Comparative sensitivities of avian neural esterases to in vitro inhibition by organophosphorus compounds

The inhibitory power of organophosphorus compounds in vitro was compared against neurotoxic esterase (also known as neuropathy target esterase, NTE), acetylcholinesterase and carboxylesterase activities in brains from chickens, turkeys, quail and rats. Brains from the species most susceptible to clinical signs of organophosphorus-induced delayed neuropathy (chicken, turkey) contained more NTE than did rat and quail. Higher concentrations of organophosphorus compounds were required to inhibit rat NTE and quail acetylcholinesterase than were necessary for inhibition of these enzymes in chicken and turkey brains. Total carboxylesterase and acetylcholinesterase activities were less in rats than in the avian species.     

Interaction of methamidophos with hen and human acetylcholinesterase and neuropathy target esterase

Methamidophos causes acute cholinergic toxicity in several species, including man, and organophosphate-induced delayed polyneuropathy which has been reported in man but not in the hen. Acetylcholinesterase (AChE) and neuropathy target esterase (NTE) are thought to be the molecular targets of acute and delayed toxicity, respectively. The rate constants of inhibition (k_a) and reactivation (k₊₃) of human and hen brain AChE and NTE by methamidophos resolved optical isomers are here reported. NTE inhibition was progressive and irreversible. Human and hen NTE k_a (M^–1·m^–1) ford-(+) methamidophos was 88 and 59, respectively, and forl-(–) methamidophos 3.2 and 3.0, respectively. AChE spontaneously reactivates after inhibition.d-(+) methamidophos 10^–3·k_a (M^–1·m^–1) for human and hen AChE was 0.24 and 0.13; 10³·k₊₃ (m^–1) was 0.83 and 0.69, respectively,l-(–) Methamidophos 10^–3·k_a (M^–1·m^–1) for human and hen AChE was 5.7 and 2.8, whereas 10³ · k₊₃ (m^–1) was 6.50 and 1.52, respectively.l-(–)-Inhibited AChE reactivated to about 60% for human and 30% for hen enzymes, respectively.d-(+)-Inhibited AChE reactivated to about 10–20% for both species. Maximal reactivation occurred within 4–6 h when a plateau was reached. The larger and faster reactivation of human AChE inhibited in vitro byl-(–) methamidophos suggests that a corresponding effect might be possible in vivo and therefore explain, in part, the relatively higher susceptibility of man to delayed polyneuropathy induced by racemic methamidophos which occurs, however, with doses always causing severe cholinergic toxicity.Part of this work was presented at the 29th Annual Meeting of the Society of Toxicology, held in Miami FL, USA, February 1990.     

Toxicodynamic analysis of the combined cholinesterase inhibition by paraoxon and methamidophos in human whole blood

Theoretical work has shown that the isobole method is not generally valid as a method for testing the absence or presence of interaction (in the biochemical sense) between chemicals. The present study illustrates how interaction can be tested by fitting a toxicodynamic model to the results of a mixture experiment. The inhibition of cholinesterases (ChE) in human whole blood by various dose combinations of paraoxon and methamidophos was measured in vitro. A toxicodynamic model describing the processes related to both OPs in inhibiting AChE activity was developed, and fit to the observed activities. This model, not containing any interaction between the two OPs, described the results from the mixture experiment well, and it was concluded that the OPs did not interact in the whole blood samples. While this approach of toxicodynamic modeling is the most appropriate method for predicting combined effects, it is not rapidly applicable. Therefore, we illustrate how toxicodynamic modeling can be used to explore under which conditions dose addition would give an acceptable approximation of the combined effects from various chemicals. In the specific case of paraoxon and methamidophos in whole blood samples, it was found that dose addition gave a reasonably accurate prediction of the combined effects, despite considerable difference in some of their rate constants, and mildly non-parallel dose-response curves. Other possibilities of validating dose-addition using toxicodynamic modeling are briefly discussed.     

Kinetic analysis of acetylcholinesterase inhibition by combinations of acephate and methamidophos

Acephate pre-exposure provided protection against the inhibition of RBC and brain acetylcholinesterase (AChE), and plasma cholinesterase (ChE) activities in rats exposed to both acephate and methamidophos. In vitro addition of acephate to AChE prior to or with methamidophos also provided complete protection against AChE inhibition by methamidophos. When acephate was added to the enzyme after methamidophos, its protective effect decreased with increasing time between the additions. Since acephate has greater affinity than does methamidophos for the AChE active site (Singh, A.K., Toxicol. Appl. Pharmacol., 81 (1985) 302), it is proposed that acephate provided protection by binding with the AChE active site and, therefore, preventing methamidophos from binding with the enzyme. It is also proposed that acephate prevented the initial competitive binding of methamidophos to the AChE active site and delayed the initial sequence of events essential for phosphorylation of AChE.     

In vitro metabolism of mirtazapine enantiomers by human cytochrome P450 enzymes

The metabolism of mirtazapine enantiomers was investigated in vitro using human lymphoblast microsomes transfected with human cDNA to overexpress either CYP1A2, CYP2C9, CYP2C19, CYP2D6 or CYP3A4 and assayed for mirtazapine enantiomers using a validated chiral method of high-performance liquid chromatography. (+)-Mirtazapine was extensively metabolised by CYP2D6 (K(m) = 9.3 +/- 3.3 &mgr;mol/l, V(max) = 40.9 +/- 7.9 &mgr;mol/h/mg, intrinsic clearance = 4.41 l/h/mg). CYP1A2 and CYP3A4 showed low metabolic activity towards (+)-mirtazapine and (-)-mirtazapine respectively. Neither CYP2C9 nor CYP2C19 appeared to be involved in the metabolism of the enantiomers of mirtazapine. Copyright 2001 John Wiley & Sons, Ltd.     

The inhibition of the high sensitive peripheral nerve soluble esterases by mipafox. A new mathematical processing for the kinetics of inhibition of esterases by organophosphorus compounds

In the study of organophosphorus (OP) sensitive enzymes, careful discrimination of specific components within a complex multienzymatic mixture is needed. However, standard kinetic analysis gives inconsistent results (i.e., apparently different kinetic constants at different inhibitor concentration) with complex multienzymatic mixtures. A strategy is now presented to obtain consistent kinetic parameters. In the peripheral nerve, soluble carboxylesterases measured with the substrate phenylvalerate (PV) are found with extremely high sensitivity to some inhibitors. Tissue preparations were preincubated with mipafox at nanomolar concentrations (up to 100 nM) for different inhibition times (up to 180 min). Inhibition data were analyzed with model equations of one or two sensitive (exponential) components, with or without resistant components. The most complex model was %act=A1e-k1It+A2e-k2It+AR (step 1). From the curve with the highest mipafox concentration (100 nM), the amplitude for the resistant component was determined as AR=15.1% (step 2). The model equation with a fixed AR value was again applied (step 3) to deduce the second-order inhibition rate constants (k1=2.6 x 10(6) M-1 min-1 and k2=0.28 x 10(6) M-1 min-1), being conserved consistently throughout all mipafox concentrations. Finally, using fixed values of AR, k1, and k2, the amplitudes for the two exponential (sensitive) components (A1 and A2) were re-estimated (A1=50.2% and A2=34.2%). The operational process was internally validated by the close similarity with values obtained by directly fitting with a three-dimensional model equation (activity versus time and inhibitor concentration) to the same inhibition data. Carboxylesterase fractions separated by preparative chromatography showed kinetic properties consistent with the kinetically discriminated components. As practical conclusion, for routine analysis of esterases in toxicological studies, a simplified procedure using the inhibition with mipafox at 30 nM, 1 microM, and 1 mM for 30 min is suggested to discriminate the main esterase components in soluble fraction preparations.     

左旋和右旋奥硝唑对犬神经毒性的比较研究

目的 本实验进行了Beagle犬静脉输注奥硝唑及其光学对映体神经毒性的比较研究.方法 Beagle犬连续静脉给药2周,观察动物的一般状况和体重变化.给药结束时进行大体解剖,血液学和血液生化学,器官重量和组织病理学检查.结果 左旋奥硝唑组(200mg/kg,iv)Beagle犬仅在给药后1～3h内出现流涎、呕吐、不自主排便、排尿等症状;动物体重增长受到轻度抑制,进食量轻微减少,但与溶媒对照组无明显差异.右旋奥硝唑组(200mg/kg,iv)动物出现流涎、呕吐、四肢无力、不能站立,抽搐等毒性反应,随给药次数增加,毒性反应表现更加明显,且恢复时间延长,对动物体重及进食量有明显抑制.消旋奥硝唑组的毒性反应介于两者之间.各组的血液学和血液生化学检查结果以及器官重量无明显差异.组织学检查结果显示右旋体组犬小脑的蒲肯野细胞数目较溶媒对照组和左旋体组明显减少,细胞呈轻度变性.结论 在本试验条件下,左旋体组Beagle犬给药后的毒性反应比右旋体组轻.右旋体组犬小脑出现可见的蒲肯野细胞变性.结果提示,奥硝唑左旋体较右旋体毒性小,临床用药应更加安全.     

Mutual inhibition between carvedilol enantiomers during racemate glucuronidation mediated by human liver and intestinal microsomes

Carvedilol is administered orally as a racemic mixture of R(+)- and S(-)-enantiomers for treatment of angina pectoris, hypertension and chronic heart failure. We have reported that enzyme kinetic parameters for carvedilol glucuronidation by human liver microsomes (HLM) differed greatly depending on the substrate form, namely, racemic carvedilol and each enantiomer. These phenomena were thought to be caused by mutual inhibition between carvedilol enantiomers during racemate glucuronidation. The aim of this study was to clarify the mechanism of these phenomena in HLM and human intestinal microsomes (HIM) and its relevance to uridine 5'-diphosphate (UDP)-glucuronosyl transferase (UGT) 1A1, UGT2B4 and UGT2B7, which mainly metabolize carvedilol directly in phase II enzymes. HLM apparently preferred metabolizing (S)-carvedilol to (R)-carvedilol in the racemate, but true activities of HLM for both glucuronidation were approximately equal. By determination of the inhibitory effects of (S)-carvedilol on (R)-carvedilol glucuronidation and vice versa, it was shown that (R)-carvedilol glucuronidation was more easily inhibited than was (S)-carvedilol glucuronidation. UGT2B7 was responsible for (S)-carvedilol glucuronidation in HLM. Ratios of contribution to (R)-carvedilol glucuronidation were approximately equal among UGT1A1, UGT2B4 and UGT2B7. However, enzyme kinetic parameters were different between the two lots of HLM used in this study, depending on the contribution ratio of UGT2B4, in which (R)-glucuronidation was much more easily inhibited by (S)-carvedilol than was (S)-glucuronidation by (R)-carvedilol. Meanwhile, HIM preferred metabolizing (R)-carvedilol, and this tendency was not different between the kinds of substrate form.     

Comparative study on in vitro inhibition of grass carp (Ctenopharyngodon idellus) and mouse protein phosphatases by microcystins

Microcystins isolated from toxic cyanobacteria are potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A). The inhibitory effects of three structural variants of microcystins (microcystin‐LR, ‐YR, and ‐RR) on protein phosphatases isolated and purified from the liver and kidney of grass carp (Ctenopharyngodon idellus) were investigated using the ³²P radiometric assay. The relationships between percentage inhibition of protein phosphatase activity and microcystin levels followed a typical dose‐dependent sigmoid curve. These results were compared to those obtained from mouse PP2A. The degree and pattern of inhibition of both fish and mouse protein phosphatases by microcystins were similar. Protein phosphatases in crude fish tissue homogenates showed similar inhibition patterns as purified fish PP2A toward microcystins. © 2000 John Wiley & Sons, Inc. Environ Toxicol 15: 71–75, 2000     

Comparative effects of medetomidine enantiomers on in vitro and in vivo microsomal drug metabolism.

The effects of dexmedetomidine, a selective alpha 2-adrenoceptor agonist, and its levo enantiomer (MPV-1441), on in vitro microsomal P450-dependent drug-metabolizing activities as well as on in vivo aminopyrine elimination and hexobarbital sleeping time were studied. Both enantiomers inhibited the oxidative metabolism of several model substrates and testosterone in rat liver microsomal incubations. Microsomal activities derived from control animals or rats pretreated with phenobarbital were more sensitive to inhibitory effects of dexmedetomidine than those from rats treated with 3-methylcholanthrene. Enzyme activities in human liver microsomes were also inhibited by dexmedetomidine. Retardation of the elimination of aminopyrine was dose-dependent; elimination was marginally retarded with doses up to 100 micrograms/kg (from 17 to 23 min.; both enantiomers). Higher doses of the levo enantiomer prolonged aminopyrine half-life to 78 (1 mg/kg) and 162 min. (10 mg/kg). The hexobarbital sleeping time was prolonged by the dose of 1 mg/kg of the levo enantiomer (128 min. versus 20 min. in controls), while the dose of 0.1 mg/kg had no effect (23 versus 20 min.). These studies indicate that both enantiomers of medetomidine are inhibitors of microsomal drug metabolism in vitro, but significant effects on aminopyrine elimination or hexobarbital sleeping time are apparent only at doses, which do not allow the use of dexmedetomidine because of excessive sedative effect.     

In vitro inhibition of human UGT isoforms by ritonavir and cobicistat

1.?Ritonavir and cobicistat are pharmacokinetic boosting agents used to increase systemic exposure to other antiretroviral therapies. The manufacturer’s data suggests that cobicistat is a more selective CYP3A4 inhibitor than ritonavir. However, the inhibitory effect of ritonavir and cobicistat on human UDP glucuronosyltransferase (UGT) enzymes in Phase II metabolism is not established. This study evaluated the inhibition of human UGT isoforms by ritonavir versus cobicistat.

2.?Acetaminophen and ibuprofen were used as substrates to evaluate the metabolic activity of the principal human UGTs. Metabolite formation rates were determined by HPLC analysis of incubates following in vitro incubation of index substrates with human liver microsomes (HLMs) at different concentrations of ritonavir or cobicistat. Probenecid and estradiol served as positive control inhibitors.

3.?The 50% inhibitory concentrations (IC₅₀) of cobicistat and ritonavir were at least 50?µM, which substantially exceeds usual clinical plasma concentrations. Probenecid inhibited the glucuronidation of acetaminophen (IC₅₀ 0.7?mM), but not glucuronidation of ibuprofen. At relatively high concentrations, estradiol inhibited ibuprofen glucuronidation (IC₅₀ 17?µM).

4.?Ritonavir and cobicistat are unlikely to produce clinically important drug interactions involving drugs metabolized to glucuronide conjugates by UGT1A1, 1A3, 1A6, 1A9, 2B4 and 2B7.     

Different in vitro activity of flurbiprofen and its enantiomers on human articular cartilage

The 2-arylpropionic acid derivatives or 'profens' are an important group of non-steroidal anti-inflammatory drugs that have been used for the symptomatic treatment of various forms of arthritis. These compounds are chiral and the majority of them are still marketed as racemate although it is known that the (S)- form is the principal effective in the cyclooxygenase inhibition. However, recent findings suggest that certain pharmacological effect of 2-arylpropionic acids cannot be attributed exclusively to the (S)-(+) enantiomer. To obtain further insights into the pharmacological effect of profens, the present study investigated the influence of racemic and pure enantiomers of flurbiprofen on the production of nitric oxide and glycosaminoglycans, key molecules involved in cartilage destruction. The culture of human articular cartilage stimulated by interleukin-1beta (IL-1beta), which plays an important role in the degradation of cartilage, has been established, as a profit experimental model, for reproducing the mechanisms involved in the pathophysiology of arthritic diseases. Our results show that mainly (S)-(+)-flurbiprofen decreases, at therapeutically concentrations, the IL-1beta induced cartilage destruction.     

In vitro study of enzymatic hydrolysis of diperodon enantiomers in blood serum by two-dimensional LC

An on-line coupled HPLC system is described for the determination of the enantiomers of diperodon in blood serum. The method involves three steps: (i) off-line preconcentration and clean-up, (ii) separation of the diperodon enantiomers from the matrix components on a reversed-phase stationary phase, and (iii) separation of the racemate from the reversed-phase column on a teicoplanin chiral stationary phase. The method is suitable for simultaneous determination of both enantiomers in serum up to 0.5 microg/ml. The degradation of diperodon enantiomers was studied in serum by an in vitro method and the experimental rate constants were determined. The enantiomeric hydrolysis rates and half-lives for diperodon in serum are different.     

Terfenadine-antidepressant interactions: an in vitro inhibition study using human liver microsomes

AIMS: Inhibition of the metabolism of terfenadine has been associated with torsades de pointes ventricular arrhythmias. The aim of this study was to assess in vitro the potency of the antidepressants nefazodone, sertraline and fluoxetine in inhibiting terfenadine biotransformation. METHODS: Human liver microsomes were incubated with terfenadine and the antidepressants at various concentrations. Formation of the two major metabolites of terfenadine was determined by h.p.l.c. RESULTS: The apparent Km for microsomes from four human livers was 11+/-5 and 18+/-3 microM (mean +/-s.e.mean) for the N-dealkylation and C-hydroxylation pathways, respectively. Nefazodone, sertraline and fluoxetine inhibited terfenadine N-dealkylation with K(i) values of 10+/-4, 10+/-3 and 68+/-15 microM respectively. Inhibition of the C-hydroxylation pathway yielded noncompetitive K(i) values of 41+/-4, 67+/-13 and 310+/-40 microM respectively. CONCLUSIONS: Nefazodone and sertraline were moderately weak in vitro inhibitors of terfenadine metabolism while fluoxetine was a very weak inhibitor. Clinically significant interaction of terfenadine is more likely with nefazodone than sertraline or fluoxetine since therapeutic plasma levels of nefazodone are comparatively higher.     

Comparative study of in vitro inhibition of activation of the classical and alternative pathways of human complement by the magnesium and sodium salts of the anti-inflammatory peptide N-acetyl-aspartyl-glutamic acid (NAAGA)

The inhibitory activity of the sodium salt of the anti-inflammatory peptide N-acetyl-aspartyl-glutamic acid (NAAGA) on activation of the classical and alternative pathways of human complement was compared with that of the clinically used magnesium salt of NAAGA (NAAGA-Mg). Sodium salt of NAAGA (NAAGA-Na) inhibited both pathways of activation in a dose-dependent manner at concentration ranging from 1 to 10 mM by acting on formation and/or function of the C3 convertases as shown by the inhibitory capacity of the peptide on the release of the C3 cleavage fragment C3b and C3a. NAAGA-Na was as effective as NAAGA-Mg in inhibiting classical pathway activation at concentration above 10 mM. NAAGA-Na was more effective than NAAGA-Mg in inhibiting the alternative pathway since the sodium salt did not interfere with Mg-dependent formation of the alternative pathway C3 convertase.     

Investigations of interactions of chlormezanone racemate and its enantiomers on human keratinocytes and human leucoytes in vitro

Chlormezanone is a centrally acting muscle relaxant introduced in human therapy as a racemic substance. The following investigation was performed in order to investigate whether the racemate and both enantiomers differ in their potential cytotoxicty in vitro. We investigated antiproliferative effects and cytotoxicity (PicoGreen and ATP assay) for human HaCaT keratinocytes, production of oxygen radicals (ROS) by human interleukin-3-stimulated leukocytes (Lucigenin assay) and production of sulfoleukotrienes (Cellular Antigen Stimulation Test - CAST) by human leukocytes. In the dosage range of 0.001 to 0.1 mg/ ml chlormezanone, no antiproliferative effects were measured with the racemate and both enantiomers. At 1.0 mg/ml, a decrease of proliferative activity was seen after 48 h incubation time of about 50% for the enantiomers and of about 80% for the racemate (PicoGreen) and 50% (enantiomers) or 21% (racemate) in the ATP assay, respectively. ROS production was significantly inhibited at concentrations < or =0.01 mg/ ml by the racemate and the (+)-enantiomer, whereas the (-)-enantiomer was less effective. There was no stimulation of sulfidoleukotrienes in human leukocytes by chlormezanone. Present data argue for absence of significant cytotoxicity against human HaCaT keratinocytes and a dose-dependent suppression of ROS production by human leukocytes that is not uniform among the racemate and its enantiomers.     

Effect of metoclopramide and ranitidine on the inhibition of human AChE by VX in vitro

The repeated misuse of highly toxic organophosphorus-type (OP) chemical warfare agents ('nerve agents') emphasizes the necessity for the development of effective medical countermeasures. The standard treatment with atropine and acetylcholinesterase (AChE) reactivators ('oximes') is considered to be ineffective with certain nerve agents due to low oxime efficacy. Therefore, pretreatment with carbamate-type compounds, e.g. pyridostigmine, was recommended to improve antidotal efficacy. Recently, the clinically used reversible AChE inhibitors metoclopramide (MCP) and ranitidine (RAN) were shown to exhibit some protective effect against the OP pesticide paraoxon in vitro and in vivo. The present study was undertaken to investigate a potential protective effect of MCP and RAN against inhibition of human AChE by the nerve agent VX (O-ethyl S-[2-(diisopropylamino)ethyl)methylphosphonothioate). Hemoglobin-free human erythrocyte membranes were incubated with various, human relevant MCP (0.5-2 microm) and RAN (0.5-5 microm) concentrations starting 1 min before addition of VX (1-40 nm). Both compounds failed to increase VX IC(50) values. In addition, human AChE was incubated with higher than human relevant therapeutic concentrations of MCP (1 microm-1 mm) and RAN (1 microm-2.0 mm) and inhibited by 40 nm VX. At concentrations higher than 100 microm MCP and RAN caused a concentration dependent increase of residual AChE activity 15 min after addition of VX. These data indicate that MCP and RAN may be ineffective in protecting human AChE against inhibition by the nerve agent VX at human relevant doses.