal preparation. The results of experiments with liver microsomes from other species indicated species differences in the rate and extent of formation of selegiline-NO. The dog and hamster liver microsomal preparations were the most active in terms of selegiline-NO production, whereas little selegiline was metabolized to its N-oxide in human liver microsomes. When selegiline-NO was incubated with rat liver microsomes, no metabolism occurred. When a short incubation time was applied in selegiline expriments no increase in the amount of selegiline-NO was detected. Accordingly, it was clear that selegiline was not metabolized to the N-dealkylated or N,N-bis-dealkylated compounds via selegiline-NO. Studies with different isoenzyme inhibitors indicated that the formation of selegiline-NO might be catalyzed at least partly by cytochrome P450 (CYP) 2D6 and CYP3A4. With the exception of hamster microsomes in the microsomal preparations in vitro, the formation of the R,S-stereoisomer of selegiline-NO was preferred.     

In vitro degradation of ziprasidone in human whole blood

A systematic study was performed into the degradation of ziprasidone in simulated postmortem blood. Fifteen potential degradation products not previously reported in the literature were observed. Four resulted from degradation in human blood, whereas the remaining products resulted from reaction with solvents: four from alkaline degradation, four from reaction with acetaldehyde, and three from reaction with acetone. To identify possible degradation products, a liquid chromatograph-diode array detector (LC-DAD) and liquid chromatograph quadrupole-time-of-flight mass spectrometer (LC-QTOF-MS) operating in auto-MS/MS mode were used. It was indicated from red-shifted UV–Vis spectra, accurate mass data, mass fragmentation data, and a deuteration experiment that the site of ziprasidone degradation, in the in vitro blood experiments, was the methylene carbon of the oxindole moiety. The major in vitro blood degradation products were proposed to be E/Z isomers of 3-ethylidene-ziprasidone. Further, another in vitro degradation product in microbially inoculated blood specimens was proposed to be 3-ethyl-ziprasidone. 3-Ethylidene-ziprasidone was hypothesized to form from the reaction of ziprasidone with acetaldehyde derived from the ethanol used to spike ziprasidone into the in vitro blood experiments. Data from two postmortem investigations were available for retrospective reanalysis. Attempts were made to detect degradation products of ziprasidone, but none were found.     

In vitro models for human skin disease

Modern tissue culture technology has made it possible to generate human skin equivalents that represent either epidermis or epidermis plus dermis (full-thickness skin) in vitro. Commercially available skin equivalents and in-house models are used for safety analysis of cosmetics and toxicity screening of various pharmaceutical compounds. Recently, tissue culture technology has also been used to develop in vitro models of skin disease, in particular to promote cutaneous drug research while sparing experimental animals. The spectrum of model diseases available covers a range from inflammatory disease to cancer. It has, thus, been possible to gain more insight into the role of active pharmaceutical ingredients of various dermatologically relevant drug classes as well as conventional and innovative formulations.     

In vitro human skin penetration of diethanolamine.

Concerns about the safety of diethanolamine (DEA) have been raised by the National Toxicology Program (NTP). Therefore, we measured the extent of DEA absorption in human skin relevant to exposures from shampoos, hair dyes and body lotions. Radiolabeled [14C]-DEA was added to two commercial products from each class and applied to excised viable and non-viable human skin in flow-through diffusion cells. The products remained on the skin for 5, 30 and 24 h for shampoos, hair dyes and body lotions, respectively. After 24 h, most of the absorbed dose was found in skin: 2.8% for shampoos, 2.9% for hair dyes and 10.0% for body lotions. Only small amounts were absorbed into the receptor fluid: 0.08%, 0.09% and 0.9% for shampoos, hair dyes and body lotions respectively. There was no significant difference in the absorption of DEA through viable and non-viable skin or from product application doses of 1, 2 or 3 mg lotion/cm2. In 72 h daily repeat dose studies with a lotion, DEA appeared to accumulate in the skin (29.2%) with little diffusing out into the receptor fluid. Therefore, skin levels of DEA should not be included in estimates of systemic absorption used in exposure assessments.     

In vitro skin irritation testing with human skin cell cultures

Cultured human skin cells are a potentially useful model for skin irritancy testing. We have evaluated the effects of chemical irritants on human epidermal keratinocytes (NHEK) and on keratinocyte-dermal fibroblast (NHEK/DF) co-cultures. Cell viability in NHEK cultures, measured as incorporation of the vital dye neutral red (NR), was reduced in a dose-dependent manner in response to the chemical irritants tested. The half-maximal effective concentration (NR₅₀) values correlated with irritation scores in human patch tests with these materials. Certain materials were found to be incompatible with this test system. NHEK/DF cultures were treated with ten prototype surfactants, and were evaluated for cell viability (MTT incorporation), cytotoxicity (release of the enzymes lactate dehydrogenase and N-acetyl glucosaminidase), metabolism (glucose utilization), and inflammatory mediator (prostaglandin E₂) release. There was a close correlation of the dose-response characteristics for all the endpoints tested, and between the in vitro responses and human patch test scores for the surfactants tested. These results demonstrate the usefulness of human skin cell cultures and of cell viability, cytotoxicity, and inflammatory mediator release as endpoints, for the in vitro assessment of skin irritancy.     

In vitro effects of soman on bronchial smooth muscle

The in vitro exposure of rat bronchial smooth muscle to the cholinesterase inhibitor soman (O-[1,2,2-trimethylpropyl]-methyl-phosphonofluoridate) potentiated the rapid and concentration dependent increase in the contraction induced by acetylcholine (ACh). There was a substantial increase in the response to ACh when soman was present in concentrations from 10 nM to 1 microM which correspond to a 65-100% inhibition of acetylcholinesterase (AChE). The apparent affinity (pD2) to ACh increased from 3.7 to 6.7 without any change in intrinsic activity (alpha) in this concentration interval. In contrast, soman did not alter the apparent affinity or intrinsic activity of carbachol, which supports the suggestion that the effect of soman is entirely due to its anticholinesterase activity. Soman by itself induced contraction which begun at 1-10 nM. This may be explained from its anticholinesterase activity and the subsequent increase in the synaptic concentration of spontaneously released ACh. The effect of soman on inhibition of cholinesterase and carboxylesterases have also been examined. The results demonstrate that low concentrations of soman induces contraction of the airway smooth muscle.     

In vitro metabolism of the analgesic bicifadine in the mouse, rat, monkey, and human.

The in vitro metabolism of [(14)C]bicifadine by hepatic microsomes and hepatocytes from mouse, rat, monkey, and human was compared using radiometric high-performance liquid chromatography and liquid chromatography/tandem mass spectrometry. Two main metabolic pathways were identified in all four species. One pathway was an NADPH-dependent pathway in which the methyl group was oxidized to form a hydroxymethyl metabolite (M2). Its formation was inhibited in human microsomes only by quinidine, a CYP2D6 inhibitor. In incubations with individual cDNA-expressed human cytochromes P450, M2 was formed only by CYP2D6 and CYP1A2, with CYP2D6 activity 6-fold greater than that of CYP1A2. M2 was oxidized further to the carboxylic acid metabolite (M3) by hepatocytes from all four species. The second major metabolic pathway was an NADPH-independent oxidation at the C2 position of the pyrrolidine ring, forming a lactam metabolite (M12). This reaction was almost completely inhibited in human hepatic microsomes and mitochondria by the monoamine oxidase (MAO)-B-specific inhibitor selegiline. Clorgyline, a specific inhibitor of MAO-A, was less effective in inhibiting M12 formation. Other metabolic pathways of variable significance among the four species included the formation of carbamoyl-O-glucuronide, hydroxymethyl lactam, and carboxyl lactam. Overall, the data indicate that the primary enzymes responsible for the primary metabolism of bicifadine in humans are MAO-B and CYP2D6.     

In vitro metabolism of the analgesic agent, tramadol-N-oxide, in mouse, rat, and human.

Tramadol-N-oxide (TNO, RWJ-38705) is a new analgesic agent, which is believed to produce its analgesic effect following metabolic conversion to tramadol. In the present study, API ionspray-MS and MS/MS techniques were used to profile the in vitro metabolism of TNO in mouse, rat, and human hepatic S9 fractions in the presence of an NADPH generating system. Unchanged TNO represented 60, 24, and 26% of the sample in mouse, rat, and human, respectively. Tramadol, and seven other metabolites were profiled and tentatively identified on the basis of MS analysis and by comparison to synthetic reference samples. TNO metabolites were formed via four Phase I reactions: (1) N-oxide reduction, (2) O-demethylation, (3) N-demethylation, and (4) cyclohexylhydroxylation. TNO was found to be substantially metabolized in hepatic S9 from all three species. The metabolism of TNO to tramadol via N-oxide reduction was greater in rat and human than in mouse.     

Isolation, anticholinesterase properties, and acute toxicity in mice of the four stereoisomers of the nerve agent soman

The four stereoisomers of the nerve agent pinacolyl methylphosphonofluoridate (soman), designated as C(+)P(+), C(+)P(-), C(-)P(+), and C(-)P(-), have different toxicologic properties due to stereospecific interactions in living organisms. We report the isolation of these stereoisomers with more than 99% optical purity. This result was realized by means of (i) complete optical resolution of pinacolyl alcohol, (ii) synthesis of C(+)- and C(-)-soman from the (+)- and (-)-enantiomers of the alcohol, (iii) optimalization of conditions for stereospecific inhibition of alpha-chymotrypsin with the P(-)-isomers of C(+)- and C(-)-soman, followed by isolation of the C(+)P(+)- and C(-)P(+)-isomers, (iv) isolation of the C(+)P(-)- and C(-)P(-)-isomers after incubation of C(+)- and C(-)-soman, respectively, in rabbit plasma, which hydrolyzes stereospecifically the P(+)-isomers. The bimolecular rate constants for inhibition of electric eel acetylcholinesterase (AChE) at pH 7.7, 25 degrees C, are at least 3.6 X 10(4) larger for the P(-)- than for the P(+)-isomers. The enzyme inhibited with C(+)P(-)-soman is much more effectively reactivated with the oximes HI-6, HGG-42, and obidoxime than AChE inhibited with C(-)P(-)-soman. The LD50 values (sc, mice) are in accordance with the P(-)/P(+) ratio of inhibition rates of AChE, i.e. 99, 38, greater than 5000, greater than 2000, 214, 133, and 156 micrograms/kg for C(+)P(-)-, C(-)P(-)-, C(+)P(+)-, C(-)P(+)-, C(+)-, C(-)-soman, and "soman", respectively. The relative LD50 values of the C(-)P(-)- and C(+)P(-)-isomers do not correspond with the small differences in their rates of inhibition of AChE, indicating that such small rate ratios may be overruled by other stereospecific effects, e.g., in vivo rates of detoxification.     

梭曼异构体气相分析及兔血中的消除动力学

建立梭曼手性异构体气相色谱分析方法以研究兔静脉染毒后血中游离梭曼的消除过程 .采用大进样量气相色谱仪及手性毛细管柱实现梭曼 4个异构体的分离 ;用二异丙基氟磷酸酯作内标 ,测定兔静脉染毒后血中游离C(± )P(- )梭曼 .结果可见梭曼 4个异构体在确定的气相色谱条件下得到了较好地分离 ;给兔iv梭曼 4 3.2 μg·kg- 1后 ,血中仅测到一对C(± )P(- )梭曼 ,其消除过程符合二室开放模型 ,Vd=2 .1L·kg- 1,t1/2α=4 .5s ,t1/2 β=72s ,AUC(15～ 3 0 0s) =2 .0 8mg·s·L- 1,CL(S) =2 1mL·kg- 1·s- 1,血中C(± )P(- )梭曼在血中消除过程反映了机体对梭曼毒性异构体的解毒过程 .结果说明 ,大进样量气相色谱分析方法测定兔静脉染毒血中C(± )P(- )梭曼消除的动力学过程 ,方法灵敏 ,可靠 ,重复性好 .     

Toxicokinetics of the four stereoisomers of the nerve agent soman in atropinized rats—Influence of a soman simulator

The toxicokinetics of the four stereoisomers of C(+/-)P(+/-)-soman were investigated in anesthetized, atropinized, and artificially ventilated rats at iv doses of 6 (495 micrograms/kg) and 3 LD50. By integration of a thermodesorption/cold trap injector into our GLC analysis, the soman stereoisomers could be followed in rat blood down to a minimum detectable concentration, i.e., 1.5 pg/ml (8.3 pM), 55-fold lower than that published previously. This new detection limit is probably near or below the minimum concentration relevant for survival. Whereas C(+)P(+)-soman disappears in vivo from rat blood within 0.25 min, the toxicokinetics of C(-)P(+)-soman could be described by a two-compartment model, with a biological half-life of 1-1.5 min. The extremely toxic C(+/-)P(-)-isomers could be followed in rat blood for greater than 4 and 2 hr at doses of 6 and 3 LD50, respectively. The toxicokinetics of the P(-)-isomers are best described with a three-compartment model, with terminal half-lives of 40-64 and 16-22 min at doses of 6 and 3 LD50, respectively. Administration of a 13.6-fold molar excess of the soman simulator 1,2,2-trimethylpropyl dimethylphosphinate (PDP) 10 min prior to administration of 6 LD50 of C(+/-)P(+/-)-soman reduces the terminal half-lives of the C(+/-)P(-)-isomers to the values measured at the dose of 3 LD50 without PDP pretreatment. Previous investigations showed that, without PDP pretreatment, rats suffer from endogenous reintoxication 4-6 hr after initially successful therapy, at C(+/-)P(+/)-soman doses greater than or equal to 6 LD50. Both this reintoxication phenomenon due to the presence of toxicologically significant C(+/-)P(-)-soman levels up to 4 hr after intoxication and its antagonism via PDP pretreatment can be understood on the basis of our toxicokinetic measurements. This shows that such investigations can contribute to insight into the toxicology of C(+/-)P(+/-)-soman and to a better treatment of intoxications with this agent.     

In vitro kinetics of styrene and styrene oxide metabolism in rat,mouse, and human

Styrene oxide (SO), a labile metabolite of styrene, is generally accepted as being responsible for any genotoxicity associated with styrene. To better define the hazard associated with styrene, the activity of the enzymes involved in the formation (monooxygenase) and destruction of SO (epoxide hydrolase and glutathione-S-transferase) were measured in the liver and lungs from naive and styrene-exposed male Sprague-Dawley rats and B6C3F1 mice (three daily 6-h inhalation exposures at up to 600 ppm styrene) and Fischer 344 rats (four daily 6-h inhalation exposures at up to 1000 ppm styrene), and in samples of human liver tissue. Additionally, the time course of styrene and SO in the blood was measured following oral administration of 500 mg styrene/kg body weight to naive Fischer rats and rats previously exposed to 1000 ppm styrene. The affinity of hepatic monooxygenase for styrene, as measured by the Michaelis constant (K _m), was similar in the rat, mouse, and human. Based on theV _max for monooxygenase activity and the relative liver and body size, the mouse had the greatest capacity and humans the lowest capacity to form SO from styrene. In contrast, human epoxide hydrolase had a greater affinity (i. e., lowerK _m) for SO than epoxide hydrolase from rats or mice while the apparent Vmax for epoxide hydrolase was similar in the rat, mouse, and human liver. However, the activity of epoxide hydrolase relative to monooxygenase activity was much greater in the human than in the rodent liver. Hepatic glutathione-S-transferase activity, as indicated by theV _max, was 6- to 33-fold higher than epoxide hydrolase activity. However, the significance of the high glutathione-S-transferase activity is unknown because hydrolysis, rather than conjugation, is the primary pathway for SO detoxification in vivo. Human hepatic glutathione-S-transferase activity was extremely variable between individual human livers and much lower than in rat or mouse liver. Prior exposure to styrene had no effect on monooxygenase activity or on blood styrene levels in rats given a large oral dose of styrene. In contrast, prior exposure to styrene increased hepatic epoxide hydrolase activity 1.6-fold and resulted in lower (0.1>P>0.05) blood SO levels in rats given a large oral dose of styrene. Qualitatively, these data indicate that the mouse has the greatest capacity and the human the lowest capacity to form SO. In addition, human liver should be more effective than rodent liver in hydrolyzing low levels of SO. Quantitative evaluation of the species differences in enzyme levels are being evaluated with the development of a physiologically based pharmacokinetic model for styrene that includes SO.