In vivo distribution of organophosphate antidotes: Autoradiography of [14C]HI-6 in the rat

ln Viwo Distribution of Organophosphate Antidotes: Autoradiography of [14C]HI-6 in the Rat DAVID

A. LIGTENSTEIN,’

GERRIT

W. H. MOES, AND SIMON

P.

KOSSEN

Prim Maurits Laboratory TNO, P.O. Box 45,228OAA Rijswijk, The Netherlands

Received ApriI 20, 1981; accepted July 28, I98 7 In Viva Distribution of Otganophosphate Antidotes: Autoradiography of [‘4C]HI-6 in the Rat. LIGTENSTEIN, D. A., MOES, G. W. H., AND KOSSEN, S. P. (1988). Toxicol. Appl. Pharmacol. 92, 324-329. In order to visualize the distribution of HI-6 in the rat after iv administration, autoradiographic experiments were carried out with [14C]HI-6, labeled at the carbon ofthe carboxamide moiety. Autoradiography clearly confirms penetration of HI-6 into the central nervous system. Considerable radioactivity was found in the cerebrum, the cerebellum, and the choroid plexus. No significant activity was detected in the pontomedullary region or the spinal cord. Peripherally, [ ?]HI-6 is observed in large amounts in kidneys, heart, liver, nose, bladder, testes,and marrow-containing bone. Thegastrointestinal tract was largely devoid ofany radioactivity. The relative absence of HI-6 in the pontomedullary region renders centrally mediated influences of HI-6 on hemodynamic and respiratory parameters less likely.

The value of a central action of cholinesterase reactivating pyridinium aldoximes (“oximes”), like pralidoxime, obidoxime, or HI6, in the therapy of intoxications with organophosphate cholinesterase inhibitors as indicated by various authors (Bodor et al., 1975; Boskovic et al., 1980; Pantelic and Maksimovic, 1982; Wolthuis et al., 1981), has been disputed by others (Lundy and Shih, 1983; Clement, 198 1, 1982; Clement and Lockwood, 1982; Ligtenstein, 1984, 1985). In order to display a central action, the primary requirement is that the oxime penetrates into the central nervous system (CNS) after peripheral administration. The oxime HI-6, which is in the center of interest because of its putative activity against soman intoxications (De Jong and Wohing, 1980; Schoene, 1973; Wolthuis et al., 1976, 198 1; Hauser and Weger, 1979), has been suggested ’ To whom all correspondence should be.addressed. 0041-008X/88

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to penetrate into the CNS (Wolthuis et al., 198 1; Clement, 198 1, 1982). Ligtenstein and Kossen (1983) described this quantitatively in the rat and concluded that in this species, significant concentrations of HI-6 in the CNS are measurable after iv administration. To obtain a qualitative impression of the distribution of HI-6 over the various anatomical structures and to verify the earlier findings obtained by HPLC, we synthesized [14C]HI6 (Fig. 1) to prepare whole body autoradiograms in the rat after iv administration. METHODS Chemicals. Pentobarbitone sodium’ was procured from Ceva (Neuilly-sur-Seine, France; 60 mg/ml). [14C]HI-6 dichloride monohydrate, labeled at the carbon atom of the carboxamide group, was synthesized at the Prins Maurits Laboratory TNO, as described below. 42 Nembutal.

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Bromopyridine hydrochloride, n-butylhthium, and pyridine-2-aldoxime were from Aldrich (Beerse, Belgium), dichlarodimethyl etherr and the TLC thin-layer plates (Cellulose F254) were obtained from Merck (Darmstadt, FRG), and Ba14COs (222 mCi/g) was procured from Amersham (UK). All other chemicals were of analytical grade. Animal procedures. Ten male “small Wistar” rats, strain WAG/MBL, weighing 180-2 10 g, bred under SPF conditions at the Medical Biological Laboratory TNO, were used. Prior to the experiment the animals were fasted for 24 hr, but had access to water ad Iibitum. The rats were anesthetized with 75 mg/kg pentobarbitone sodium. [“C]HI-6 dichloride monohydrate was moistened with a drop of ethanol in order to avoid fly up of the substance. It was then dissolved in water, without further mixing with “cold” HI-6, in a concentration of 50 mg/ ml (0.1325 mmol/ml: 135 &i/ml). Through the dorsal penile vein I ml/kg (50 mg/kg) of this solution was injected. After 20 min the animals were frozen by immersion in a mixture of acetone and CO2 (-30°C). The sudden change in temperature causes the animals to curl, which would hamper microtome section. In order to prevent this the animals were, prior to immersion into the cooling mixture, stretched and fixed in that position by means of a simple metal holder. Autoradiography. In order to evaporate the acetone from the skin, the animals were covered with solid carbon dioxide for 6 hr and placed in a well-ventilated hood. Care was taken that the cadavers were kept well frozen. When the smell of acetone had disappeared, they were stored at - 15°C. Sagittal sections of 30 pm were made at - 1s”C by a LKB Microtome 2250 and stuck on a cardboard by means of Scotch tape No. 8 10. After lyophilization at -15°C for 24 hr, an Agfa-Gevaert D-7FW film was pressed against the preparation and exposed for 2 1 days. The autoradiograms were subsequently developed for 6 min in Agfa-Gevaert G-230 developer and fixed with G-334 fixative. The detection limit is approximately I nCi/g tissue, equaling approximately 1 nmd/g tissue (0.377 mg/g tissue) for HI-6. The procedure is modified from Van der Kleijn (1969). Synthesis of[“cjZiI-6. A solution of 4-bromopyridine (55 mg/ml) in ether was prepared by neutralizing a solution of 4-bromopyridine hydrochloride (8 g) in water (5 ml) with NaHCOx at 0°C followed by extraction with ether (100 ml). Water was removed by repeatedly distilling off ether at reduced pressure. The final solution (0.01% water) was stored at -20°C for several weeks, without noticeable decomposition. Circa 4 g of I-chloromethoxymethyl pyridinium-2-aldoxime chloride was

3 Dichlorodimethyl ether is a carcinogen. Special precautionary measures are necessary for appropriate laboratory handling.

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prepared from pyridine-f-aldoxime and dichlorodimethyl ether according to the method of Schoene ( 1967). The product showed only minor impurities upon TLC analysis. n-Butyllithium was handled under dry argon to increase the yield in the carbonation step in the synthesis of the intermediate [‘4C]isonicotinic acid. Prior to use Ba14C03 ( 112 mg; 25 mCi) was mixed with coarsely powdered unlabeled BaCO, (394 mg). TLC analysis was carried out on cellulose thin-layer plates with fluorescence indicator by elution with n-propanolfacetic acid/water (80/ 10/50, v/v). The plates were scanned on radioactivity distribution with a Berthold LB 2723 thin-layer scanner, equipped with a windowless proportional counting tube. For the synthesis of [‘4C]isonicotinic acid, an apparatus modified from Murray et al. (1948) was used. In a flask Ba”C03 (506 mg; 2.56 mmol; 25 mCi) was reacted with concentrated sulphuric acid asdesciibed by Murray and Langham (I 952). The 14C02 produced was dried by passage through a trap, which was cooled in dry ice/acetone, and collected by cooling with liquid nitrogen. A solution of n-butyllithium in hexane (3.2 ml, 5.1 mmol) was mixed with dry ether (20 ml). From a funnel a solution of 4-bromopyridine in ether (25 ml, 8 mmol) cooled to -30°C was added dropwise under stirring at -7O’C over 10 min. After the reaction mixture was stirred for 30 min it was frozen in liquid nitrogen and evacuated. Carbonation was then performed at -80°C. followed by hydrolysis of the reaction mixture with 6 N hydrochloric acid (10 ml) at - 10°C. Next, the mixture was made dkaline with NaOH in water (4 g in 4 ml) at O’C and finally warmed to room temperature. Excess 4-bromopyridine and other contaminants were removed by means of continuous extraction with ether for 12 hr. Subsequently the remaining water layer was evaporated to dryness at reduced pressure. The dry salt mixture, containing sodium [‘%]isonicotinate and small amounts of sodium [‘“Clvalerate as byproduct, was used without further purihcation. The salt mixture was refluxed with SCQ (I 5 ml4 for 1 hr and the escaping radioactive gases were trapped in active carbon. After cooling, the mixture was filtered on a G3 glass filter, the solids were washed on the filter with SGCl, (10 ml). and the combined filtrates were evaporated to dryness. The acid chloride was ester&d by refluxing with anhydrous methanol (4 ml) for 30 min. After evaporation to dryness, the residue was taken up into water (2 ml) and the solution was neutralized with solid NaHCO, and saturated with KCl. The ester was isolated by extraction with ether (4 X 10 ml), followed by evaporation, filtration, and distillation at 7o”C/ 13 Pa, yielding 200 mg methyl [‘4C]isonicotinate (57%, based on BaC03) as a colorless oil. The methyl isonicotinate was dissolved in dry methanol (3 ml) and the solution was saturated with dry NH3. After the solution was stirred for 5 hr at room tempera-

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ture, evaporated at reduced pressure, and dried, 178 mg of [ “Clisonicotinamide ( 100%) was obtained as a white solid (total act 14.7 mCi; sp act 10.0 mCi/mmol). TLC analysis showed a radiochemical purity of 83% (R/0.80); the only detectable impurity was unreacted methyl isonicotinate (17%; Rf0.92). [‘4C]Isonicotinamide was diluted with unlabeled isonicotinamide (672 mg), yielding 7 mmol. 1-Chloromethoxymethyl pyridinium-2-aldoxime chloride ( 1650 mg; 7 mmol) and acetonitrile ( 10 ml) were added, after which the reaction mixture was stirred at room temperature. After 24 hr another portion of acetonitrile (10 ml) was added. The progress of the reaction was monitored by TLC analysis. Stirring was continued for 1 week, after which no further reaction was observed; neither was a reaction observed after heating to 45°C for one day. The reaction mixture consisted of HI-6 (54%; R/0.40), isonicotinic amide (37%; R/0.82), and an unidentified impurity (8%; Rf0.60). Ether (20 ml) was added to the mixture, and after the precipitate was stirred for 10 min it was filtered off and washed with ether (2 X 10 ml). Recrystallization from boiling ethanol/water (10/6, v/v), followed by extraction with boiling ethanol (5 ml), yielded 1290 mg of [14CJHI-6 (chemical yield of the last step, 50%) with a total activity of 3.4 mCi (radiochemical yield last step, 23%) and a specific activity of0.97 mCi/mmol. Final TLC analysis showed the product to contain [‘4C]HI-6 (95.7%; R/0.43), [‘4C]isonicotinamide (2.4%; RfO.8 I), and an unidentified radioactive impurity (1.9%; R/0.62). No additional nonradioactive impurities could be detected on TLC, either upon exposure to iodine vapour or under ultraviolet light (254 nm). The structures of the products, obtained in parallel cold runs, were confirmed by means of infrared spectrometry, proton nuclear magnetic resonance spectrometry, and mass spectrometry.

RESULTS Introduction of 14C at the carboxamide moiety was found to be a convenient method to obtain radioactively labeled HI-6 The desired product was synthesized by reaction of 1chloromethoxymethyl pyridinium-2-aldoxime chloride with [ 14C]isonicotinamide. The latter product was obtained from methyl[ “C]isonicotinate, which was prepared according to the method described by Murray and Langham (1952). This method was modified by deletion of the time-consuming extraction procedure for the isolation of the intermediate [‘4C]isonicotinic acid.

t

Br --EIUL,

COOH

=coz

.[I/0 CHsOtl

.c3 0 NH3_ CHJ-OH

FIG. 1. The reaction route for the synthesis of [‘4C]HI-6.

The synthesis route, as indicated in Fig. 1, yielded 1290 mg of [‘4C]HI-6 with a specific activity of0.97 mCi/mmol and a radiochemical purity exceeding 95%. Distribution of radioactivity 20 min after iv administration of [14C]HI-6 (50 mg/kg; 0.1325 mmol/kg; 0.13 mCi/kg) is depicted in Fig. 2. Peripherally, above-average activity is found in the testes, bladder, kidneys, liver, heart, nose, trachea (not shown on the autoradiograms represented here), and marrowcontaining bone (vertebrae and costals). The gastrointestinal tract is largely devoid of radioactivity. In the CNS, activity was found in the cerebrum, the cerebellum, and the choroid plexus. Neither in the pontomedullary region, nor in the spinal cord, could any activity be observed. All 10 rats showed essentially the same distribution of radioactivity, with all the abovementioned characteristics. DISCUSSION The detection limit of the autoradiographic experiments is in the same range as that of the HPLC methods used for the description of the pharmacokinetic profile of HI-6 (Ligtenstein and Kossen, 1983). The autoradiograms found, therefore, are an illus-

AUTORADIOGRAPHY

FIG. 2. Autoradiograms and lower autoradiograms, Bladder, pl. char., choroid ach; kidn., kidney; thym.,

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of [“‘C]HI-6 in the rat, after iv administration of 50 mgjkg (135 &i/kg). Upper left laterosagittal section. Middle autoradiogram, mediosagittal section. blad.. plexus; cereb., cerebellum; sal. gl., salivary glands; intest., intestine; stom., stomthymus; nas. c., nasal cavity; vert., vertebra.

tration of the distribution of HI-6 over the whole body where, at similar concentrations, the pharmacokinetic profile only describes a distribution over blood and brain tissue.

The 20-min interval between administration of HI-6 and the actual sampling was based on three considerations. First, pharmacokinetic results obtained by Ligtenstein and

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Kossen (1983) show that after 20 min the elimination phase is reached, but that reasonably high concentrations in blood and in brain tissue are still present. Second, pilot studies showed shorter intervals to yield considerable interindividual differences in radioactivity distribution, an observation that is in accordance with the former consideration. Third, HI-6, being a quatemary ammonium compound, is unextractable from aqueous samples, as are two of its major metabolites (Ligtenstein et al., 1987). This property renders an experiment in tissue to judge the contribution of non-HI-6 radioactivity to the outcome of the autoradiography experiments impracticable. However, an experiment conducted with “cold” HI-6 in three rats showed that after 20 min the concentration of these two metabolites in blood was still undetectable. The rapid elimination of HI-6 by the kidney (Ligtenstein and Kossen, 1983; Ligtenstein, 1984) is consistent with the high activity found in the urogenital tract. The relatively large concentrations found in wellperfused structures, like lungs, heart, and liver, are well in accordance with the high water solubility of the compound. Furthermore, the absence of HI-6 in feces as found by Ligtenstein and Kossen (1983) is confirmed by the lack of radioactivity observed in the gastrointestinal tract. The presence of activity in marrow-containing bone and in the tracheal and nasal cartilage is striking and unexplained. Although marrow-containing bone is a well-perfused tissue, the measured activity is too large to be attributed only to the extreme water solubility of HI-6. The penetration of HI-6 into the CNS, as measured by means of high-performance liquid chromatography (Ligtenstein and Kossen, 1983) is confirmed by the autoradiograms. The unexpected penetration into the CNS of a quaternary ammonium compound like HI-6 can be attributed to the existence of a “rapidly equilibrating space,” bypassing the blood-brain barrier. For a further discussion of this phenomenon we refer to Ligtenstein

and Kossen (1983). Measurable activity is found in the cerebrum, the cerebellum, and the choroid plexus. The activity found in the cerebellum and in the diencephalon is in accordance with the anti-convulsive action of HI-6, as found by Ligtenstein ( 1984, 1985). No significant activity is found in the pontomedullary region, rendering an interaction of HI-6 with the vasomotor and respiratory centers that are located in this part ofthe CNS less likely. In overall cerebral tissue, HI-6 concentrations, 20 min after iv administration of 0.1325 mmol/kg, were found to be approximately 8 nmol/g wet wt tissue (Ligtenstein and Kossen, 1983). Apparently the radioactivity in most brain areas was only just above the detection limit of the method applied (1 nmol/g, w/w). A few well-defined spots with larger activity may account for the slightly higher concentrations found in brain by means of HPLC. ACKNOWLEDGMENT The department of Clinical Pharmacy of the Sint Radboud Hospital, Nijmegen, The Netherlands, is gratefully acknowledged for their help with the processing of the autoradiograms.

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