In vitro degradation of the stereoisomers of soman in guinea-pig, mouse and human skin

The fate of the four stereoisomers of soman [C(-)P(+), C(+)P(+), C(+)P(-) and C(-)P(-)] was studied by incubating 10 microM C(+/-)P(+/-)-soman at pH 7.4 and 37 degrees for various periods in the presence or absence of homogenates (1:10 and 1:20 w/v) of guinea-pig, mouse or human skin. The remaining concentrations of the soman isomers were determined gas chromatographically. Similar rates of spontaneous (non-enzymatic) hydrolysis (K = 0.005 min-1) were found for the four isomers of soman. Hydrolysis of the toxic (C(+/-)P(-)-isomers is not accelerated in the presence of the skin homogenates. In contrast, the non-toxic C(+/-)P(+)-isomers are enzymatically hydrolysed. As the amount of proteins present in the homogenates varied the rate constants for enzyme hydrolysis per protein concentration were calculated. Except for the high hydrolysis rate constant of greater than 0.127/min.g.l for C(+)P(+) in human skin, these values were almost similar (0.031-0.045/min.g.l) for the skin homogenates tested. Irreversible binding sites for the four soman-stereoisomers are only found in homogenates of mouse skin; 122-195 pmol soman-isomer are bound per mg protein. After preincubation of mouse homogenate with 10 microM soman during 18 hr at 0-4 degrees no further binding of the isomers was detected. It is concluded that skin of the three species tested does not contain enzymes that degrade the toxic C(+/-)P(-)-isomers of soman, whereas phosphorylphosphatase activity for the C(+/-)P(+)-isomers is present in the skin of all three species. Binding sites for all four soman isomers are only present in mouse skin.     

Stereoselective hydrolysis of soman in human plasma and serum

The contribution of various human serum and plasma fractions to the total hydrolysis rate constants of the four isomers of soman is studied. Spontaneous hydrolysis (as measured in buffer) occurs at a faster rate for the C(+)P(+)- and C(-)P(-)-isomers. A stereoselectively catalyzed hydrolysis of soman occurs in serum fractions IV and V (albumin). In fraction V the C(+)P(+)- and C(-)P(-)-isomers are hydrolyzed at a faster rate than their respective epimers, while in fraction IV-1 a stereoselective effect towards C(+)P(+)-soman is found. All the forementioned contributions, however, are negligible in comparison with the stereoselective enzymatic hydrolysis of the P(+)-isomers. The latter reaction is characterized by a significant lowering of the activation energy as compared with the spontaneous hydrolysis of the P(+)-isomers. Such a lowering in activation energy is not found for the hydrolysis of the P(-)-isomers in whole serum or plasma; hence it can be concluded that a phosphorylphosphatase hydrolyzes the P(+)-isomers in a stereoselective way, the P(-)-isomers either not being affected by this (these) enzyme(s) or the mechanism of catalysis being fundamentally different. This conclusion is in agreement with the observations on the influence of Hg2+ on the hydrolysis of soman in serum; the hydrolysis of the P(+)-isomers is significantly inhibited by 1 mM of Hg2+ while the P(-)-hydrolysis is unaffected by this concentration of Hg2+. The action of some potential inhibitors on this phosphorylphosphatase activity was studied. Iodoacetate did not inhibit nor did Ba2+, Sr2+, Co2+ or Mn2+ show a significant effect on the hydrolysis of the P(+)-isomers. On the other hand the hydrolytic activity in serum was nearly completely inhibited by EDTA but restored upon addition of Ca2+. These findings suggest that this enzymatic activity can be classified as an arylesterase (paraoxonase). Finally, the influence of pH on the hydrolytic activity shows a different pattern for C(+)P(+)- and C(-)P(+)-soman, which may suggest that more than one enzyme is involved in the degradation of soman.     

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.     

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.     

Phosphonylation of purified human, canine and porcine cholinesterase by soman. Stereoselective aspects

Cholinesterases (EC 3.1.1.8, acylcholine acylhydrolase) from the sera of man, dog and pig were purified 400-600-fold using a combination of ion-exchange and affinity chromatography. In a first approach, phosphonylation by soman was studied by using the half-resolved epimers C(+)P(+/-)-soman and C(-)P(+/-)-soman. The degradation of soman at the nanomolar level was followed in time by determining the remaining soman by capillary gas chromatography with NP detection. In the three sera investigated the P-(-)-epimer phosphonylates at a higher rate than its corresponding P(+)-counterpart and the stereoselectivity is greater for the C(+)-epimers than for the C(-)-epimers. Individual soman isomers were isolated from C(+)- and C(-)-epimers and quantified by gas chromatography. Second-order rate constants were determined for the phosphonylation of purified cholinesterase by isolated soman isomers. The C(+)P(-)-isomer has the highest phosphonylation rate for the three species; the other toxic isomer, C(-)P(-), has a five to ten-fold lower rate. The overall stereoselectivity is more marked in human cholinesterase than in canine. Porcine serum cholinesterase is phosphonylated by the P(-)-isomers at a slightly higher rate than the human enzyme.     

Stereoisomers of soman (pinacolyl methylphosphonofluoridate): inhibition of serum carboxylic ester hydrolase and potentiation of their toxicity by CBDP (2-(2-methylphenoxy)-4H-1,3,2-benzodioxaphosphorin-2-oxide) in mice

The interaction of C(+/-)P(+/-)-soman (pinacolyl methylphosphonofluoridate) and its individual stereoisomers with serum carboxylic-ester hydrolase and potentiation of their toxicity by a carboxylic-ester hydrolase inhibitor CBDP (2-(2-methylphenoxy)-4H-1,3,2-benzodioxaphosphorin-2-oxide) was investigated. C(+/-)P(+/-)-Soman and the individual stereoisomers all inhibited purified mouse serum carboxylic-ester hydrolase to different degrees. C(+/-)P(+/-)-Soman and the C(-)P(-)- and C(+)P(-)-isomers had Ki values of 30.6, 18.7, and 35.7 nM, respectively, and C(-)P(+)- and C(+)P(+)-isomers had Ki values of 1412 and 2523 nM, respectively. In toxicity experiments CBDP (0.5 mg/kg; iv 1 hr prior to soman) pretreatment potentiated the toxicity of C(+/-)P(+/-)-, C(+)P(-)-, and C(-)P(-)-soman to a similar degree. Thus, it appears that the toxic stereoisomers of soman have a similar affinity for mouse serum carboxylic-ester hydrolase, and CBDP pretreatment does not enhance selectively the toxicity of one stereoisomer over the other.     

Stereoselective phosphonylation of human serum proteins by soman

Phosphonylation has been reported as part of the degradation of soman in human serum. The concentration of phosphonylation sites can be quantified by comparing the degradation in serum, preincubated with soman (all sites occupied), with the degradation in serum not preincubated. The mean value of 73 nM of phosphonylation sites is in agreement with the concentration of active sites of butyrylcholinesterase (EC 3.1.1.8.), which is known to be phosphonylated by soman. Hence, it is concluded that butyrylcholinesterase accounts for all the phosphonylation sites present in human serum. The stereoselectivity of the reaction was investigated by using epimeric pairs of soman, in casu C(+)P(+/-)- and C(-)P(+/-)-soman. In a first approach enzymatic hydrolysis was blocked and the ratios of phosphonylation rate constants, C(+)P(+)/C(+)P(-) and C(-)P(+)/C(-)P(-), were determined to be 0.15 and 0.31, respectively. In a second approach, in untreated serum, the bimolecular phosphonylation rate constants of C(+)P(-)- and C(-)P(-)-soman were determined, neglecting their small hydrolysis rate and taking advantage of the fast enzymatically catalysed disappearance of their respective P(+)-epimeric counterparts. Values for C(+)P(-)- and C(-)P(-)-soman are 3.6 X 10(7) and 0.6 X 10(7) M-1.min-1, respectively. Using a combination of both approaches, a relative ranking of phosphonylation rates of the four isomers was found to be C(+)P(-) much greater than C(+)P(+) approximately equal to C(-)P(-) greater than C(-)P(+).     

Stereospecific reactivation by some Hagedorn-oximes of acetylcholinesterases from various species including man, inhibited by soman

Reactivation by bispyridinium mono-oximes (Hagedorn-oximes) and some classical oximes (0.03 or 1mM) was studied in vitro of rat, bovine and human erythrocyte acetylcholinesterase and of electric eel acetylcholinesterase inhibited by soman. Relative reactivating potencies of the oximes are similar for the three inhibited erythrocyte enzymes. In general, Hagedorn-oximes are more potent than the classical oximes. Among the Hagedorn-oximes, HI-6 is the most potent reactivator for the three inhibited enzymes. Relative reactivating potencies for the inhibited erythrocyte acetylcholinesterases and electric eel acetylcholinesterase, however, clearly differ. Since the reactivation experiments were carried out with racemic soman, a mixture of the two inhibited enzymes may be formed, which may cause additional problems in the comparison of various results. In order to get more detailed information on differences between human erythrocyte and electric eel acetylcholinesterase, reactivation of these enzymes inhibited with the P(-)-isomers of C(+)- and C(-)-soman were studied separately. Reactivation appeared to be dependent on the chirality of the alpha-carbon atom in the pinacolyl group. HI-6 is by far the most potent reactivator for the human enzyme inhibited by the two P(-)-isomers. It is suggested that electric eel acetylcholinesterase is not a reliable model for in vitro testing of therapeutic potencies of oximes against soman intoxication in mammals. Rate constants of aging of the four acetylcholinesterases inhibited with racemic soman and of the human and eel enzyme inhibited by the P(-)-isomers of C(+)- and C(-)-soman were also determined. The aging of the inhibited rat enzymes proceeds remarkably slowly (t1/2 = 21 min). The rate of aging is not affected by the chirality on the alpha-carbon atom in the pinacolyl group. Consequences of the present results are discussed in view of extrapolation of reactivation data of a series of reactivators to their relative therapeutic effect, ultimately in man. It is speculated that the more rapid aging of the human inhibited enzyme may hamper oxime-therapy in man more seriously than in rat.     

Development of a physiologically based model for the toxicokinetics of C(±)P(±)-soman in the atropinized guinea pig

A physiologically based model was developed which describes the in vivo toxicokinetics of the highly reactive nerve agent C(±)P(±)-soman at doses corresponding to 0.8–6 LD₅₀ in the atropinized guinea pig. The model differentiates between the summated highly toxic C(±)P(−)-soman stereoisomers at supralethal doses and the individual nontoxic C(±)P(+)-isomers. Several toxicant-specific parameters for the soman stereoisomers were measured in guinea pig tissue homogenates. Cardiac output and blood flow distribution were measured in the atropinized, anesthetized, and artificially ventilated guinea pig. The model was validated by comparison of the time courses for the blood concentrations of the two pairs of stereoisomers in the guinea pig after i.v. bolus administration with the blood concentrations predicted by the model. The predictions put forward for the summated C(±)P(−)-isomers are in reasonable agreement with the experimental data obtained after doses corresponding to 2 and 6 LD₅₀. In view of large differences in the rates of hydrolysis of the C(±)P(+)-isomers, these two isomers had to be differentiated for satisfactory modeling of both isomers. In order to model the toxicokinetics of C(±)P(−)-soman at a dose of 0.8 LD₅₀, the almost instantaneous elimination of the C(+)P(−)-isomer at that dose had to be taken into account. The sensitivity of the predictions of the model to variations in the parameters has been studied with incremental sensitivity analysis. The results of this analysis indicate that extension to a model involving four individual stereoisomers is desirable in view of large differences in the biochemical characteristics of the two C(±)P(−)- and C(±)P(+)-isomers. Received: 8 July 1996 / Accepted: 30 October 1996     

Formation of soman (1,2,2-trimethylpropyl methylphosphonofluoridate) via fluoride-induced reactivation of soman-inhibited aliesterase in rat plasma

After incubation (37°) of rat blood or plasma with the nerve agent soman, (CH₃)₃C(CH₃)C(H)O(CH₃)P(O)F (7.7 μM), for 10 min, only a small amount of this organophosphate (7 or 1%, respectively) is left, as determined enzymatically (acetylcholinesterase) and gas chromatographically. Comparison of the results obtained with both analyses shows that this residual soman consists only of its P(?)-isomers. Incubation (25°) at pH 4.8–6.1 of such soman-treated rat blood or plasma with sodium fluoride (2.5 mM) for 0.5 min leads to (i) a substantial increase of the P(?)-soman concentration, and (ii) a (partial) reactivation of the soman-inhibited aliesterase, proportional to the amount of generated P(?)-soman. These results indicate strongly that added fluoride ions regenerate soman by a reversal of the inhibition reaction. From the relationship between percentage of reactivation and increase of soman concentration the aliesterase concentration in rat plasma is calculated as 2.6 μM.Sodium fluoride has a similar effect in blood taken from rats to which soman was administered intravenously.The increase of the P(?)-soman concentration is higher with higher sodium fluoride concentrations and at lower pH values.In accordance with the absence of aliesterase, addition of sodium fluoride does not induce an increase of the P(?)-soman concentration in soman-treated human plasma.     

Stereoselective oxidation of nilvadipine, a new dihydropyridine calcium antagonist, in rat and dog liver

The stereoselective oxidation of nilvadipine (NV), a new 1,4-dihydropyridine calcium antagonist, to the corresponding pyridine analog was studied after incubation of (+)- and (-)-NV with rat and dog liver microsomes. The rates of formation of the pyridine analog and disappearance of NV were similar for each species, indicating that aromatization of NV is the primary metabolic step. Formation of the corresponding pyridine required the presence of an NADPH-generating system and was significantly inhibited by carbon monoxide and metyrapone, indicating the participation of cytochrome P-450. In male rat liver microsomes, the apparent Km values for the formation of the pyridine from (+)- and (-)-NV were 11.2 and 8.1 microM, and the Vmax values were 7.48 and 3.37 nmol/mg of protein/min, respectively. Therefore, the Vmax/Km value, which is equivalent to the intrinsic clearance of the drug, for the oxidation of (+)-NV was 1.59-fold greater than that for the oxidation of the (-)-enantiomer. In female rats, (-)-NV oxidation exhibited two distinct apparent Km values, whereas the that of the (+)-enantiomer did not. The (+)/(-) ratio of Vmax/Km was 1.23. On the other hand, in male dog microsomes the Km values for (+)- and (-)-NV were 21.9 and 12.2 microM, and Vmax values were 3.02 and 2.45 nmol/mg of protein/min, respectively; the (+)/(-) ratio of Vmax/Km was 0.69. These results indicate that the stereo-selective oxidation of NV is species dependent and is sex related in rat liver.     

Characterization of deltamethrin metabolism by rat plasma and liver microsomes

Deltamethrin, a widely used type II pyrethroid insecticide, is a relatively potent neurotoxicant. While the toxicity has been extensively examined, toxicokinetic studies of deltamethrin and most other pyrethroids are very limited. The aims of this study were to identify, characterize, and assess the relative contributions of esterases and cytochrome P450s (CYP450s) responsible for deltamethrin metabolism by measuring deltamethrin disappearance following incubation of various concentrations (2 to 400 microM) in plasma (esterases) and liver microsomes (esterases and CYP450s) prepared from adult male rats. While the carboxylesterase metabolism in plasma and liver was characterized using an inhibitor, tetra isopropyl pyrophosphoramide (isoOMPA), CYP450 metabolism was characterized using the cofactor, NADPH. Michaelis-Menten rate constants were calculated using linear and nonlinear regression as applicable. The metabolic efficiency of these pathways was estimated by calculating intrinsic clearance (Vmax/Km). In plasma, isoOMPA completely inhibited deltamethrin biotransformation at concentrations (2 and 20 microM of deltamethrin) that are 2- to 10-fold higher than previously reported peak blood levels in deltamethrin-poisoned rats. For carboxylesterase-mediated deltamethrin metabolism in plasma, Vmax=325.3+/-53.4 nmol/h/ml and Km=165.4+/-41.9 microM. Calcium chelation by EGTA did not inhibit deltamethrin metabolism in plasma or liver microsomes, indicating that A-esterases do not metabolize deltamethrin. In liver microsomes, esterase-mediated deltamethrin metabolism was completely inhibited by isoOMPA, confirming the role of carboxylesterases. The rate constants for liver carboxylesterases were Vmax=1981.8+/-132.3 nmol/h/g liver and Km=172.5+/-22.5 microM. Liver microsomal CYP450-mediated biotransformation of deltamethrin was a higher capacity (Vmax=2611.3+/-134.1 nmol/h/g liver) and higher affinity (Km=74.9+/-5.9 microM) process than carboxylesterase (plasma or liver) detoxification. Genetically engineered individual rat CYP450s (Supersomes) were used to identify specific CYP450 isozyme(s) involved in the deltamethrin metabolism. CYP1A2, CYP1A1, and CYP2C11 in decreasing order of importance quantitatively, metabolized deltamethrin. Intrinsic clearance by liver CYP450s (35.5) was more efficient than that by liver (12.0) or plasma carboxylesterases (2.4).     

Stereoselective sulphate conjugation of salbutamol in humans: comparison of hepatic, intestinal and platelet activity.

1. The oral bioavailability of the beta 2-adrenoceptor agonist salbutamol has been proposed to be stereoselective, presumably due to presystemic sulphate conjugation. In the present study we examined the stereochemistry of the sulphation reaction in vitro using human tissue preparations. 2. Sulphation of salbutamol was studied with partially purified hepatic M and P form phenol sulphotransferases (PSTs), 100,000 g cytosol of jejunal mucosa and platelet homogenate. The cosubstrate PAP35S was used as the sulphate donor. The acceptor substrate was either (+)-, (-)-or (+/)-salbutamol. 3. Sulphation was catalyzed by the M form PST of the liver but not the P form. The sulphation efficiency (Vmax/Km) was 11.9-fold greater for the (-)- than for the (+)- enantiomer, due entirely to a lower apparent Km for (-)-salbutamol, 103 microM, than for (+)-salbutamol, 1394 microM. 4. Sulphation by the jejunal mucosa (n = 3) was very similar to that of the M form PST with the efficiency being 9.8-fold greater for the (-)-enantiomer and apparent Km values 95 microM and 889 microM for (-)- and (+)-salbutamol, respectively. 5. Sulphation by the platelet (n = 3) was also very similar to that of the M form PST with the efficiency being 9.9-fold greater for the (-)-enantiomer and apparent Km values 141 microM and 1190 microM for (-)- and (+)-salbutamol, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereoselective hydrolysis of O-acetyl propranolol as prodrug in rat tissue homogenates.

The stereochemical characteristics of the hydrolysis of O-acetyl propranolol were studied using phosphate buffer (pH 7.4), rat plasma, and rat tissue homogenates. In the phosphate buffer, no difference was observed in the hydrolysis rate between the esters of (R)- and (S)-propranolol. In rat plasma and tissue homogenates, hydrolysis of the ester was both accelerated and stereoselective. Hydrolysis of O-acetyl (R)-propranolol was five times faster than that of the (S)-isomer in rat plasma. However, in the liver and intestine homogenates, the (S)-isomer was hydrolyzed faster than the (R)-isomer. Interconversion between the (R)- and (S)-isomers was not observed under the experimental conditions. The same stereoselective hydrolysis was also observed with racemic O-acetyl propranolol. However, observed rate constants for the hydrolysis were lower than those for the pure isomers. These results indicate that enzymatic hydrolysis of O-acetyl propranolol occurred stereoselectively and the selectivity of the plasma enzyme was different from those of liver and intestine enzymes.     

Partial characterization of an enzyme that hydrolyzes sarin, soman, tabun, and diisopropyl phosphorofluoridate (DFP)

The properties of a rat liver enzyme that hydrolyzes organophosphorus (OP) inhibitors of cholinesterases were studied. The rates of hydrolysis of OP inhibitors were determined by continuous titration of released hydrogen ions, using a pH stat method. Centrifugation of homogenates at 205,000 g for 30 min demonstrated that the activity was in the soluble fraction. Hydrolysis of sarin, soman, and diisopropyl phosphorofluoridate (DFP), but not of tabun, was stimulated by the addition of Mn2+ and Mg2+. Hydrolysis of sarin greater than soman greater than tabun greater than DFP. Unlike other OP hydrolases that preferentially hydrolyze the non-toxic isomers of soman, this enzyme hydrolyzed all four soman isomers at approximately the same rate. This result was obtained in vitro by gas chromatographic analysis of enzyme-catalyzed soman hydrolysis and confirmed in vivo by demonstrating reduced toxicity in mice of soman partially hydrolyzed by this enzyme. Km and Vmax were determined by fitting V vs [S] to a hyperbolic function using regression analysis. Km values ranged from 1.1 mM for soman to 8.9 mM for tabun. Vmax values ranged from 54 nmol/min/mg protein for DFP to 2694 for sarin. The enzyme was stable for at least 2 months at -90 degrees but was inactivated by heating at 100 degrees for 5 min. Elution profiles from gel filtration by high pressure liquid chromatography showed that the hydrolytic activity for the OP inhibitors eluted in a single peak, suggesting that a single enzyme was responsible for the observed hydrolysis. Further purification and characterization of this enzyme should prove useful for the development of methods for detection, detoxification, and decontamination of these cholinesterase inhibitors.     

Further studies on the metabolism of methylamine by semicarbazide-sensitive amine oxidase activities in human plasma, umbilical artery and rat aorta

An ion exchange radiochemical assay has been developed to study the deamination of [14C]methylamine (MA) in homogenates of rat aorta and human umbilical artery, as well as in samples of human plasma. MA metabolism was found to be inhibited almost completely by 1 mM semicarbazide, but virtually unaffected by 0.1 mM clorgyline, suggesting that MA is a substrate for the semicarbazide-sensitive amino oxidase (SSAO) activities which also metabolize benzylamine (BZ) in these sources. Mean Km values for MA metabolism by aorta, umbilical artery and plasma were 182, 832 and 516 microM, respectively, with corresponding Vmax values in aorta and umbilical artery of 100 and 590 nmol (mg prot.)-1 h-1, and in plasma of 48 nmol (mL serum)-1 h-1. Kinetic constants determined for [14C]BZ metabolism in plasma (by an organic solvent extraction assay) and in umbilical artery (by the ion exchange assay) yielded mean Km values of 225 microM (plasma), 222 microM (umbilical artery), and Vmax values of 28 nmol (mL serum)-1 h-1 (plasma) and 377 nmol (mg prot.)-1 h-1 (umbilical artery). The deamination of [14C]MA was inhibited competitively by unlabelled BZ, with Ki values in umbilical artery and plasma of 220 and 172 microM, respectively. Also, metabolite formation from mixtures of [14C]BZ (200 microM) and [14C]MA (800 microM) was extremely close to that predicted for a single enzyme capable of metabolizing two alternative substrates in a competitive fashion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of CYP3A4 and CYP2C19 in the stereoselective metabolism of lansoprazole by human liver microsomes

OBJECTIVE: The aim of this investigation was to clarify the stereoselective properties in lansoprazole metabolism by monitoring the metabolic consumption for each enantiomer and the formation of the main metabolites, lansoprazole sulfone and 5-hydroxylansoprazole, in the presence of human liver microsomal enzymes. METHODS: Human liver microsomes or recombinant cytochrome P450 (CYP) enzymes were incubated with either (+/- )-, (+)-, or (-)-lansoprazole in the presence of reduced nicotinamide adenine dinucleotide phosphate. The metabolic consumption of lansoprazole enantiomers was estimated from the amounts of enantiomers consumed by microsomal enzymes after incubation at 37 degrees C for 60 min. Metabolites of lansoprazole, lansoprazole sulfone, and 5-hydroxylansoprazole were determined after incubation at 37 degrees C for 20 min, and kinetic parameters [Michaelis constant (Km) and maximum velocity (Vmax)] were obtained using Eadie-Hofstee plots. RESULTS: (-)-Lansoprazole was metabolized more preferentially than (+)-lansoprazole in human liver microsomes. Stereoselective sulfoxidation and hydroxylation [(+) > (-)] were observed in human liver microsomes. Strikingly, in sulfoxidation, a significantly higher intrinsic clearance (Vmax,l/Km,l) of (-)-lansoprazole (0.023 +/- 0.001 ml/min/mg) than (+)-lansoprazole (0.006 +/- 0.000 ml/min/mg) was observed. Consequently, sulfoxidation is likely to play an important role in the stereoselective metabolism of lansoprazole enantiomers. P450-isoform specificity for each enantiomer was evident. CYP3A4, which mainly catalyzed sulfoxidation, was more active toward (-)-lansoprazole in either a chiral or racemic drug as a substrate. CYP2C19, which catalyzed hydroxylation, preferentially metabolized (+)-lansoprazole. The consumption of (+)-lansoprazole was markedly inhibited by (-)-lansoprazole, indicating a metabolic enantiomer/enantiomer interaction. However, this alteration of recombinant CYP2C19 specificity for (+)-lansoprazole did not appear in metabolism in human liver microsomes. CONCLUSIONS: Stereoselective metabolism was observed in human liver microsomes, and this stereoselectivity was mainly based on CYP3A4 specificity for preferable metabolism of (-)-lansoprazole.     

Metabolism of (-)-fenchone by CYP2A6 and CYP2B6 in human liver microsomes

The in vitro metabolism of (-)-fenchone was examined in human liver microsomes and recombinant enzymes. The biotransformation of (-)-fenchone was investigated by gas chromatography-mass spectrometry. (-)-Fenchone was found to be oxidized to 6-exo-hydroxyfenchone, 6-endo-hydroxyfenchone and 10-hydroxyfenchone by human liver microsomal P450 enzymes. The formation of metabolites was determined by the relative abundance of mass fragments and retention times on gas chromatography (GC). CYP2A6 and CYP2B6 were major enzymes involved in the hydroxylation of (-)-fenchone by human liver microsomes, based on the following lines of evidence. First, of 11 recombinant human P450 enzymes tested, CYP2A6 and CYP2B6 catalysed the oxidation of (-)-fenchone. Second, oxidation of (-)-fenchone was inhibited by thioTEPA and (+)-menthofuran. Finally, there was a good correlation between CYP2A6, CYP2B6 contents and (-)-fenchone hydroxylation activities in liver microsomes of 11 human samples. CYP2A6 may be more important than CYP2B6 in human liver microsomes. Kinetic analysis showed that the Vmax/Km values for (-)-fenchone 6-endo-, 6-exo- and 10-hydroxylation catalysed by liver microsomes of human sample HG-03 were 24.3, 44.0 and 1.3nM(-1)min(-1) , respectively. Human recombinant CYP2A6 and CYP2B6 catalysed (-)-fenchone 6-exo-hydroxylation with Vmax values of 2.7 and 12.9 nmol min(-1) nmol(-1) P450 and apparent Km values of 0.18 and 0.15 mM and (-)-fenchone 6-endo-hydroxylation with Vmax values of 1.26 and 5.33nmolmin(-l) nmol(-1) P450 with apparent Km values of 0.29 and 0.26mM. (-)-Fenchone 10-hydroxylation was catalysed by CYP2B6 with Km and Vmax values of 0.2 mM and 10.66 nmol min(-1) nmol(-1) P450, respectively.     

Metabolism of bradykinin agonists and antagonists by plasma aminopeptidase P.

In addition to angiotensin I converting enzyme (ACE; EC 3.4.15.1) and carboxypeptidase N (CPN; EC 3.4.17.3), other peptidases contribute to bradykinin (BK) degradation in plasma. Rat plasma degraded BK by hydrolysis of the N-terminal Arg1-Pro2 bond, and the characteristics of hydrolysis are consistent with identification of aminopeptidase P (APP; EC 3.4.11.9) as the responsible enzyme. BK and BK[1-5] N-terminal hydrolysis was optimal at neutral pH, was inhibited by 2-mercaptoethanol, dithiothreitol, o-phenanthroline and EDTA, but was unaffected by the aminopeptidase inhibitors amastatin, puromycin and diprotin A, the endopeptidase-24.11 inhibitors phosphoramidon and ZINCOV, and the ACE and CPN inhibitors captopril and D,L-mercapto-methyl-3-guanidinoethylthiopropanoic acid (MERGETPA), respectively. Although kallidin (Lys-BK) was not metabolized directly by APP, conversion to BK by plasma aminopeptidase M (EC 3.4.11.2) resulted in subsequent degradation by APP. BK analogs containing N-terminal Arg1-Pro2 bonds, including [Tyr8-(OMe)] BK and [Phe8 psi(CH2NH)Arg9]BK (B2 agonists), des-Arg9-BK and [D-Phe8]des-Arg9-BK (B1 agonists), and [Leu8]des-Arg9-BK (B1 antagonist), were degraded by APP with Km and Vmax values comparable to those found for BK (Km = 19.7 +/- 2.6 microM; Vmax = 12.1 +/- 1.2 nmol/min/mL). In contrast, B2 antagonists containing D-Arg0 N-termini, including D-Arg[Hyp3,Thi5.8,D-Phe7]BK and D-Arg[Hyp3,D-Phe7,Phe8 psi(CH2NH)Arg9]BK, were resistant to APP-mediated hydrolysis. These data support a role for plasma aminopeptidase P in the degradation of circulating kinins, and a variety of B2 and B1 kinin agonists and antagonists. However, APP does not participate in the degradation of D-Arg0-containing antagonists.     

Kinetic modeling of beta-chloroprene metabolism: I. In vitro rates in liver and lung tissue fractions from mice, rats, hamsters, and humans.

Beta-chloroprene (2-chloro-1,3-butadiene, CD) is carcinogenic by inhalation exposure to B6C3F1 mice and Fischer F344 rats but not to Wistar rats or Syrian hamsters. The initial step in metabolism is oxidation, forming a stable epoxide (1-chloroethenyl)oxirane (1-CEO), a genotoxicant that might be involved in rodent tumorigenicity. This study investigated the species-dependent in vitro kinetics of CD oxidation and subsequent 1-CEO metabolism by microsomal epoxide hydrolase and cytosolic glutathione S-transferases in liver and lung, tissues that are prone to tumor induction. Estimates for Vmax and Km for cytochrome P450-dependent oxidation of CD in liver microsomes ranged from 0.068 to 0.29 micromol/h/mg protein and 0.53 to 1.33 microM, respectively. Oxidation (Vmax/Km) of CD in liver was slightly faster in the mouse and hamster than in rats or humans. In lung microsomes, Vmax/Km was much greater for mice compared with the other species. The Vmax and Km estimates for microsomal epoxide hydrolase activity toward 1-CEO ranged from 0.11 to 3.66 micromol/h/mg protein and 20.9 to 187.6 microM, respectively, across tissues and species. Hydrolysis (Vmax/Km) of 1-CEO in liver and lung microsomes was faster for the human and hamster than for rat or mouse. The Vmax/Km in liver was 3 to 11 times greater than in lung. 1-CEO formation from CD was measured in liver microsomes and was estimated to be 2-5% of the total CD oxidation. Glutathione S-transferase-mediated metabolism of 1-CEO in cytosolic tissue fractions was described as a pseudo-second order reaction; rates were 0.0016-0.0068/h/mg cytosolic protein in liver and 0.00056-0.0022 h/mg in lung. The observed differences in metabolism are relevant to understanding species differences in sensitivity to CD-induced liver and lung tumorigenicity.