PRALIDOXIME MESYLATE ABSORPTION AND HEART RATE RESPONSE TO ATROPINE
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1 Br. J. clin. Pharmac. (1975), 2, PRALIDOXIME MESYLATE ABSORPTION AND HEART RATE RESPONSE TO ATROPINE SULPHATE FOLLOWING INTRAMUSCULAR ADMINISTRATION OF SOLUTION MIXTURES P. HOLLAND, D.C. PARKES & R.G. WHITE Medical Division, Chemical Defence Establishment, Porton Down, Salisbury, Wiltshire 1 The oxime pralidoxime mesylate (P2S) and atropine sulphate, alone and mixed, have been administered intramuscularly to forty-four human subjects. Doses of 750 mg and 500 mg P2S and 2.0 mg atropine sulphate were used. 2 The presence of P2S had no significant influence, as judged by effect on heart rate, on the absorption of atropine although there was a tendency for atropine to exert its effects more rapidly when administered mixed with P2S. 3 No significant difference in the rate of uptake of P2S as judged by plasma levels following injection, between a combined (plus atropine) and single (P2S alone) intramuscular injection was found. Introduction The drug therapy of poisoning by organophosphorus anticholinesterase compounds is two fold viz atropine sulphate to counteract the muscarinic effects of accumulated acetylcholine and oximes to reactivate the inhibited phosphorylated cholinesterase (Ellin & Wills, 1964). This double therapy has proved of benefit in many instances of poisoning in humans (Namba, Nolte, Jackrel & Grob, 1971; Quinby, 1964; Grob & Johns, 1958). The oxime currently available in this country is pralidoxime mesylate (P2S) and it is recommended (Martindale, 1972) that it be given by slow intravenous injection in a dose of 1.0 g repeated at intervals if necessary. In severe cases of poisoning, where convulsions are likely to be present this route of administration is impractical and the intramuscular (i.m.) route is to be preferred. With some of the more potent anticholinesterase compounds e.g., the nerve gases, there is little delay between poisoning and onset of symptoms and therefore immediate treatment is essential. Rapidity in instituting treatment is also necessary due to the ageing process of the phosphorylated cholinesterase which in some instances becomes rapidly non-reactivatable by oxime. In all recorded cases of poisoning when both drugs have been used oximes and atropine have been administered by a skilled attendant as separate injections. In certain field conditions however medical aid is not readily available and then self aid is recommended (Medical manual of defence against chemical agents, 1972). For self aid by means of an automatic injector, the administration by the poisoned subject, to himself of two separate injections, one of atropine and one of oxime, is not the most efficient method and a simpler approach would be to combine the oxime and atropine into one injection administered i.m. Automatic self-injection devices containing atropine sulphate (2.0 mg) have been developed in America (Rodana Research Corporation), Sweden (Astra autoinjector) and in this country (Sinclair & Watson, 1966). The possibility of incorporating an oxime together with atropine into such a device has occured to workers in both Sweden and the UK Ṫhe study to be described has been carried out to determine the effect addition of the oxime P2S to the atropine solution would have on the absorption of either drug following i.m. injection in human subjects. The doses of P2S chosen were those that have previously been shown to produce, rapidly, plasma P2S levels of 4.0 ;g/ml, i.e., 500 mg and 750 mg P2S/man (Holland, Parkes & Shakespeare, 1972). This plasma level has been shown to be necessary for the treatment of poisoning by anticholinesterase compounds (Sundwall, 1960; Sundwall, 1961; Crook, Goodman, Colbourne, Zvirblis, Oberst & Wills, 1962). The dose of atropine sulphate was kept constant at 2.0 mg/man. This quantity of atropine
2 334 P. HOLLAND, D.C. PARKES & R.G. WHITE is widely recommended as the basic dose and is incorporated in all currently available automatic injectors. Methods Subjects A total of forty-four male volunteers, having had the purpose of the trial explained to them, consented to take part. Materials Oxime (P2S) injections (1.0 g in 2.0 ml) were obtained from Koch Light Laboratories. Atropine sulphate injections (2.0 mg in 1.0 ml) were obtained from Evans Medical Supplies. Procedure The study was carried out in two parts. First to determine the effect on atropine absorption in the presence of P2S following i.m. administration of a mixture of the two drugs, 22 healthy volunteers took part. The uptake of atropine following injection was measured by reference to change in heart rate recorded continuously from a single chest lead e.c.g. This procedure was chosen, because, in the absence of ultrasensitive analytical techniques and the restrictions imposed on administering isotopes to normal healthy service volunteers, it was thought more convenient to measure the subjects response to atropine administration rather than to measure blood atropine levels. All records were taken from resting subjects in the following manner. After application of e.c.g electrodes, each subject lay quietly and undisturbed on a bed for 1 h before commencement of the experiment. For the last 5 min of this period a continuous control record of the e.c.g., paper speed 10 mm/s, was made. The i.m. injections were then given, 2 h after a light breakfast, into the upper aspect of the thigh and the e.c.g. then continuously recorded for 2 hours. The air temperature lay between C and 24.0 C and smoking was forbidden. All injections were formulated with water for injection BP, immediately before use, from the readily available injection solutions mentioned above. Consequently minimum volumes for injection were 2.5 ml for the 750 mg dose and 2.0 ml for the 500 mg dose. The injection procedure employed permitted a sufficient time interval between single and combined drug administration to prevent any overlap of drug effect. The single and combined injections were administered to a single blind cross over design, the volunteers having no knowledge of the type of drug they were receiving or symptoms to expect. Of the twenty-two subjects, twelve (1-12) received atropine sulphate (2.0 mg) both alone and combined with P2S (750 mg) and ten (13-22) received a similar dose of atropine sulphate alone and combined with P2S (500 mg) with a 5 day interval between injections in each instance. Five of the subjects, who subsequently received the 750 mg dose, also received injections of P2S (750 mg) and water for injection BP to determine if these alone had any effect on resting heart rate. For analytical purposes measurements were made, for each individual, of the time taken for the heart rate to reach maximum bradycardia, return to control heart rate and reach maximum tachycardia. Secondly, to determine the effect of the presence of atropine on P2S absorption from an i.m. injection of solution containing both P2S and atropine an experiment involving twenty-two volunteers was conducted. Two dose levels of P2S 500 mg and 750 mg/man) were used, formulated in water for injection BP as above. Twelve volunteers received the 750 mg dose both alone and combined with atropine and ten volunteers the 500 mg dose. A two day interval occurred between single and combined injections. Injections were made i.m. into the upper outer aspect of the thigh. Blood samples for P2S analysis were obtained from the medium cubital vein immediately prior to each injection (control) and at 3, 10, 20 and 30 min following injection. Plasma P2S levels were estimated by making the sample alkaline and measuring the resultant oxamate ion at 335 nm using a spectrophotometer 500 (Creasey & Green, 1959). Statistical analysis (a) Values for time to reach maximum bradycardia, return to control heart rate and reach maximum tachycardia for each individual following administration of the mixed injection were subtracted from the corresponding values for the same individual for the atropine alone injection. A mean difference was then calculated at the P2S dose level of 750 mg for twelve subjects and at 500 mg for ten subjects. To assess the overall effect of oxime a similar calculation was made for all twenty-two subjects. From these mean differences, t values were calculated and the level of significant difference, if any, obtained from the Student's t test tables. (b) Plasma P2S levels obtained for each individual at the set times following injection with the
3 OXIME AND ATROPINE ABSORPTION IN MAN [ 50L Time(min) Figure 1 Mean heart rate changes following i.m. injection of water for injection BP (2.5 ml, n = 5, o), P2S (750 mg in 2.5 ml water for injection, n = 5, A), atropine sulphate (2.0 mg in 2.5 ml water for injection, n = 12, *) and atropine sulphate (2.0 mg) plus P2S (750 mg) in 2.5 ml water for injection (n = 12, *). o, resting heart rate, no injection. combined injection were subtracted from similar values for the same individuals obtained with the single injection and the t test then applied to the mean difference within each group. This procedure was carried out for both dose levels and for each of the time intervals mentioned above. Results Effects on atropine absorption of a combined im. injection of P2S and atropine Figure 1 shows mean changes in heart rate with time in twelve subjects following i.m. injection of atropine sulphate (2.0 mg) alone and combined with P2S (750 mg). Similar changes were seen with P2S (500 mg). Changes in heart rate response with time were measured every 30 s but for clarity of presentation only 10 min readings are shown in Figure 1. Water for injection BP alone and P2S alone had no effect on resting heart rate. Superimposition of the curves for heart rate response with time for the single and combined injections shows them to be practically identical. An initial bradycardia being characteristic of the atropine response. Table 1 shows the mean differences in time (min) to reach maximum bradycardia, return to control heart rate and reach maximum tachy- Table 1 Statistical analysis of mean time differences to reach maximum bradycardia, control heart rate and maximum tachycardia following i.m. injection of atropine sulphate alone and combined with P2S 22 bradycardia (min) Time to reach control heart rate (min) tachycardia (min) Mean Dose P2S atropine-atropine (mg) Subjects plus P2S (min) s.d. p
4 336 P. HOLLAND, D.C. PARKES & R.G. WHITE Figure 2 Mean (n = 12) plasma P2S concentrations following i.m. injections of (a) P2S (500 mg, o) of P2S (500 rng) plus atropine (2.0 mg, *) and (b) P2S (750 mg o) or (750 mg) plus atropine (2.0 mg, u). cardia, standard deviations and significance of difference between the combined and single injections. Atropine sulphate produced a maximum bradycardia in 15.3 and 18.7 min in subjects 1-12 and respectively whereas when combined with P2S this was shortened to 12.2 min and 12.9 minutes. Return to control heart rate was seen in 29.5 min and 35.1 min with atropine sulphate alone but when combined with P2S this time was shortened to 24.8 min and 30.6 min for the 750 mg and 500 mg P2S doses respectively. Times to reach maximum tachycardia were 51.4 min and 58.3 min with atropine alone and when combined with P2S 56.5 min and 56.1 min for the two groups of subjects respectively. Statistical analysis shows that at P = 0.05 there is no significant differences in the rate of absorption of atropine from either the single or combined injection. When all twenty-two subjects are treated as one group, irrespective of the dose of P2S, statistical analysis shows that at P = 0.05, only in the time to reach maximum bradycardia is there any significant difference P < 0.01 (Table 2). This finding indicates that the initial absorption of atropine is possibly enhanced when given in combination with P2S. Effect of P2S absorption of a combined i.m. injection of P2S and atropine Figure 2 shows changes in P2S plasma concentrations with time following i.m. administration of P2S (750 mg or 500 mg) alone and combined with Table 2 Statistical analysis of the differences in the rates of absorption of atropine sulphate single and combined (with P2S i.m. injection in twenty-tv from both a bradycardia (min) Time to control heart rate (min) tachycardia (min) Mean Subjects atropine-atropine plus P2S (mini s.d. p < >
5 OXIME AND ATROPINE ABSORPTION IN MAN 337 atropine sulphate. Statistical analysis of the data shows that there is no significant difference between plasma P2S concentrations obtained following injections of P2S alone or combined with atropine sulphate (Table 3). Mean P2S plasma levels of 4.0,ug/ml were reached within 3 min and 6 min following i.m. administration of 750 mg and 500 mg P2S respectively. Discussion In situations where therapy for poisoning by some organo-phosphorus anticholinesterase compounds needs to be administered rapidly an i.m. injection of a mixture of atropine sulphate and P2S is desirable. For speed of administration, especially where no medical aid is immediately available, self injection devices may be used. The automatic injectors currently available in this country and the USA contain only atropine sulphate (2.0 mg); in Sweden however collaboration between Astra Pharmaceuticals and the Swedish military and civil authorities has resulted in the production of a device containing a mixture of the oxime Toxogonin and atropine sulphate (Barkman, Edgren & Sundwall, 1963; Barkman, 1973). The oxime advocated in this country is pralidoxime mesylate (P2S). This study indicates that this oxime combined with atropine sulphate can be administered i.m. to human subjects without significant effect on the absorption characteristics of either drug. The figures presented disclose a tendency for atropine to exert its initial slowing effect on the heart rate more rapidly when combined with P2S as judged by the time interval between injection and maximum bradycardia. This is probably due to an enhanced absorption of atropine rather than a slowing effect of the P2S, since P2S did not slow the heart rate below that seen when the subjects were resting. It can be argued that a more rational approach to verify this finding would be to correlate the atropine levels in the blood with heart rate response but owing to the restrictions imposed on the use of isotopes in service volunteers this was not possible in this study; nevertheless the finding that the i.m. administration of a combined P2S and atropine solution did not produce any detrimental effect on the absorption of atropine is encouraging. There remains some doubt about the effect that the presence of other oximes might have on the absorption of atropine when combined in the same injection solution. For example it has been shown that 2-hydroxyimino-methyl-N methyl pyridinium chloride (PAM Cl) might delay the absorption of atropine by as much as 7 min (Sidell, Magness & Bollen, 1970) possibly due to a local vasoconstriction produced by the oxime (O'Leary, Kunkle, Murthe & Somers, 1962) mediated by catecholamine release (Zarro & di Palma, 1965). lt is appreciated that in this study the subjects remained at rest throughout the investigation but in practical situations might be active. However the effect of exercise on the absorption charactertistics of P2S and atropine, following i.m. injection of the mixed solution has been studied (Martin, 1973) and it was shown that with moderate exercise the absorption of P2S and atropine, in combination, was not significantly different from that when either drug was administered alone. The finding that neither of the drugs in this mixture has a detrimental effect on the absorption of the other when given i.m. indicates that single injection emergency treatment of organophosphorus poisoning is feasible and possibly desirable. Table 3 Statistical analysis of mean difference in P2S plasma levels produced by i.m. injections of P2S alone and combined with atropine sulphate P2S (750 mg) 3 Mean difference in _ 19 plasma P2S levels (jg/ml) ± s.d. 2.48E t p Mean difference in P2S plasma P2S levels (,g/ml) mg ±s.d (500 mg) t p B Time (min)
6 338 P. HOLLAND, D.C. PARKES & R.G. WHITE References BARKMAN, R., EDGREN, B. & SUNDWALL, A. (1963). Self administration of pralidoxime in nerve gas poisoning with a note on the stability of the drug. J. Pharm Pharmac., 15, BARKMAN, R. (1973). Self administration of antidotes with the autoinjector. Communication to the 33rd International Congress of Pharmaceutical Sciences, Stockholm, September. CREASEY, N.H. & GREEN, A.L. (1959). 24hydroxyiminomethyl-N-methyl pyridinium methane sulphonate (P2S) on antidote to organo-phosphorus poisoning. Its preparation estimation and stability. J. Pharnt Pharmac., 11, CROOK, J.W., GOODMAN, A.I., COLBOURNE, J.H., ZVIRBLIS, P., OBERST, F.W. & WILLS. J.H. (1962). Adjunctive value of oral prophylaxis with the oximes 2-PAM lactate and 2-PAM methane sulphonate to therapeutic administration of atropine in dogs poisoned by inhaled sarin vapour. J. Pharmac. exp. Ther., 136, ELLIN, R.1. & WILLS, H.J. (1964). Oximes antagonistic to inhibitors of cholinesterase. J. Pharm. Sci., 53, ELLIN, R.I. & WILLS, H.J. (1964). Oximes antagonists to inhibitors of cholinesterase. J. Pharm. Sci., 53, GROB, D. & JOHNS, R.J. (1958). Use of oximes in the treatment of intoxication by anticholinesterase compounds in normal subjects. Am. J. Med., 24, HOLLAND, P., PARKES, D. & SHAKESPEARE, J. (1972). Concentrations of the oxime (P2S) after intramuscular injection in humans. Br. J. Pharmac., 44, 368 P. MARTIN, H. de V. (1973). Atropine sulphate absorption from an intramuscular injection of a mixture of the oxime P2S and atropine in exercising humans. Br. J. Pharmac., 47, 619 P. MARTINDALE, W. (1972). The Extra Pharmacopoeia. Edition 26, 412. MEDICAL MANUAL OF DEFENCE AGAINST CHEMICAL AGENTS. (1972). H.M.S.O. Publication JSP 312. NAMBA, T., NOLTE, C.T., JACKREL, J. & GROB, D. (1971). Poisoning due to organophosphate insecticides. Am. J. Med., 50, O'LEARY, J.F., KUNKLE, A.M., MURTHA, E.F. & SOMERS, L.M. (1962). Sympathomimetic actions of 2-formyl-1-methylpyridinium chloride oxime (2-PAM Cl) Fed. Proc., 21, 112. QUINBY, G.E. (1964). Further therapeutic experience with pralidoxime in organophosphorus poisoning. J. Am med. Assoc., 187, SIDELL, F.R., MAGNESS, J.S. & BOLLEN, T.E. (1970). Modification of the effects of atropine on human heart rate by pralidoxime. Clin. Pharmac. Ther., 11, SINCLAIR, K. & WATSON, C.D. Patent Nos , London: Patent Office. SUNDWALL, A. (1960). Plasma concentrations of the oxime (P2S) after intravenous, intramuscular and oral administration in man. Biochem Phiarmac., 5, SUNDWALL, A. (1961). Minimum concentrations of the oxime (P2S) which reverse neauromuscular block. BiochermL Pharmac., 8, ZARRO, V.J. & di PALMA, J.R. (1965).The sympathomimetic effects of 2-pyridine aldoxime methyl chloride (2-PAM Cl). J. Pharmac. exp. Ther., 147, (Received October 25, 1974)
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