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Effects of pretreatment with 8018 on the toxicokinetics of soman in rabbits and distribution in mice
Life Sciences 73 (2003) 1053 – 1062 www.elsevier.com/locate/lifescie
Effects of pretreatment with 8018 on the toxicokinetics of soman in rabbits and distribution in mice Jin-Tong Li *, Jin-Xiu Ruan, Zhen-Qing Zhang, Shu-Lan Yuan, Wei-Dong Yu, Zheng-Yan Song Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850, People’s Republic of China Received 25 September 2002; accepted 10 February 2003
Abstract The effects of 8018 [3-(2V-phenyl-2V-cyclopentyl-2V-hydroxyl-ethoxy)quinuclidine] on the elimination of soman in rabbits blood and distribution in mice brain and diaphragm were investigated using the chirasil capillary gas chromatographic analysis method. In all experiments, the concentration of P(+)soman was below the detection limit ( < 0.1 ngmL 1). 8018 (1 mgkg 1, im, 10 min pre-treated) could significantly reduce the concentration of P(-)soman in rabbit blood from 53.6 F 13.3 to 26.2 F 9.70 ngmL 1 blood as compared to soman-treated control animal at 15 s following soman injection(43.2 Agkg 1, iv). Toxicokinetic parameters showed 8018 could increase clearance (CL(S)) from 20.8 F 1.54 to 38.2 F 15.3 mLkg 1s 1 and reduce AUC of P(-)soman from 2.08 F 0.151 to 1.30 F 0.564 mgsL 1. 8018 could reduce the concentration P(-)soman in diaphragm from 74.7, 70.5, 88.7 ngg 1 to 54.5 45.6, 50.0 ngg 1 at the time of 30, 90, 120 s after intoxication of soman subcutaneously vs. soman control respectively, but it had no influence on the concentration of free P(-)soman in brain. Isotope trace experiments showed that it could significantly increase the distribution amount of bound [3H]soman in mice plasma and small intestine during 0 –120 min after mice received [3H]soman (0.544 GBq119 Agkg 1, sc) compared to soman control group. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Soman; 8018(3-2(2-Cyclopentyl-2-Hydroxy-2-Phenylethoxy)); Toxicokinetics; Detoxification; Elimination; Distribution
* Corresponding author. Tel.: +86-10-6687-4610; fax: +86-10-6821-1656. E-mail address: [email protected] (J.-T. Li). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00371-0
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Introduction Soman (O-1,2,2-trimethylpropyl methylphosphonofluoridate) is an extremely toxic organphosphorus agent whose action is believed to interfere with the central and peripheral cholinergic nervous system and result in hyperactivity such as salivation, bradycardia, hypotension, muscle tremor, convulsion and respiratory depression. Due to the presence of an asymmetry center at carbon in pinacoloyl moiety as well as the phosphorus atom, four steroisomers can be designated as C(+)P(+), C(+)P(-), C(-)P(+), C(-)P(-). These isomers possess widely different toxicological and toxicokinetic properties (Benschop et al., 1984; Langenberg et al., 1997). After absorption through respiratory tract or skin, soman could distribute to brain and diaphragm and other important target organs to exert its toxic effects through pulmonary circulation and system circulation. The defensive mechanisms of the organism occur at same time while soman poisoning. The relatively nontoxic P(+)-isomers are rapidly eliminated through hydrolyzing by A-esterase [also known as phosphorylphosphatases (EC 3.1.8.1)] in vivo. Otherwise, detoxification of high toxic P(-)-isomers are accomplished through several different reactions, including reactions with proteins, particularly with carboxylesterase (CaE; EC 3.1.1.1) and hydrolysis, distribution to tissue depots etc. (Benschop et al., 1984; Maxwell, 1992; De Jong et al., 1993). It had been postulated that only a little amount of soman was transported to target organs to inhibit functional acetylcholinesterase. The different organs contribute to detoxification process to an extent which is related to the blood flow through the organ. Soman could induce peripheral vasoconstriction and reduce blood to peripheral organs indicated that it was likely to exert the existing endogenous capacities for detoxification (Maxwell et al., 1987). Karlsson’s study and ours all showed that nimodipine, a calcium antagonist, whose reduce the initial concentration of soman in the blood was presumably by reducing the peripheral vasoconstriction and thereby increasing the peripheral blood flow through the detoxifying organs such as liver and small intestine (Karlsson et al., 1994, 1997; Li et al., 2002a). Oxime in combination with anticholinergic drugs are considered to be the standard antidotal treatment of soman toxicity currently, but the rapid aging of soman-phosphonylated AChE results in low reactivating rate of oxime, therefore, the anticholinergic drugs may play very important role in anti-toxicity of soman (Shih et al., 1993; Kassa, 1995; Kassa and Fusek, 1998, 2000). 8018, a new designed anticholinergic drug, shows more efficacy in counteracting with the toxicity of soman than atropine (Niu et al., 1990; Gao et al., 1990). Pretreatment with 8018 could also normalize the soman induced disturbance of cardiovascular system (pre-investigated in our institute, unpublished data). So, we were concerned whether 8018 could accelerate the elimination of soman in blood as nimodipine to provide to efficacy to a certain extent. Otherwise, it should be more significant in toxicology if 8018 could reduce toxicity of soman to important target organs such as brain and diaphragm. So the effects of 8018 on the elimination of soman in rabbits blood and distribution in mice brain and diaphragm were investigated by determining the concentration of P(-)soman using the chirasil capillary gas chromatographic analysis method in order to infer the metabolic detoxification of 8018 to soman. Furthermore, [3H]soman trace method was used to study the effect of 8018 on distribution of soman in mice to infer its metabolic detoxification to soman.
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Materials and methods Animals Nippon large-ear strain white rabbits and U, 2–2.5 kg; Kunming stain mice, U, 18–22 g, were provided by the Laboratory Animal Center of Academy Military Medical Sciences (Beijing, China), and were housed under standard laboratory conditions with free access to water and food. Chemicals and reagents Soman (O-1,2,2-trimethylpropylmethyl-phosphonofluoridate), [3H]Soman (more than 95% pure by thin-layer chromatography, TLC, 839.9 GBqmmol 1, labeled in pinacolyl) and DFP (Diisopropyl Phosphorofluoridate) was 96% pure by gas chromatography, 8018 (3-2(2-Cyclopentyl-2-Hydroxy-2Phenylethoxy), greater than 99% purity, were all obtained from Beijing Institute of Pharmacology and Toxicology (Beijing, China). n-pentane, diethyl ether, pentobarbitone sodium and perchloric acid were of analytical grade and all from Beijing Chemical Reagent Company (Beijing, China). 2,5-diphenyloxazole (PPO) and 1,4-bis-[5-phenyl oxazoyl-2]-benzene (POPOP) were purchased from Sigma. The structure of 8018 and soman (labeled site of [3H]) are showed as below.
(1) Chemical structure of 8018 (C20H29NO2 HCl, FW 351.9).
(2) Chemical structure of soman and the labeled site of [3H] (C7H16 FP O2, FW 182.2) Animal experiments The rabbits were anaesthetized with intravenous penobarbitone, 30 mgkg 1. A catheter was introduced into the carotid artery and the animals were tracheostomized to keep the upper airway free.
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8018 (1 mgmL 1 dissolved in saline, 1 mgkg 1, which is effective pharmacological dose) or saline was injected intramuscularly 10 min before C( F )P( F )soman (43.2 Agkg 1, equivalent to 4 LD50 on rabbits) intoxication via the marginal ear vein. 1 mL blood samples were withdrawn from the carotid artery in 15, 30, 60, 90, 120, 180, 240, 300 s after soman administration respectively for determined of free soman concentration. In mice distribution experiment, 8018 (1 mgkg 1 dissolved in saline) or saline was injected ip 10 min before mice received C( F )P( F )soman via sc administration (1 mgkg 1, equivalent to 5 LD50 on mice). 30 s, 90 s and 120 s after intoxication with soman, mice were decapitated,brain and diaphragm were removed, weighed, and homogenized with a high-speed homogenizer in 2 ml ice-cold perchloric acid (1 molL 1) for 2 min and free soman concentration in tissue homogenates were determined. In isotope trace experiment, 8018 (1 mgkg 1 dissolved in saline) or saline was injected ip 10 min before administered [3H]soman (0.544 GBq119 Agkg 1, equivalent to 0.6 LD50 on mice, dissolved in saline) sc in the neck. 10 min, 30 min, 1 h and 2 h after treatment, mice were decapitated and blood samples from the cervical wound were collected into heparinized tubes and centrifuged at 1000 g. 100–150 mg tissue were removed, weighed, and homogenized with a high-speed homogenizer in 600 AL ice-cold saline for 2 min, homogenate were centrifuged at 8000 g for 10 min in a refrigerated centrifuge (0–4 jC) and supernatants were removed, the pellets were resuspended in 600 AL ice-cold saline and centrifuged again. These procedure were performed for 3 times. After the last centrifugation, the pellets were resuspended in 100 AL ice-cold saline and dropped to the small round filter paper uniformly. The filter papers were dried and added to spectrofluor 0.5% PPO and 0.02% POPOP in xylene for determination of bound [3H]soman concentration by liquid scintillation spectrometry (L1409, Wallac company). Counting efficiency was determined by external standardization. Work-up procedure and gas chromatography Blood samples and tissue homogenates for gas chromatographic analysis of soman isomers were stabilized and worked up as described before (Li et al., 2001a,b). The instructive summary of processing of blood samples before analysis and measurement procedure was given below: Extraction and sample preparation 1 mL blood samples as well as 2 ml tissue homogenates were immediately mixed with 0.5 mL icecold perchloric acid (1 molL 1) and 50 ng DFP (prepared in hexane and stored at 20 jC) was added as internal standard. The samples were centrifuged at 10 000 g for 10 min using a TLL-C refrigerated centrifuge (Beijing Sihuan Science Instrument Factory) at 4 jC–0 jC. The supernatants were extracted with 2 mL n-pentane: diethyl ether (5/1, V/V) twice, the extracts were concentrated to 200 AL for GC analysis. Blood samples for blank and recovery experiments were taken immediately before the start of the experiments. Gas chromatography Gas chromatography was performed on a Hewlett-Packard 6890 gas chromatograph equipped with a large volume injector (Injection volume: 50 AL 3, using programmable temperature vaporization technique, isothermally at 50 jC for 0.6 min, the increasing at 700 jC/min to 180 jC and holding for 5 min), Alltech Chirasil-Val analytical column(length, 50 m; i.d., 0.32 mm; film thickness, 0.2 Am) and a NP detector were used. For each chromatographic run, samples were
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introduced by splitless injection. Oven temperature program is as follow: isothermal at 60 jC for 1.6 min, then increasing at 5 jC/min to 90 jC and holding for 10 min, then increasing at 5 jC/min to 130 jC and holding for 1 min, at last step, increasing at 10 jC/min to 200 jC and holding for 1 min. Detector temperature was held at 250 jC and the carrier gas helium was used at a flow of 1 mL/min, whereas flow of air and hydrogen through the detector were 40 and 3.5 mL/min respectively. Under these conditions, The four stereoisomers of standard racemic soman were well separated and the retention time for the soman steroisomers was 19–20 min. Peak areas were measured with Hewlett-Packard chemical station. Following the iv or ip injection of soman, only the C( F )P(-)soman was detected during the first few minutes after administration in our study. So we used a pair of C( F )P(-)soman (Isolation of C( F )P(-)soman was according to Benschop et al in 1984)on making the calibration curve with rabbit blood and mice tissue homogenates. The linearity of the calibration curves was well (R>0.999) in which the concentration range was 1 f 100 AgL 1 blood and 0.25–5 AgL 1 tissue homogenates respectively. The lower limit of detection was 0.1 AgL 1 blood or tissue homogenates. The precision was less than 10% and averaged percentage extraction recovery was 55% approximately. Data analysis and statistical evaluation The 3p87 pharmacokinetic program (Chinese Pharmacological Society) was used to generate the toxicokinetic parameters for soman iv administration. The area under the curve [AUC(0 – 300 s)] for C( F )P(-)soman in rabbits plasma, AUC(0 – 120 min) for bound [3H]soman in mice plasma, and different tissue were calculated by the linear trapezoidal rule to the last blood or tissue concentration. The concentration of bound [3H]soman in tissue per 100 mg was a relative value which compared to each experiment’s bound [3H]soman counting in 100 AL plasma of soman control group, so AUC(0 – 120 min) for bound [3H]soman in mice plasma and different tissue didn’t
Fig. 1. Influence of 8018 (1 mgkg 1, im, 10 min, pre-treated) on elimination of free P(-)soman in rabbit blood after injection of soman (43.2 Agkg 1, iv). **p < 0.01 vs. soman control. x¯ F s.d., n = 4 – 6. n-indicated soman control; 5-indicated 8018 pretreated + soman.
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Table 1 Influence of 8018 (1 mgkg 1, im, 10 min, pre-treated) on toxicokinetic parameters of free P(-)soman in rabbit blood after injection of soman (43.2 Agkg 1, iv) Toxicokinetic parameters -1
Vd/Lkg t1/2a/s t1/2h/s AUC(0 – 300 s)/mgsL CL(S)/mLkg 1s 1
1
Soman 2.14 4.46 71.9 2.08 20.8
F F F F F
Penehyclidine hydrochloride + Soman 0.52 3.17 20.9 0.154 1.51
3.83 19.5 78.6 1.30 38.2
F F F F F
2.74 21.7 69.1 0.564* 15.3*
* p < 0.01 vs soman control, x¯ F s.d., n = 4 – 6.
have unit. The unpaired Student’s t-test was used for the comparisons of 8018-pretreated group and soman control group. Results Soman intoxication induced hyperactivity such as miosis, salivation, fasciculation, muscle tremors and convulsions, and respiratory failure through interfering with the cholinergic nervous system were observed soon after soman injection in soman intoxication control. Pretreatment with 8018 ether via im to rabbits or via ip to mice, the intoxication signs described above were all significant alleviate at certain. The effects of 8018 on the elimination of P(-)soman in rabbit blood after intoxication of C(F)P(F)soman via iv administration 15–300 s after rabbits were received C( F )P( F )soman via iv administration, P(+)soman was below the detection limit( < 0.1 ngmL 1), otherwise, the toxicokinetics of P(-)soman in rabbit could
Fig. 2. Influence of 8018 (1mgkg 1, ip, 10 min, pre-treated) on concentration of free P(-)soman in mice diaphragm after intoxication with soman via sc (1mgkg 1). **p < 0.01 vs. Soman control. x¯ F s, n = 5. n-indicated soman control; 5-8018 pre-treated + soman.
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Fig. 3. Influence of 8018 (1mgkg 1, ip, 10 min, pre-treated) on concentration of free P(-)soman in mice brain after intoxication with soman via sc (1mgkg 1). x¯ F s.d., n = 5. n-indicated soman control; 5-indicated 8018 pre-treated + soman.
be described by a two-compartment model, first a fast distribution phase, then a slow elimination phase. 8018 (1 mgkg 1 im, 10 min. pretreated) could significantly reduce the concentration of P(-)soman in rabbit blood from 53.6 F 13.3 to 26.2 F 9.70 ngml 1 blood as compared to C( F )P( F )somantreated rabbits at 15 s following soman injection. But during 30 s to 300 s after soman intoxification, 8018 didn’t significantly influence the concentration of P(-)soman in rabbit blood compared to soman control group(see Fig. 1).
Fig. 4. Influence of 8018 (1mgkg 1, ip, 10 min, pre-treated) on AUC(0 – 120 min) of bound [3H]soman in mice plasma and different tissues after injection of [3H]soman (0.544 GBq119 Agkg 1, sc). **p < 0.01 vs. soman control. x¯ F s.d., n = 5. n-indicated soman control; 5-indicated 8018 pre-treated + soman.
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Toxicokinetic parameters showed that: 8018 could significantly increase clearance rate(CL(S)) of P(-)soman from 20.8 F 1.51 to 38.2 F 15.3 mLkg 1s 1 and reduce AUC of P(-)soman from 2.08 F 0.151 to 1.30 F 0.564 mgsL 1 as compared to soman-treated rabbits (see Table 1). The influence of 8018 on distribution of P(-)soman in mice brain and diaphragm Only P(-)soman was detected in mice brain and diaphragm after intoxication of C( F )P( F ) soman(1 mgkg 1) via sc administration. and the amount of free P(-)soman per gram tissue distributed in diaphragm was 10 times as high as distributed in brain. 8018 (1 mgkg 1 ip, 10 min pretreated) could reduce the concentration P(-)soman in diaphragm from 74.7, 70.5, 88.7 ngg 1 to 54.5, 45.6, 50.0 ngg 1 at the time of 30, 90, 120 s after intoxication of C( F )P( F )soman subcutaneously vs. soman control respectively(see Fig. 2), but it had no influence on the concentration of free P(-)soman distributed in brain(see Fig. 3). The influence of 8018 on distribution of bound [3H]soman in mice different tissues 8018 treatment could significantly increase the tissue distribution of bound [3H]soman in mice plasma and small intestine after injection of [3H]soman (0.544 GBq119 Agkg 1, sc) as compared to soman control group (Fig. 4).
Discussion In all experiments, P(+)-isomers were rapidly eliminated through hydrolyzing by A-esterase and the concentration of them was below the detection limit following administration of C( F )P( F )soman via iv in rabbits or via sc in mice, only P(-)soman were detected during the first several minutes in our study. This was observed in many studies on soman’s toxicokinetics (Due et al., 1994; Langenberg et al., 1998). Soman was rapidly eliminated in vivo through different processes involving of metabolism detoxification of protein and enzyme located in blood and other detoxification tissues (Langenberg et al., 1997). The large differences in acute toxicity of C( F )P( F )soman in various species appear to be due primarily to interspecies difference in the amounts of CaE in plasma (Maxwell, 1992; Maxwell and Brecht, 2001). In present experiment, mice have a high LD50 (200 Agkg 1, sc), while rabbits have a relatively lower LD50 (10.8 Agkg 1, iv). These relative toxicities correlate with the relative amounts of CaE in plasma of two species. at the lower dosing (0.8 LD50) intoxication of soman via iv administration, P(-) soman was rapidly eliminated mainly by reacting with covalent binding sites in blood during the rapid distribution phase according with the concept for a highly reactive toxicant on a first come, first served basis (Benschop and De Jong, 1991; Langenberg et al., 1997). But at the high dosing (2–6 LD50) intoxication of soman via iv administration, while the dose of P(-) soman exceeds the capacity of blood binding sites, large blood flow volume tissues such as liver and intestine played a very important role in elimination the free P(-)soman in blood maybe through hydrolyzing effect of A-esterase and combination effect of CaE (Langenberg et al., 1997; Li et al., 2002b). So, biodisposition and metabolism play important role in the toxicity and detoxification of soman (Reynolds et al., 1985). Simultaneously, soman could cause a disturbance of cardiovascular system which could affect the detoxifying processes. As described before, the acetylcholine accumulation of soman intoxication could induce an increase in
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total peripheral vascular resistance which can affect the blood flow in different organs, soman intoxication could also increase the blood pressure and heart rate in rat (Kentera et al., 1982; Maxwell et al., 1987). It was conceivable that the effects of decreased blood flow in different tissues especially in detoxification tissues which was induced by soman intoxication maybe result in a lower ability to degrade the soman. If the effect on the cardiovascular system could be diminished and the circulation normalized, the more soman distributed in peripheral detoxification tissue, the more soman could be detoxified. 8018 pretreatment could antagonize the toxicity of soman mainly though it’s relative strong central and peripheral anticholinergic effects to soman (pre-investigated in our institute, unpublished data). In present study, it could significantly reduce the concentration of P(-)soman in rabbit blood at 15 s following soman injection as similar as nimodipine described before (Karlsson et al., 1994; 1997; Li et al., 2002a). 15 s after soman injection intravenously was just in the fast distribution phase, so it was presumed that 8018 could alter the distribution of soman by reducing the peripheral vasoconstriction and thereby increase the peripheral blood flow through the detoxifying organs such as small intestine which contained abundant CaE, the very important detoxifying enzyme to soman to accelerate the soman’s elimination. The presumption was convinced in our isotope trace that 8018 treatment could significantly increase the tissue distribution of bound [3H]soman in small intestine. The increased mice plasma bound [3H]soman maybe due to it’s rise the availability of covalent detoxification enzyme in plasma which needs further research to confirm. Inhibition of functional AChE located in target tissues such as brain and diaphragm is very crucial to soman induced toxicity, so, the concentration of high toxic P(-)soman in these tissue could reflect the intoxication degree of soman at some extent. In this study, the concentration of free P(-)soman per g tissue distributed in diaphragm was 10 times as high as distributed in brain, This phenomena was observed by Reynolds using [3H]soman trace method (Reynolds et al., 1985). 8018 could reduce the distribution of P(-)soman in diaphragm following soman intoxication via sc in our study which had toxicology significance, but 8018 didn’t influence the concentration of free P(-)soman in brain. In conclusion, other than the anticholinergic effects to soman, 8018 could accelerated the elimination of P(-)soman in the rabbits blood and reduced the distribution of P(-)soman in the mice diaphragm. Moreover, it could significantly increase bound [3H]soman distribution in small intestine as well as in plasma. More details and exact mechanisms of 8018’s antitoxicity of soman await deeply investigation.
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Effects of pretreatment with 8018 on the toxicokinetics of soman in rabbits and distribution in mice Jin-Tong Li *, Jin-Xiu Ruan, Zhen-Qing Zhang, Shu-Lan Yuan, Wei-Dong Yu, Zheng-Yan Song Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850, People’s Republic of China Received 25 September 2002; accepted 10 February 2003
Abstract The effects of 8018 [3-(2V-phenyl-2V-cyclopentyl-2V-hydroxyl-ethoxy)quinuclidine] on the elimination of soman in rabbits blood and distribution in mice brain and diaphragm were investigated using the chirasil capillary gas chromatographic analysis method. In all experiments, the concentration of P(+)soman was below the detection limit ( < 0.1 ngmL 1). 8018 (1 mgkg 1, im, 10 min pre-treated) could significantly reduce the concentration of P(-)soman in rabbit blood from 53.6 F 13.3 to 26.2 F 9.70 ngmL 1 blood as compared to soman-treated control animal at 15 s following soman injection(43.2 Agkg 1, iv). Toxicokinetic parameters showed 8018 could increase clearance (CL(S)) from 20.8 F 1.54 to 38.2 F 15.3 mLkg 1s 1 and reduce AUC of P(-)soman from 2.08 F 0.151 to 1.30 F 0.564 mgsL 1. 8018 could reduce the concentration P(-)soman in diaphragm from 74.7, 70.5, 88.7 ngg 1 to 54.5 45.6, 50.0 ngg 1 at the time of 30, 90, 120 s after intoxication of soman subcutaneously vs. soman control respectively, but it had no influence on the concentration of free P(-)soman in brain. Isotope trace experiments showed that it could significantly increase the distribution amount of bound [3H]soman in mice plasma and small intestine during 0 –120 min after mice received [3H]soman (0.544 GBq119 Agkg 1, sc) compared to soman control group. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Soman; 8018(3-2(2-Cyclopentyl-2-Hydroxy-2-Phenylethoxy)); Toxicokinetics; Detoxification; Elimination; Distribution
* Corresponding author. Tel.: +86-10-6687-4610; fax: +86-10-6821-1656. E-mail address: [email protected] (J.-T. Li). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00371-0
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Introduction Soman (O-1,2,2-trimethylpropyl methylphosphonofluoridate) is an extremely toxic organphosphorus agent whose action is believed to interfere with the central and peripheral cholinergic nervous system and result in hyperactivity such as salivation, bradycardia, hypotension, muscle tremor, convulsion and respiratory depression. Due to the presence of an asymmetry center at carbon in pinacoloyl moiety as well as the phosphorus atom, four steroisomers can be designated as C(+)P(+), C(+)P(-), C(-)P(+), C(-)P(-). These isomers possess widely different toxicological and toxicokinetic properties (Benschop et al., 1984; Langenberg et al., 1997). After absorption through respiratory tract or skin, soman could distribute to brain and diaphragm and other important target organs to exert its toxic effects through pulmonary circulation and system circulation. The defensive mechanisms of the organism occur at same time while soman poisoning. The relatively nontoxic P(+)-isomers are rapidly eliminated through hydrolyzing by A-esterase [also known as phosphorylphosphatases (EC 3.1.8.1)] in vivo. Otherwise, detoxification of high toxic P(-)-isomers are accomplished through several different reactions, including reactions with proteins, particularly with carboxylesterase (CaE; EC 3.1.1.1) and hydrolysis, distribution to tissue depots etc. (Benschop et al., 1984; Maxwell, 1992; De Jong et al., 1993). It had been postulated that only a little amount of soman was transported to target organs to inhibit functional acetylcholinesterase. The different organs contribute to detoxification process to an extent which is related to the blood flow through the organ. Soman could induce peripheral vasoconstriction and reduce blood to peripheral organs indicated that it was likely to exert the existing endogenous capacities for detoxification (Maxwell et al., 1987). Karlsson’s study and ours all showed that nimodipine, a calcium antagonist, whose reduce the initial concentration of soman in the blood was presumably by reducing the peripheral vasoconstriction and thereby increasing the peripheral blood flow through the detoxifying organs such as liver and small intestine (Karlsson et al., 1994, 1997; Li et al., 2002a). Oxime in combination with anticholinergic drugs are considered to be the standard antidotal treatment of soman toxicity currently, but the rapid aging of soman-phosphonylated AChE results in low reactivating rate of oxime, therefore, the anticholinergic drugs may play very important role in anti-toxicity of soman (Shih et al., 1993; Kassa, 1995; Kassa and Fusek, 1998, 2000). 8018, a new designed anticholinergic drug, shows more efficacy in counteracting with the toxicity of soman than atropine (Niu et al., 1990; Gao et al., 1990). Pretreatment with 8018 could also normalize the soman induced disturbance of cardiovascular system (pre-investigated in our institute, unpublished data). So, we were concerned whether 8018 could accelerate the elimination of soman in blood as nimodipine to provide to efficacy to a certain extent. Otherwise, it should be more significant in toxicology if 8018 could reduce toxicity of soman to important target organs such as brain and diaphragm. So the effects of 8018 on the elimination of soman in rabbits blood and distribution in mice brain and diaphragm were investigated by determining the concentration of P(-)soman using the chirasil capillary gas chromatographic analysis method in order to infer the metabolic detoxification of 8018 to soman. Furthermore, [3H]soman trace method was used to study the effect of 8018 on distribution of soman in mice to infer its metabolic detoxification to soman.
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Materials and methods Animals Nippon large-ear strain white rabbits and U, 2–2.5 kg; Kunming stain mice, U, 18–22 g, were provided by the Laboratory Animal Center of Academy Military Medical Sciences (Beijing, China), and were housed under standard laboratory conditions with free access to water and food. Chemicals and reagents Soman (O-1,2,2-trimethylpropylmethyl-phosphonofluoridate), [3H]Soman (more than 95% pure by thin-layer chromatography, TLC, 839.9 GBqmmol 1, labeled in pinacolyl) and DFP (Diisopropyl Phosphorofluoridate) was 96% pure by gas chromatography, 8018 (3-2(2-Cyclopentyl-2-Hydroxy-2Phenylethoxy), greater than 99% purity, were all obtained from Beijing Institute of Pharmacology and Toxicology (Beijing, China). n-pentane, diethyl ether, pentobarbitone sodium and perchloric acid were of analytical grade and all from Beijing Chemical Reagent Company (Beijing, China). 2,5-diphenyloxazole (PPO) and 1,4-bis-[5-phenyl oxazoyl-2]-benzene (POPOP) were purchased from Sigma. The structure of 8018 and soman (labeled site of [3H]) are showed as below.
(1) Chemical structure of 8018 (C20H29NO2 HCl, FW 351.9).
(2) Chemical structure of soman and the labeled site of [3H] (C7H16 FP O2, FW 182.2) Animal experiments The rabbits were anaesthetized with intravenous penobarbitone, 30 mgkg 1. A catheter was introduced into the carotid artery and the animals were tracheostomized to keep the upper airway free.
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8018 (1 mgmL 1 dissolved in saline, 1 mgkg 1, which is effective pharmacological dose) or saline was injected intramuscularly 10 min before C( F )P( F )soman (43.2 Agkg 1, equivalent to 4 LD50 on rabbits) intoxication via the marginal ear vein. 1 mL blood samples were withdrawn from the carotid artery in 15, 30, 60, 90, 120, 180, 240, 300 s after soman administration respectively for determined of free soman concentration. In mice distribution experiment, 8018 (1 mgkg 1 dissolved in saline) or saline was injected ip 10 min before mice received C( F )P( F )soman via sc administration (1 mgkg 1, equivalent to 5 LD50 on mice). 30 s, 90 s and 120 s after intoxication with soman, mice were decapitated,brain and diaphragm were removed, weighed, and homogenized with a high-speed homogenizer in 2 ml ice-cold perchloric acid (1 molL 1) for 2 min and free soman concentration in tissue homogenates were determined. In isotope trace experiment, 8018 (1 mgkg 1 dissolved in saline) or saline was injected ip 10 min before administered [3H]soman (0.544 GBq119 Agkg 1, equivalent to 0.6 LD50 on mice, dissolved in saline) sc in the neck. 10 min, 30 min, 1 h and 2 h after treatment, mice were decapitated and blood samples from the cervical wound were collected into heparinized tubes and centrifuged at 1000 g. 100–150 mg tissue were removed, weighed, and homogenized with a high-speed homogenizer in 600 AL ice-cold saline for 2 min, homogenate were centrifuged at 8000 g for 10 min in a refrigerated centrifuge (0–4 jC) and supernatants were removed, the pellets were resuspended in 600 AL ice-cold saline and centrifuged again. These procedure were performed for 3 times. After the last centrifugation, the pellets were resuspended in 100 AL ice-cold saline and dropped to the small round filter paper uniformly. The filter papers were dried and added to spectrofluor 0.5% PPO and 0.02% POPOP in xylene for determination of bound [3H]soman concentration by liquid scintillation spectrometry (L1409, Wallac company). Counting efficiency was determined by external standardization. Work-up procedure and gas chromatography Blood samples and tissue homogenates for gas chromatographic analysis of soman isomers were stabilized and worked up as described before (Li et al., 2001a,b). The instructive summary of processing of blood samples before analysis and measurement procedure was given below: Extraction and sample preparation 1 mL blood samples as well as 2 ml tissue homogenates were immediately mixed with 0.5 mL icecold perchloric acid (1 molL 1) and 50 ng DFP (prepared in hexane and stored at 20 jC) was added as internal standard. The samples were centrifuged at 10 000 g for 10 min using a TLL-C refrigerated centrifuge (Beijing Sihuan Science Instrument Factory) at 4 jC–0 jC. The supernatants were extracted with 2 mL n-pentane: diethyl ether (5/1, V/V) twice, the extracts were concentrated to 200 AL for GC analysis. Blood samples for blank and recovery experiments were taken immediately before the start of the experiments. Gas chromatography Gas chromatography was performed on a Hewlett-Packard 6890 gas chromatograph equipped with a large volume injector (Injection volume: 50 AL 3, using programmable temperature vaporization technique, isothermally at 50 jC for 0.6 min, the increasing at 700 jC/min to 180 jC and holding for 5 min), Alltech Chirasil-Val analytical column(length, 50 m; i.d., 0.32 mm; film thickness, 0.2 Am) and a NP detector were used. For each chromatographic run, samples were
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introduced by splitless injection. Oven temperature program is as follow: isothermal at 60 jC for 1.6 min, then increasing at 5 jC/min to 90 jC and holding for 10 min, then increasing at 5 jC/min to 130 jC and holding for 1 min, at last step, increasing at 10 jC/min to 200 jC and holding for 1 min. Detector temperature was held at 250 jC and the carrier gas helium was used at a flow of 1 mL/min, whereas flow of air and hydrogen through the detector were 40 and 3.5 mL/min respectively. Under these conditions, The four stereoisomers of standard racemic soman were well separated and the retention time for the soman steroisomers was 19–20 min. Peak areas were measured with Hewlett-Packard chemical station. Following the iv or ip injection of soman, only the C( F )P(-)soman was detected during the first few minutes after administration in our study. So we used a pair of C( F )P(-)soman (Isolation of C( F )P(-)soman was according to Benschop et al in 1984)on making the calibration curve with rabbit blood and mice tissue homogenates. The linearity of the calibration curves was well (R>0.999) in which the concentration range was 1 f 100 AgL 1 blood and 0.25–5 AgL 1 tissue homogenates respectively. The lower limit of detection was 0.1 AgL 1 blood or tissue homogenates. The precision was less than 10% and averaged percentage extraction recovery was 55% approximately. Data analysis and statistical evaluation The 3p87 pharmacokinetic program (Chinese Pharmacological Society) was used to generate the toxicokinetic parameters for soman iv administration. The area under the curve [AUC(0 – 300 s)] for C( F )P(-)soman in rabbits plasma, AUC(0 – 120 min) for bound [3H]soman in mice plasma, and different tissue were calculated by the linear trapezoidal rule to the last blood or tissue concentration. The concentration of bound [3H]soman in tissue per 100 mg was a relative value which compared to each experiment’s bound [3H]soman counting in 100 AL plasma of soman control group, so AUC(0 – 120 min) for bound [3H]soman in mice plasma and different tissue didn’t
Fig. 1. Influence of 8018 (1 mgkg 1, im, 10 min, pre-treated) on elimination of free P(-)soman in rabbit blood after injection of soman (43.2 Agkg 1, iv). **p < 0.01 vs. soman control. x¯ F s.d., n = 4 – 6. n-indicated soman control; 5-indicated 8018 pretreated + soman.
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Table 1 Influence of 8018 (1 mgkg 1, im, 10 min, pre-treated) on toxicokinetic parameters of free P(-)soman in rabbit blood after injection of soman (43.2 Agkg 1, iv) Toxicokinetic parameters -1
Vd/Lkg t1/2a/s t1/2h/s AUC(0 – 300 s)/mgsL CL(S)/mLkg 1s 1
1
Soman 2.14 4.46 71.9 2.08 20.8
F F F F F
Penehyclidine hydrochloride + Soman 0.52 3.17 20.9 0.154 1.51
3.83 19.5 78.6 1.30 38.2
F F F F F
2.74 21.7 69.1 0.564* 15.3*
* p < 0.01 vs soman control, x¯ F s.d., n = 4 – 6.
have unit. The unpaired Student’s t-test was used for the comparisons of 8018-pretreated group and soman control group. Results Soman intoxication induced hyperactivity such as miosis, salivation, fasciculation, muscle tremors and convulsions, and respiratory failure through interfering with the cholinergic nervous system were observed soon after soman injection in soman intoxication control. Pretreatment with 8018 ether via im to rabbits or via ip to mice, the intoxication signs described above were all significant alleviate at certain. The effects of 8018 on the elimination of P(-)soman in rabbit blood after intoxication of C(F)P(F)soman via iv administration 15–300 s after rabbits were received C( F )P( F )soman via iv administration, P(+)soman was below the detection limit( < 0.1 ngmL 1), otherwise, the toxicokinetics of P(-)soman in rabbit could
Fig. 2. Influence of 8018 (1mgkg 1, ip, 10 min, pre-treated) on concentration of free P(-)soman in mice diaphragm after intoxication with soman via sc (1mgkg 1). **p < 0.01 vs. Soman control. x¯ F s, n = 5. n-indicated soman control; 5-8018 pre-treated + soman.
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Fig. 3. Influence of 8018 (1mgkg 1, ip, 10 min, pre-treated) on concentration of free P(-)soman in mice brain after intoxication with soman via sc (1mgkg 1). x¯ F s.d., n = 5. n-indicated soman control; 5-indicated 8018 pre-treated + soman.
be described by a two-compartment model, first a fast distribution phase, then a slow elimination phase. 8018 (1 mgkg 1 im, 10 min. pretreated) could significantly reduce the concentration of P(-)soman in rabbit blood from 53.6 F 13.3 to 26.2 F 9.70 ngml 1 blood as compared to C( F )P( F )somantreated rabbits at 15 s following soman injection. But during 30 s to 300 s after soman intoxification, 8018 didn’t significantly influence the concentration of P(-)soman in rabbit blood compared to soman control group(see Fig. 1).
Fig. 4. Influence of 8018 (1mgkg 1, ip, 10 min, pre-treated) on AUC(0 – 120 min) of bound [3H]soman in mice plasma and different tissues after injection of [3H]soman (0.544 GBq119 Agkg 1, sc). **p < 0.01 vs. soman control. x¯ F s.d., n = 5. n-indicated soman control; 5-indicated 8018 pre-treated + soman.
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Toxicokinetic parameters showed that: 8018 could significantly increase clearance rate(CL(S)) of P(-)soman from 20.8 F 1.51 to 38.2 F 15.3 mLkg 1s 1 and reduce AUC of P(-)soman from 2.08 F 0.151 to 1.30 F 0.564 mgsL 1 as compared to soman-treated rabbits (see Table 1). The influence of 8018 on distribution of P(-)soman in mice brain and diaphragm Only P(-)soman was detected in mice brain and diaphragm after intoxication of C( F )P( F ) soman(1 mgkg 1) via sc administration. and the amount of free P(-)soman per gram tissue distributed in diaphragm was 10 times as high as distributed in brain. 8018 (1 mgkg 1 ip, 10 min pretreated) could reduce the concentration P(-)soman in diaphragm from 74.7, 70.5, 88.7 ngg 1 to 54.5, 45.6, 50.0 ngg 1 at the time of 30, 90, 120 s after intoxication of C( F )P( F )soman subcutaneously vs. soman control respectively(see Fig. 2), but it had no influence on the concentration of free P(-)soman distributed in brain(see Fig. 3). The influence of 8018 on distribution of bound [3H]soman in mice different tissues 8018 treatment could significantly increase the tissue distribution of bound [3H]soman in mice plasma and small intestine after injection of [3H]soman (0.544 GBq119 Agkg 1, sc) as compared to soman control group (Fig. 4).
Discussion In all experiments, P(+)-isomers were rapidly eliminated through hydrolyzing by A-esterase and the concentration of them was below the detection limit following administration of C( F )P( F )soman via iv in rabbits or via sc in mice, only P(-)soman were detected during the first several minutes in our study. This was observed in many studies on soman’s toxicokinetics (Due et al., 1994; Langenberg et al., 1998). Soman was rapidly eliminated in vivo through different processes involving of metabolism detoxification of protein and enzyme located in blood and other detoxification tissues (Langenberg et al., 1997). The large differences in acute toxicity of C( F )P( F )soman in various species appear to be due primarily to interspecies difference in the amounts of CaE in plasma (Maxwell, 1992; Maxwell and Brecht, 2001). In present experiment, mice have a high LD50 (200 Agkg 1, sc), while rabbits have a relatively lower LD50 (10.8 Agkg 1, iv). These relative toxicities correlate with the relative amounts of CaE in plasma of two species. at the lower dosing (0.8 LD50) intoxication of soman via iv administration, P(-) soman was rapidly eliminated mainly by reacting with covalent binding sites in blood during the rapid distribution phase according with the concept for a highly reactive toxicant on a first come, first served basis (Benschop and De Jong, 1991; Langenberg et al., 1997). But at the high dosing (2–6 LD50) intoxication of soman via iv administration, while the dose of P(-) soman exceeds the capacity of blood binding sites, large blood flow volume tissues such as liver and intestine played a very important role in elimination the free P(-)soman in blood maybe through hydrolyzing effect of A-esterase and combination effect of CaE (Langenberg et al., 1997; Li et al., 2002b). So, biodisposition and metabolism play important role in the toxicity and detoxification of soman (Reynolds et al., 1985). Simultaneously, soman could cause a disturbance of cardiovascular system which could affect the detoxifying processes. As described before, the acetylcholine accumulation of soman intoxication could induce an increase in
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total peripheral vascular resistance which can affect the blood flow in different organs, soman intoxication could also increase the blood pressure and heart rate in rat (Kentera et al., 1982; Maxwell et al., 1987). It was conceivable that the effects of decreased blood flow in different tissues especially in detoxification tissues which was induced by soman intoxication maybe result in a lower ability to degrade the soman. If the effect on the cardiovascular system could be diminished and the circulation normalized, the more soman distributed in peripheral detoxification tissue, the more soman could be detoxified. 8018 pretreatment could antagonize the toxicity of soman mainly though it’s relative strong central and peripheral anticholinergic effects to soman (pre-investigated in our institute, unpublished data). In present study, it could significantly reduce the concentration of P(-)soman in rabbit blood at 15 s following soman injection as similar as nimodipine described before (Karlsson et al., 1994; 1997; Li et al., 2002a). 15 s after soman injection intravenously was just in the fast distribution phase, so it was presumed that 8018 could alter the distribution of soman by reducing the peripheral vasoconstriction and thereby increase the peripheral blood flow through the detoxifying organs such as small intestine which contained abundant CaE, the very important detoxifying enzyme to soman to accelerate the soman’s elimination. The presumption was convinced in our isotope trace that 8018 treatment could significantly increase the tissue distribution of bound [3H]soman in small intestine. The increased mice plasma bound [3H]soman maybe due to it’s rise the availability of covalent detoxification enzyme in plasma which needs further research to confirm. Inhibition of functional AChE located in target tissues such as brain and diaphragm is very crucial to soman induced toxicity, so, the concentration of high toxic P(-)soman in these tissue could reflect the intoxication degree of soman at some extent. In this study, the concentration of free P(-)soman per g tissue distributed in diaphragm was 10 times as high as distributed in brain, This phenomena was observed by Reynolds using [3H]soman trace method (Reynolds et al., 1985). 8018 could reduce the distribution of P(-)soman in diaphragm following soman intoxication via sc in our study which had toxicology significance, but 8018 didn’t influence the concentration of free P(-)soman in brain. In conclusion, other than the anticholinergic effects to soman, 8018 could accelerated the elimination of P(-)soman in the rabbits blood and reduced the distribution of P(-)soman in the mice diaphragm. Moreover, it could significantly increase bound [3H]soman distribution in small intestine as well as in plasma. More details and exact mechanisms of 8018’s antitoxicity of soman await deeply investigation.
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