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Ganglion-blocking, neuromuscular-blocking and other actions of 2-imino-4-ethyloxycarbonyl-1,3-diazacycloheptine hydrochloride (OG 30), a ganglionic stimulant
EUROPEAN JOURNAL OF PHARMACOLOGY 11 (1970) 75-83. NORTH-HOLLAND PUBLISHING COMPANY
GANGLION-BLOCKING,
NEUROMUSCULAR-BLOCKING
AND OTHER ACTIONS
OF 2-IMINO-4-ETHYLOXYCARBONYL-1,3-DIAZACYCLOHEPTANE HYDROCHLORIDE
(OG 30), A GANGLIONIC
STIMULANT
Pramod R. SAXENA Pharmacological Research Department, N. V. Organon, Oss, The Netherlands
Received 18 November 1969
Accepted 11 March 1970
P.R. Saxena, Ganglion-blocking, neuromuscular-blocking and other actions o f 2-imino-4-ethyloxycarbonyl-l,3-diazacycloheptane hydrochloride (OG 30), a ganglionic stimulant, European J. Pharmacol. 11 (1970) 000-000. 2-Imino-4-ethyloxycarbonyl-l,3-diazacycloheptane hydrochloride (OG 30) was previously found to be a ganglion-stimulating agent acting on nicotinic receptors. The purpose of the present investigation was to study further the pharmacological actions of OG 30. After initial stimulation, OG 30 caused a brief period of "non-depolarizing" ganglion block, in cats. It also impaired neuromuscular transmission in several in vivo and in vitro preparations by "depolarization". Although OG 30 stimulated the dog heart in vivo, it had no appreciable activity on the isolated guinea pig heart in contrast to DMPP. The LDso of OG 30 in mice by the i.v. and i.p. routes was 10.25 and 57.75 mg/kg, respectively. It was, however, non-toxic by the oral route even in large doses probably because of poor absorption. On the basis of the toxicity pattern, it has been argued that the compound causes death mainly by blocking neuromuscular transmission. OG 30
Ganglion stimulation
Neuromuscular block
1. INTRODUCTION Panneman (to be published) synthesized several compounds bearing a c o m m o n 2-imino-l,3-diazacycloalkane ring structure. One of these compounds, 2-imino-4-ethyloxycarbonyl-1,3-diazacycloheptane hydrochloride (OG 30), whose structure is shown in fig. 1, produced an increase in blood pressure and a contraction o f the nictitating membrane in anaesthetised cats. These effects of OG 30 were due to the stimulation of sympathetic ganglia (Saxena, 1969).
DMPP
Ganglion block
The compound was found to act on the nicotinic receptors of the ganglion cells and thus had a mode of action similar to 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), a recognised ganglionic stimulant (Chen et al., 1951). Since DMPP has also been shown to have a blocking action on ganglia (Chen and Portman, 1954; Leach, 1957) and neuromuscular junction (Kaller, 1956; Ling, 1959), it was thought interesting to study further the pharmacological actions o f OG 30.
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2.1. Ganglion-blockade 2.1.1. Intravenous (i.v.) administration of OG 30 Sixteen cats ( 2 - 4 kg) of either sex anaesthetised with pentobarbital sodium (30 mg/kg, i.p.) were employed for these experiments. They were kept on
76
P.R.Saxena, Actions of OG 30
positive pressure ventilation except for three cats in which spontaneous respiratory excursions were recorded with a Grass PT 5 volumetric transducer. Blood pressure was measured with a Statham P23AC transducer via a polyethylene cannula inserted in one of the femoral arteries. The semi-isometric contractions of the nictitating membrane, which had 4 g initial tension, was recorded with a Grass FT03 transducer. Heart rate was recorded on a Grass tachograph preamplifier (7P4A) triggered by ECG signals. The parameters were recorded on a model 7 Grass polygraph. Responses to adrenaline (3/ag/kg), bilateral occlusion of common carotid arteries for 30 sec (C.O.), and to stimulation (10 V, 1 msec, 25/sec for 15 sec) of the peripheral end of the sectioned vagus and the preganglionic cervical sympathetic nerves were obtained before and after administration of compound, OG 30. A Grass stimulator, model $4, was used. All drugs were injected i.v. into a cannulated femoral vein. 2.1.2. Direct injection of OG 30 to superior cervical ganglion Blood pressure and tension of the nictitating membrane were recorded, as stated above, in 5 cats anaesthetised with pentobarbital sodium (30 mg/kg) and ventilated artificially. A cannula was inserted into the central end of the cut lingual artery to inject the compounds directly to the superior cervical ganglion (Trendelenburg, 1954) of the corresponding side. 2.2. Neuromuscular blockade 2.2.1. Sciatic nerve-gastrocnemius preparation in cats Neuromuscular preparations were made in 8 cats anaesthetised with pentobarbital sodium (30 mg/kg, i.p.) and artificially ventilated. The peripheral end of the sectioned sciatic nerve received supramaximal electrical stimuli of 1 msec duration every 10 see from a model $4 stimulator. The semi-isometric contractions of the gastrocnemius muscle were recorded on the Grass polygraph by a thread connecting the tendon to a Grass FT 10 transducer. The effects of both i.v. and intra-arterial (i.a.) administration of the compound were studied. The i.a. injection was given peripherally into the femoral artery of the same side by a cannula inserted through one of its branches close to the iliac artery.
2.2.2. Common peroneal nerve-tibialis anterior preparation in cats (Brown, 1938) This preparation was set up in 6 cats in a manner similar to that described in 2.2.1. Close i.a. injections were delivered to the tibialis anterior muscle through the anterior tibial artery. 2.2.3. Isolated phrenic nerve diaphragm preparation of rat (Bulbring, 1946) Twelve experiments were set up in a 25 ml bath containing Krebs-Henseleit solution bubbled with a mixture of 95% 02 and 5% CO2 at 37°C. Direct or indirect stimuli of supramaximal voltage, 1 msec duration and 0.1/sec frequency derived from a Grass model S 4 stimulator were applied to the muscle. The contractions of the diaphragm were recorded either semi-isometrically with a Grass FT 03 transducer on the Grass polygraph or isotonically with a transducer on a Harvard recorder. 2.2.4. Isolated rectus abdominis muscle of frog (Jindal and Deshpande, 1960) The effect of compound OG 30 was studied in 10 experiments on the above muscle set up in a 5 or 25 ml bath containing frog-Ringer solution equilibrated with 95% 02 and 5% CO2 at 22°C. The contractions of the muscle were recorded on a smoked drum through a lever arrangement or by a transducer on a Harvard recorder. 2.3. Action on heart 2.3.1. Open chest preparation of dogs In three dogs anaesthetised with pentobarbital sodium (35 mg/kg, i.v.), the chest was split in the midline and the heart was exposed by cutting the pericardium. The force of contraction of the heart muscle was measured by a strain gauge arch (Boniface et al., 1953) attached to the right ventricle. Blood pressure was measured as in 2.1.1. 2.3.2. Perfused guinea-pig heart preparation (Langendorff, 1895) Six experiments were conducted on the heart perfused with Meyler's solution (Meyler et al., 1959) bubbled with 5% CO2 and 95% O2 at 37°C. Contractile force of the heart was recorded by a Grass FT 03 transducer attached via a spring and thread to its
77
P.R.Saxena, Actions of OG 30
2.6. Drugs The following compound dissolved in normal saline were used: acetylcholine chloride (Roche); adrenaline bitartrate; 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP); d-tubocurarine chloride (Fluka); 2-imino-4-ethyloxycarbonyl- 1,3-diazacycloheptane hydrochloride (OG 30); 4-(m-chlorophenylcarbamoyloxy)-2- but yniltrimethylammonium chloride (MeN-A-343); neostigmine methylsulphate (Prostigmine, Roche); physostigmine salicylate (BDH); potassium chloride (KC1), and succinylcholine chloride (suxamethonium). All doses are expressed in terms of the salt.
apex. The outflowing fluid was collected in a U tube with a side arm through which the fluid escaped every 20 see. The other limb was connected to a Grass PT5 volumetric transducer. Displacement of air in this limb have the measurement of the outflow every 20 sec. The events were registered on a model 7 Grass Polygraph. All drugs were injected directly into the aorta with the perfusion fluid through a rubber tube. 2.4. Acute toxicity The acute toxicity of compound OG 30 was determined in albino mice by the oral, i.p. and i.v. routes. Groups of ten mice were injected with different doses of the compound by the different routes of administration. The mice were observed continuously for 3 hr and later left overnight. The total number of mice dying during the 24 hr-period was recorded. The data were analysed by the method of Miller and Tainter (1944) to determine LDso.
3. RESULTS 3.1. Ganglion-blocking action
3.1.1. Effect on i.v. administration Administration of OG 30 (1 mg/kg) in 5 cats caused an increase in blood pressure and tension of the nictitating membrane similar to that described previously (Saxena, 1969). This dose of OG 30 did not modify the pressor effects of adrenaline (3/ag/kg) or of bilateral carotid occlusion (C.O.). Similarly, the bradycardia and hypotension caused by the stimulation of peripheral end of the divided vagus nerve, and the nictitating membrane contraction elicited by preganglionic cervical sympathetic nerve stimulation were unaffected showing lack of ganglionic block
2.5. Administration o f OG 30 into gastrointestinal tract or into a portal venous tributary Five cats were prepared for blood pressure measurement as described in 2.1.1. In 3 cats, OG 30 was injected by a needle after laparotomy into stomach, duodenum or small intestine. In the 2 other cats, the pressor effects of OG 30 injected through the femoral vein and through a tributary of the superior mesentric vein were compared.
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Fig. 2. Effect of OG 30 on the responses to simultaneous stimulation (10 V, 1 msec, 25/sec for 1S sec) of preganglionic cervical sympathetic and peripheral vagus nerves (S), bilateral occlusion of carotid arteries for 30 sec (C), and adrenaline, 3 ~ag/kg (A). Cat, 3.6 kg, 9. Records from above downwards; heart rate/rain, blood pressure (ram Hg), nictitating membrane tension (g). First panel, control responses; middle panel, drug effect; last panel; recovery after 10 min. Note the secondary fall in blood pressure and blockade of S by OG 30.
78
P.R.Saxena, Actions of OG 30
with the above dose. However, in 5 other cats, a higher dose (10mg/kg) caused, after an initial increase in heart rate, blood pressure and tension of the nictitating membrane, a definite fall in blood pressure accompanied by a short-lasting blockade of the responses produced by C.O., vagal stimulation, and preganglionic cervical sympathetic stimulation (see fig. 2). All these responses returned to normal within 10 min. These results indicated a short-lived ganglionblocking action of OG 30. In order to find the threshold dose blocking ganglionic transmission, the effects of different doses of OG 30 on the responses (see above) elicited by stimulation of the vagus and the preganglionic cervical sympathetic nerves were studied in three cats. OG 30 (up to 2 mg/kg) did not affect nictitating membrane contraction, hypotension or bradycardia,
but a dose of 2.5 mg]kg produced transient inhibition of these responses. Higher doses of OG 30 (5 and 10 mg/kg) completely blocked these effects. It was also observed in three spontaneously-breathing cats that when two doses of OG 30 (20 mg/kg) were administered shortly after each other, the second was inactive in causing a rise in blood pressure. Instead, bradycardia and hypotension occurred, and the animal died later; respiration failed before the heart (fig. 3). 3.1.2. Effect on i.a. injection to ganglion As has been reported earlier (Saxena, 1969), OG 30 caused a contraction of the nictitating membrane when injected directly to the superior cervical ganglion. However, when injected repeatedly in a sequence of 0.125, 0.25, 0.5, 0.5 mg every 1 to
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0.5 mg) on the nictitating membrane contraction in response to the stimulation (10V, 1 msec, 2S/sec for 10sec) of preganglionic cervical sympathetic nerve (S), and to i.a. injection of McN-A-343, 20 ug (M) and KC1, 4 mg (K) to the ganglion. Cat, 4 kg, C~.First panels, control responses; middle panels, drug effect; last panels, recovery in 10 min. Note that repeated injection of OG 30 blocks its own stimulatory action, and suppressed the responses to S and M without affecting those to K.
P.R.Saxena, Actions of OG 30
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Fig. 5. Effect of OG 30, 0.5 mg/kg (O) on the blood pressure (upper records) and gastrocnemius muscle contractions (lower records) in response to sciatic nerve stimulation (2 V, 1 msec, 0.1/sec). C~t, 2.75 kg, 9- Note that neostigmine slightly enhanced the neuromuscular block by OG 30. 2 rain, the last dose failed to contract the nictitating membrane (fig. 4) confirming that OG 30 can block its own ganglion-stimulating action. The effects of preganglionic cervical sympathetic stimulation and o f i.a. injections of McN-A-343, but not KC1, were reduced during OG 30-induced ganglion block (fig. 4).
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6 3.2. Neuromuscular-blocking action 3.2. l. Cat sciatic nerve-gastrocnemius preparation OG 30 (up to 2 mg/kg, i.v.) did not significantly affect the tension developed by the gastrocnemius muscle in response to sciatic nerve stimulation. When administered i.a, into the femoral artery of the same side, OG 30 (0.5 mg/kg) repeatedly reduced the contractions o f the muscle to nerve stimulation. The effect was more marked with high doses o f OG 30. The cholinesterase inh~itors, neostigmine ( 0 . 1 - 0 . 2 5 mg/kg, i.v.) or physostigmine (0.1 mg/kg, i.v.) slightly facilitated neuromuscular block induced by OG 30 (fig. 5). In two experiments DMPP (0.05 mg/kg, i.a.) was tested and found to be 3 0 - 4 0 times as effective in blocking the gastrocnemius contractions as OG 30. 3.2.2. Cat c o m m o n peroneal nerve-tibialis anterior muscle preparation On close i.a. injection, both acetylcholine (10/sg) and OG 30 (1 rag) caused a twitch response in the muscle without blockade o f the nerve stimulated muscle contractions (fig. 6, upper panels). Higher
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Fig. 6. Effect of close i.a. injection of OG 30 (O), acetylcholine (Ach) and suxamethonium (S) on the common peroneal nerve-tibialis anterior muscle preparation. Cat, 3.6 kg, q?. Peripheral end of the cut nerve was stimulated (3 V, 1 msec, 0.1/sec) except at upper bars where no stimulation was applied. T, tetanic stimulation (50/see for 5 sec). Note that both OG 30 and Ach caused a contraction of the non-stimulated muscle and that the OG 30-induced block of neuromuscular transmission was preceded by a facilitated contraction of the muscle. Tetanus was sustained (not clearly observed in the Figure because of slow chart speed) for the period of stimulation during the block caused by both OG 30 and suxamethonium and no post-tetanic twitch potentiation is evident.
80
P.R.Saxena, Actions o f OG 30
amounts of OG 30 (5 mg) caused a brief period of fasciculation and a potentiation of the indirectlyelicited twitch response before causing neuromuscular transmission block (fig. 6, upper panel). A tetanus elicited for 5 sec was well sustained and the post° tetanic twitch potentiation was not observed. Similar effects occurred with suxamethonium although the fasciculations were more marked and twitch potentiation was not evident. Tetanic response was also sustained (fig. 6, lower panel) and the post-tetanic twitch potentiation was absent. 3.2.3. Isolated phrenic nerve-diaphragm preparation of rat OG 30 (0.04-0.2 mg/ml) blocked contractions of the diaphragm elicited indirectly by stimulation of the phrenic nerve without appreciably affecting those produced by direct musclar stimulation. The neuromuscular blockade caused by OG 30 was not antagonised by neostigmine (2-10tag/ml) but was readily reversible upon changing the bath fluid. 3.2.4. Rectus abdominis muscle preparation of frog Three concentrations of OG 30 (5, 20 and 80 tag/ ml) potentiated contractions of the rectus muscle to acetyicholine (fig. 7). OG 30 did not cause any appreciable contraction of the muscle at these dose levels. High doses of OG 30 (160-1000 tag/ml) elicited muscle contractions that could be completely antagonised by d-tubocurarine (1 tag/ml).
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Fig. 7. Mean (+ S.E.M.) percentage potentiation by OG 30 of the acetylcholine (Ach)-induced contractions of the frog rectus abdominis muscle suspended in a 25 ml bath containing frog Ringer solution. Number of observations is given in the brackets. All values differ significantly (p ,(0.01) when compared to the control responses to acetylcholine in the absence of OG 30. 3.3. E f f e c t on heart
3.3.1. Open chest preparation of dog Both OG 30 and DMPP increased the blood pressure and the force of myocardial contraction in the open chest preparation of dog (fig. 8). Hexamethonium abolished the effects of both compounds.
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Fig. 8. Effect of OG 30, 0.5 mg/kg (O) and DMPP (D) on myocardial contractile force and blood pressure. Dog, 7.5 kg, d. Upper trace, time mark 5 sec; middle trace, myocardial contractile force; lower trace, blood pressure. OG 30 and DMPP increased the blood pressure and myocardial force; these effects were blocked after 5 mg/kg hexamethonium (at arrow); compare the two panels.
P.R.Saxena, Actions ofOG 30
81
Table 1 Acute toxicity of OG 30 in mice. Route of administration i.v.
i.p.
oral
Dose* (mg/kg) 7.5 11.25 15.0 30.0
% Mortality 20 80 100 100
25.0 50.0 75.0 100.0
0 30 70 100
50.0 300.0 1000.0 2000.0
0 0 0 0
LD~ff (mg/kg) 10.25
Toxicity symptoms clonic convulsion, loss of righting reflex, respixatory failure and cyanosis before stopping of the heart As above
57.75
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* The number of mice injected at each dose level was 10. ** Calculated using the method of Miller and Tainter (1944).
3.3.2. Perfused guinea-pig heart On the isolated perfused guinea-pig heart, OG 30 (up to 5 mg) had no appreciable effect except for a small transient increase in coronary outflow or an occasional slight decrease in heart contraction. DMPP (10-20/ag) always increased the heart contractions after a slight initial decrease. 3.4. Acute toxicity The results presented in table 1 establish the LDso of OG 30 as 10.25 and 57.75 mg/kg by the i.v. and i.p. routes respectively. However, the compound was non-toxic orally, even in a dose of 2 g/kg. Under similar circumstances, the LDso of DMPP was 1.6 and 18.75 mg/kg by the i.v. and i.p. routes respectively. The toxic symptoms were the same as in the case of OG 30. 3.5. Effect of OG 30 after intra-intestinal injection Since OG 30 was non-toxic orally in mice it was injected into stomach, duodenum or small intestine of anaesthetised cats to see, ff it was ineffective by the enteral route in the cat. Injections of OG 30 (up to 60 mg/kg) did not raise the blood pressure when observed for up to four hours; the effective intravenous dose was 0.2 to 0.5 mg/kg.
3.6. Comparison of the effects of OG 30 by systemic or portal venous injection in cat To rule out the possibility that breakdown of OG 30 in the liver was responsible for its lack of activity after oral or enteral administration, the effects of injections into the femoral vein and into a tributary of the superior mesenteric vein were compared in two cats. OG 30 (1 mg/kg) produced a similar pressor response when injected by either route.
4. DISCUSSION Study of the ganglion-blocking action of OG 30 was prompted because its actions generally resembled those of DMPP which, in higher amounts, can block ganglia (Chen and Portman, 1954; Leach, 1957). It is evident from the results that OG 30 could also depress the ganglion-cells following an initial stimulant action. This ganglion-blockade by OG 30 was shortlived presumably due to rapid elimination of the compound from the site of action as is likely with DMPP (Chen and Portman, 1954). The impairment of ganglionic-transmission resulting from high doses of OG 30 does not appear to be due to depolarization
82
P.R.Saxena, Actions of OG 30
since responsiveness to KCI was unaffected (Trendelenburg, 1957). However, an initial depolarizing block, as has been conclusively demonstrated for nicotine (Paton and Perry, 1953; Trendelenburg, 1966a, 1966b) and suggested for DMPP (Leach, 1957), may have passed off even before KC1 could be injected, but, pending an electrophysiological study, it was concluded that OG 30 produced a "nondepolarizing" block. Suppression of McN-A-343 response by OG 30 may be similar to that observed between DMPP and McN-A-343 (Smith, 1966). The neuromuscular-blocking action of OG 30 was clearly demonstrated in both in vivo and in vitro experiments. This effect was mainly due to a depolarizing action, because of: (i) quick contraction of the non-stimulated tibialis anterior muscle on close i.a. injection, (ii) short period of fasciculation and potentiation of the maximal twitch before the block of neuromuscular transmission (see fig. 6), (iii) sustained tetanus and lack of post-tetanic twitch potentiation during neuromuscular block, (iv) slight potentiation (and not antagonism) of neuromuscular blocking action of OG 30 by anti-cholinesterases in cat gastrocnemius preparation, (v) a potentiation by OG 30 of acetvlcholine-induced frog rectus abdominis muscle contractions, (vi) a contracture of the rectus muscle by OG 30 itself and (vii) blockade by d-tubocurarine of the OG 30-induced rectus contractions. Furthermore, it has been observed (Saxena et al., 1970) that OG 30 resembled the actions of McN-A-343 on a new in vitro sciatic nerve-tibialis anterior muscle preparation (Van Riezen, 1968a, 1968b) in blocking the stimulant action of the depolarizing drugs in a lower concentration; higher amounts of OG 30 did increase the chick muscle tension and blocked the nervestimulated muscle contractions. The latter effect also points to a depolarizing action of OG 30. The other ganglionic stimulants, nicotine (Thesleff, 1955) and DMPP (Kaller, 1956; Ling, 1959) also cause neuromuscular blockade by depolarization. Like DMPP, OG 30 stimulated the dog heart, in vivo by an action at ganglia. However, unlike DMPP and other ganglion stimulants, OG 30 did not have a stimulant effect on isolated guinea pig heart. The in vitro cardiostimulant action of nicotine, DMPP and McN-A-343 is known but the existence of sympa-
thetic ganglia in the heart has been denied (Lee and Shideman, 1959; Leaders and Long, 1962; Bhagat, 1966). Bhagat (1966) concluded that these ganglionstimulants produce cardiac stimulation by liberating catecholamines directly from the stores associated with postganglionic sympathetic nerve endings. DMPP has further been shown to have a tyramine-like effect on isolated atria (Lindmar and Muscholl, 1961; Bhagat et al., 1967). OG 30 thus seems to act more specifically on the ganglia and lacks this extraganglionic action. An inhibitory effect on the heart associated with parasympathetic ganglia was also not consistently seen, again indicating a weaker parasympathetic ganglion stimulation with OG 30 (see Saxena, 1969). In view of the similar toxicity symptoms of OG 30 and DMPP (loss of righting reflex, respiratory arrest before cardiac arrest and cyanosis) and also because of the observation of Chen et al. (1951) that artificial respiration could prevent the toxicity of DMPP in rabbits, it is possible that death due to parenteral administration of these compounds is mainly, if not entirely, the result of respiratory failure due to neuromuscular block. Observations in spontaneously breathing anaesthetised cats, where a very high dose of OG 30 caused cessation of respiration before hypotension and stoppage of heart (fig. 3), also support the above statement. It was interesting to observe that even very large amounts of OG 30, a ganglionstimulant which lacks quaternary-ammonium character, were ineffective orally in mice or when injected in the alimentary canal of anaesthetised cats. Whether OG 30 is destroyed in the gut, is not absorbed orally, or is broken down in the liver after absorption, are points for consideration. OG 30 was inactive after administration at three different sites (stomach, duodenum and intestine), but since it was stable at acid pH, it was unlikely to be destroyed in the gut. Its destruction during passage through the liver after oral absorption was also unlikely since i.p. injections following which absorption into the blood takes place through the visceral rather than the parietal peritoneum (Goodman and Gilman, 1965), were effective. Moreover, administration of the same quantities of OG 30 through a systemic vein and a portal vein had similar effects. Thus, ineffectiveness of oral doses of OG 30 were probably due to poor absorption.
P.R.Saxena, Actions o f OG 30 ACKNOWLEDGEMENTS Grateful acknowledgement is made to Dr. H.J.Panneman for making available sufficient OG 30 for the study, and to Mr. P.van Houwelingen and Mr. H.Spierenburg for technical assistance in some of the experiments.
REFERENCES Bhagat, B., 1966, Response of isolated guinea-pig atria to various ganglion-stimulating agents, J. Pharmacol. Exptl. Therap. 154, 264-270. Bhagat, B., l.N.Robinson and W.L.West, 1967, Mechanism of sympathomimetic responses of isolated guinea-pig atria to nicotine and dimethylphenylpiperazinium iodide, Brit. J. Pharmacol. 30, 470-477. Boniface, K.J., O.J.Brodie and R.P.Walton, 1953, Resistance strain gauge arches for direct measurements of heart contractile force in animals, Proc. Soc. Exptl. Biol. Med. 84, 263-266. Brown, G.L., 1938, The preparation of tibialis anterior (cat) for close arterial injection, J. Physiol. (London) 92, 22-23P. Biilbring, E., 1946, Observations on the isolated phrenic nerve diaphragm preparation of the rat, Brit. J. Pharmacol. 1, 38-61. Chen, G. and R.Portman, 1954, Effect of 1,1-dimethyl4-phenylpiperazinium iodide on peristaiytic reflexes of isolated guinea pig ileum, Proc. Soc. Exptl. Biol. Med. 85, 245 -248. Chen, G., R.Portman and A.Wickel, 1951, Pharmacology of 1,1-dimethyl-4-phenylpiperazinium iodide, a ganglion stimulating agent, J. Pharmacol. Exptl. Therap. 103, 330-336. Goodman, L.S. and A.Gilman, 1965, The Pharmacological Basis of Therapeutics, 3rd ed. (Macmillan, New York) p. 8. Jindal, M.N. and V.R.Deshpande, 1960, Neuromuscular blockade by streptomycin and dihydrostreptomycin, Brit. J. Pharmacol. 15,506-509. Kaller, H., 1956, Pharmakologische Untersuchungen an der quergestreiften Augenmuskulatur der Ratte, Arch. Exptl. Pathol. Pharmakol. 229,297-304. Langendorff, O., 1895, Untersuchungen am fiberlebenden S~iugetierherzen,Arch. Ges. Physiol. 61, 291. Leach, G.D.H., 1957, Ganglion-blocking action of dimethylphenyl piperazinium (DMPP), J. Pharm. Pharmacol. 9, 747-751. Leaders, F.E. and J.P.Long, 1962, Mechanism of positive chronotropic response to nicotine, J. Pharmacol. Exptl. Therap. 137, 207-212. Lee, W.C. and F.E.Shideman, 1959, Mechanism of positive
83
inotropic response to certain ganglionic stimulants, J. Pharmacol. Exptl. Therap. 126,239-249. Lindmar, R. and E.Muscholl, 1961, Wirkung von Cocain, Guanethidin, Reserpin, Hexamethonium, Tetracain und Psicain auf die Noradrenalin-Freisetzung aus dem Herzen, Arch. Exptl. Pathol. Pharmakol. 242, 214-227. Ling, H.W., 1959, Actions of dimethyl phenylpiperazinium, Brit. J. Pharmacol. 14,505-511. Meyler, F.L., F.G.J.Offerijns, A.F.Willebrands and J.Groen, 1959, Contractie mechanisme en elektrolythuishouding van het geisoleerde, overlevende rattenhart, mede in verband met de werking van insuline, Ned. T. Geneeskunde 103, 1479-1486. Miller, L.C. and M.L.Tainter, 1944, Estimation of EDso and its error by means of logarithmic-probit graph paper. Proc. Soc. Exptl. Biol. Med. 57,261-264. Paton, W.D.M. and W.L.M.Perry, 1953, The relationship between depolarization and block in the cat's superior cervical ganglion, J. Physiol. (London) 119, 43-57. Saxena, P.R., 1969, A new ganglion-stimulating compound: 2-imino-l,3-diaza-4-carboxyethyl cycloheptane hydrochloride (OG 30), European J. Pharmacol. 7, 159-169. Saxena, P.R., H. Van Riezen and H.J. Panneman, 1970. Pharmacological properties of some analogues of 2-imino4-ethyloxy-carbonyl- 1,3-rliazacycloheptane hydrochloride (OG 30), a ganglionic stimulant, Arch. Intern. Pharmacodyn. Therap. 185, in press. Smith, J.C., 1966, Pharmacologic interactions with 4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium chloride, a sympathetic ganglion stimulant, 1. Pharmacol. Exptl. Therap. 153,276-284. Thesleff, S., 1955, Mode of neuromuscular block cau~d by acetylcholine, decamethonium, nicotine and succinylcholine, Acta Physiol. Scand. 34,218-231. Trendelenburg, U., 1954, The action of histamine and pilocarpine on the superior cervical ganglion and the adrenal ~lands of the cat, Brit. J. Pharmacol. 9,481-487. Trendelenburg, U., 1957, Reaktion sympathischer Ganglien wh~rend der Ganglienblockade durch Nicotin, Arch. Ex~tl. Pathol. Pharmakol. 230, 448-456. Trendelenburg, U., 1966a, Observations on the ganglion stimulating action of angiotensin and bradykinin, J. Pharmacol. Exptl. Therap. 154,418-425. Trendelenburg, U., 1966b, Transmission of preganglionic impulses through the muscarinic receptors of the superior cervical ganglion of the cat, J. Pharmacol. Exptl. Therap. 154,426-440. Van Riezen, H., 1968a, Classification of neuromuscular blocking agents in a new neuromuscular preparation of the chick in vitro, European J. Pharmacol. 5, 29-36. Van Riezen, H., 1968b, Effect of cholinergic and anticholinergic drugs on a new neuromuscular preparation of the chick, European J. Pharmacol. 5, 37-48.
GANGLION-BLOCKING,
NEUROMUSCULAR-BLOCKING
AND OTHER ACTIONS
OF 2-IMINO-4-ETHYLOXYCARBONYL-1,3-DIAZACYCLOHEPTANE HYDROCHLORIDE
(OG 30), A GANGLIONIC
STIMULANT
Pramod R. SAXENA Pharmacological Research Department, N. V. Organon, Oss, The Netherlands
Received 18 November 1969
Accepted 11 March 1970
P.R. Saxena, Ganglion-blocking, neuromuscular-blocking and other actions o f 2-imino-4-ethyloxycarbonyl-l,3-diazacycloheptane hydrochloride (OG 30), a ganglionic stimulant, European J. Pharmacol. 11 (1970) 000-000. 2-Imino-4-ethyloxycarbonyl-l,3-diazacycloheptane hydrochloride (OG 30) was previously found to be a ganglion-stimulating agent acting on nicotinic receptors. The purpose of the present investigation was to study further the pharmacological actions of OG 30. After initial stimulation, OG 30 caused a brief period of "non-depolarizing" ganglion block, in cats. It also impaired neuromuscular transmission in several in vivo and in vitro preparations by "depolarization". Although OG 30 stimulated the dog heart in vivo, it had no appreciable activity on the isolated guinea pig heart in contrast to DMPP. The LDso of OG 30 in mice by the i.v. and i.p. routes was 10.25 and 57.75 mg/kg, respectively. It was, however, non-toxic by the oral route even in large doses probably because of poor absorption. On the basis of the toxicity pattern, it has been argued that the compound causes death mainly by blocking neuromuscular transmission. OG 30
Ganglion stimulation
Neuromuscular block
1. INTRODUCTION Panneman (to be published) synthesized several compounds bearing a c o m m o n 2-imino-l,3-diazacycloalkane ring structure. One of these compounds, 2-imino-4-ethyloxycarbonyl-1,3-diazacycloheptane hydrochloride (OG 30), whose structure is shown in fig. 1, produced an increase in blood pressure and a contraction o f the nictitating membrane in anaesthetised cats. These effects of OG 30 were due to the stimulation of sympathetic ganglia (Saxena, 1969).
DMPP
Ganglion block
The compound was found to act on the nicotinic receptors of the ganglion cells and thus had a mode of action similar to 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), a recognised ganglionic stimulant (Chen et al., 1951). Since DMPP has also been shown to have a blocking action on ganglia (Chen and Portman, 1954; Leach, 1957) and neuromuscular junction (Kaller, 1956; Ling, 1959), it was thought interesting to study further the pharmacological actions o f OG 30.
2. METHODS -cooc~s
HN
-,y/
Nil
=,
~t Fig. 1. Strnctural formula of Ot3 30.
2.1. Ganglion-blockade 2.1.1. Intravenous (i.v.) administration of OG 30 Sixteen cats ( 2 - 4 kg) of either sex anaesthetised with pentobarbital sodium (30 mg/kg, i.p.) were employed for these experiments. They were kept on
76
P.R.Saxena, Actions of OG 30
positive pressure ventilation except for three cats in which spontaneous respiratory excursions were recorded with a Grass PT 5 volumetric transducer. Blood pressure was measured with a Statham P23AC transducer via a polyethylene cannula inserted in one of the femoral arteries. The semi-isometric contractions of the nictitating membrane, which had 4 g initial tension, was recorded with a Grass FT03 transducer. Heart rate was recorded on a Grass tachograph preamplifier (7P4A) triggered by ECG signals. The parameters were recorded on a model 7 Grass polygraph. Responses to adrenaline (3/ag/kg), bilateral occlusion of common carotid arteries for 30 sec (C.O.), and to stimulation (10 V, 1 msec, 25/sec for 15 sec) of the peripheral end of the sectioned vagus and the preganglionic cervical sympathetic nerves were obtained before and after administration of compound, OG 30. A Grass stimulator, model $4, was used. All drugs were injected i.v. into a cannulated femoral vein. 2.1.2. Direct injection of OG 30 to superior cervical ganglion Blood pressure and tension of the nictitating membrane were recorded, as stated above, in 5 cats anaesthetised with pentobarbital sodium (30 mg/kg) and ventilated artificially. A cannula was inserted into the central end of the cut lingual artery to inject the compounds directly to the superior cervical ganglion (Trendelenburg, 1954) of the corresponding side. 2.2. Neuromuscular blockade 2.2.1. Sciatic nerve-gastrocnemius preparation in cats Neuromuscular preparations were made in 8 cats anaesthetised with pentobarbital sodium (30 mg/kg, i.p.) and artificially ventilated. The peripheral end of the sectioned sciatic nerve received supramaximal electrical stimuli of 1 msec duration every 10 see from a model $4 stimulator. The semi-isometric contractions of the gastrocnemius muscle were recorded on the Grass polygraph by a thread connecting the tendon to a Grass FT 10 transducer. The effects of both i.v. and intra-arterial (i.a.) administration of the compound were studied. The i.a. injection was given peripherally into the femoral artery of the same side by a cannula inserted through one of its branches close to the iliac artery.
2.2.2. Common peroneal nerve-tibialis anterior preparation in cats (Brown, 1938) This preparation was set up in 6 cats in a manner similar to that described in 2.2.1. Close i.a. injections were delivered to the tibialis anterior muscle through the anterior tibial artery. 2.2.3. Isolated phrenic nerve diaphragm preparation of rat (Bulbring, 1946) Twelve experiments were set up in a 25 ml bath containing Krebs-Henseleit solution bubbled with a mixture of 95% 02 and 5% CO2 at 37°C. Direct or indirect stimuli of supramaximal voltage, 1 msec duration and 0.1/sec frequency derived from a Grass model S 4 stimulator were applied to the muscle. The contractions of the diaphragm were recorded either semi-isometrically with a Grass FT 03 transducer on the Grass polygraph or isotonically with a transducer on a Harvard recorder. 2.2.4. Isolated rectus abdominis muscle of frog (Jindal and Deshpande, 1960) The effect of compound OG 30 was studied in 10 experiments on the above muscle set up in a 5 or 25 ml bath containing frog-Ringer solution equilibrated with 95% 02 and 5% CO2 at 22°C. The contractions of the muscle were recorded on a smoked drum through a lever arrangement or by a transducer on a Harvard recorder. 2.3. Action on heart 2.3.1. Open chest preparation of dogs In three dogs anaesthetised with pentobarbital sodium (35 mg/kg, i.v.), the chest was split in the midline and the heart was exposed by cutting the pericardium. The force of contraction of the heart muscle was measured by a strain gauge arch (Boniface et al., 1953) attached to the right ventricle. Blood pressure was measured as in 2.1.1. 2.3.2. Perfused guinea-pig heart preparation (Langendorff, 1895) Six experiments were conducted on the heart perfused with Meyler's solution (Meyler et al., 1959) bubbled with 5% CO2 and 95% O2 at 37°C. Contractile force of the heart was recorded by a Grass FT 03 transducer attached via a spring and thread to its
77
P.R.Saxena, Actions of OG 30
2.6. Drugs The following compound dissolved in normal saline were used: acetylcholine chloride (Roche); adrenaline bitartrate; 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP); d-tubocurarine chloride (Fluka); 2-imino-4-ethyloxycarbonyl- 1,3-diazacycloheptane hydrochloride (OG 30); 4-(m-chlorophenylcarbamoyloxy)-2- but yniltrimethylammonium chloride (MeN-A-343); neostigmine methylsulphate (Prostigmine, Roche); physostigmine salicylate (BDH); potassium chloride (KC1), and succinylcholine chloride (suxamethonium). All doses are expressed in terms of the salt.
apex. The outflowing fluid was collected in a U tube with a side arm through which the fluid escaped every 20 see. The other limb was connected to a Grass PT5 volumetric transducer. Displacement of air in this limb have the measurement of the outflow every 20 sec. The events were registered on a model 7 Grass Polygraph. All drugs were injected directly into the aorta with the perfusion fluid through a rubber tube. 2.4. Acute toxicity The acute toxicity of compound OG 30 was determined in albino mice by the oral, i.p. and i.v. routes. Groups of ten mice were injected with different doses of the compound by the different routes of administration. The mice were observed continuously for 3 hr and later left overnight. The total number of mice dying during the 24 hr-period was recorded. The data were analysed by the method of Miller and Tainter (1944) to determine LDso.
3. RESULTS 3.1. Ganglion-blocking action
3.1.1. Effect on i.v. administration Administration of OG 30 (1 mg/kg) in 5 cats caused an increase in blood pressure and tension of the nictitating membrane similar to that described previously (Saxena, 1969). This dose of OG 30 did not modify the pressor effects of adrenaline (3/ag/kg) or of bilateral carotid occlusion (C.O.). Similarly, the bradycardia and hypotension caused by the stimulation of peripheral end of the divided vagus nerve, and the nictitating membrane contraction elicited by preganglionic cervical sympathetic nerve stimulation were unaffected showing lack of ganglionic block
2.5. Administration o f OG 30 into gastrointestinal tract or into a portal venous tributary Five cats were prepared for blood pressure measurement as described in 2.1.1. In 3 cats, OG 30 was injected by a needle after laparotomy into stomach, duodenum or small intestine. In the 2 other cats, the pressor effects of OG 30 injected through the femoral vein and through a tributary of the superior mesentric vein were compared.
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Fig. 2. Effect of OG 30 on the responses to simultaneous stimulation (10 V, 1 msec, 25/sec for 1S sec) of preganglionic cervical sympathetic and peripheral vagus nerves (S), bilateral occlusion of carotid arteries for 30 sec (C), and adrenaline, 3 ~ag/kg (A). Cat, 3.6 kg, 9. Records from above downwards; heart rate/rain, blood pressure (ram Hg), nictitating membrane tension (g). First panel, control responses; middle panel, drug effect; last panel; recovery after 10 min. Note the secondary fall in blood pressure and blockade of S by OG 30.
78
P.R.Saxena, Actions of OG 30
with the above dose. However, in 5 other cats, a higher dose (10mg/kg) caused, after an initial increase in heart rate, blood pressure and tension of the nictitating membrane, a definite fall in blood pressure accompanied by a short-lasting blockade of the responses produced by C.O., vagal stimulation, and preganglionic cervical sympathetic stimulation (see fig. 2). All these responses returned to normal within 10 min. These results indicated a short-lived ganglionblocking action of OG 30. In order to find the threshold dose blocking ganglionic transmission, the effects of different doses of OG 30 on the responses (see above) elicited by stimulation of the vagus and the preganglionic cervical sympathetic nerves were studied in three cats. OG 30 (up to 2 mg/kg) did not affect nictitating membrane contraction, hypotension or bradycardia,
but a dose of 2.5 mg]kg produced transient inhibition of these responses. Higher doses of OG 30 (5 and 10 mg/kg) completely blocked these effects. It was also observed in three spontaneously-breathing cats that when two doses of OG 30 (20 mg/kg) were administered shortly after each other, the second was inactive in causing a rise in blood pressure. Instead, bradycardia and hypotension occurred, and the animal died later; respiration failed before the heart (fig. 3). 3.1.2. Effect on i.a. injection to ganglion As has been reported earlier (Saxena, 1969), OG 30 caused a contraction of the nictitating membrane when injected directly to the superior cervical ganglion. However, when injected repeatedly in a sequence of 0.125, 0.25, 0.5, 0.5 mg every 1 to
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Fig. 3. Effect of two injections of 20 mg/kg, OG 30 (O) given at a short interval. Cat 2.6 kg, 9. Tracings from above downwards; respiration, blood pressure and heart rate/min. Note that the second dose of OG 30 fails to produce a pressor effect, but caused complete stoppage of respiration immediately followed by hypotension and bradycardia.
0.5 mg) on the nictitating membrane contraction in response to the stimulation (10V, 1 msec, 2S/sec for 10sec) of preganglionic cervical sympathetic nerve (S), and to i.a. injection of McN-A-343, 20 ug (M) and KC1, 4 mg (K) to the ganglion. Cat, 4 kg, C~.First panels, control responses; middle panels, drug effect; last panels, recovery in 10 min. Note that repeated injection of OG 30 blocks its own stimulatory action, and suppressed the responses to S and M without affecting those to K.
P.R.Saxena, Actions of OG 30
79
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Fig. 5. Effect of OG 30, 0.5 mg/kg (O) on the blood pressure (upper records) and gastrocnemius muscle contractions (lower records) in response to sciatic nerve stimulation (2 V, 1 msec, 0.1/sec). C~t, 2.75 kg, 9- Note that neostigmine slightly enhanced the neuromuscular block by OG 30. 2 rain, the last dose failed to contract the nictitating membrane (fig. 4) confirming that OG 30 can block its own ganglion-stimulating action. The effects of preganglionic cervical sympathetic stimulation and o f i.a. injections of McN-A-343, but not KC1, were reduced during OG 30-induced ganglion block (fig. 4).
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6 3.2. Neuromuscular-blocking action 3.2. l. Cat sciatic nerve-gastrocnemius preparation OG 30 (up to 2 mg/kg, i.v.) did not significantly affect the tension developed by the gastrocnemius muscle in response to sciatic nerve stimulation. When administered i.a, into the femoral artery of the same side, OG 30 (0.5 mg/kg) repeatedly reduced the contractions o f the muscle to nerve stimulation. The effect was more marked with high doses o f OG 30. The cholinesterase inh~itors, neostigmine ( 0 . 1 - 0 . 2 5 mg/kg, i.v.) or physostigmine (0.1 mg/kg, i.v.) slightly facilitated neuromuscular block induced by OG 30 (fig. 5). In two experiments DMPP (0.05 mg/kg, i.a.) was tested and found to be 3 0 - 4 0 times as effective in blocking the gastrocnemius contractions as OG 30. 3.2.2. Cat c o m m o n peroneal nerve-tibialis anterior muscle preparation On close i.a. injection, both acetylcholine (10/sg) and OG 30 (1 rag) caused a twitch response in the muscle without blockade o f the nerve stimulated muscle contractions (fig. 6, upper panels). Higher
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Fig. 6. Effect of close i.a. injection of OG 30 (O), acetylcholine (Ach) and suxamethonium (S) on the common peroneal nerve-tibialis anterior muscle preparation. Cat, 3.6 kg, q?. Peripheral end of the cut nerve was stimulated (3 V, 1 msec, 0.1/sec) except at upper bars where no stimulation was applied. T, tetanic stimulation (50/see for 5 sec). Note that both OG 30 and Ach caused a contraction of the non-stimulated muscle and that the OG 30-induced block of neuromuscular transmission was preceded by a facilitated contraction of the muscle. Tetanus was sustained (not clearly observed in the Figure because of slow chart speed) for the period of stimulation during the block caused by both OG 30 and suxamethonium and no post-tetanic twitch potentiation is evident.
80
P.R.Saxena, Actions o f OG 30
amounts of OG 30 (5 mg) caused a brief period of fasciculation and a potentiation of the indirectlyelicited twitch response before causing neuromuscular transmission block (fig. 6, upper panel). A tetanus elicited for 5 sec was well sustained and the post° tetanic twitch potentiation was not observed. Similar effects occurred with suxamethonium although the fasciculations were more marked and twitch potentiation was not evident. Tetanic response was also sustained (fig. 6, lower panel) and the post-tetanic twitch potentiation was absent. 3.2.3. Isolated phrenic nerve-diaphragm preparation of rat OG 30 (0.04-0.2 mg/ml) blocked contractions of the diaphragm elicited indirectly by stimulation of the phrenic nerve without appreciably affecting those produced by direct musclar stimulation. The neuromuscular blockade caused by OG 30 was not antagonised by neostigmine (2-10tag/ml) but was readily reversible upon changing the bath fluid. 3.2.4. Rectus abdominis muscle preparation of frog Three concentrations of OG 30 (5, 20 and 80 tag/ ml) potentiated contractions of the rectus muscle to acetyicholine (fig. 7). OG 30 did not cause any appreciable contraction of the muscle at these dose levels. High doses of OG 30 (160-1000 tag/ml) elicited muscle contractions that could be completely antagonised by d-tubocurarine (1 tag/ml).
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Fig. 7. Mean (+ S.E.M.) percentage potentiation by OG 30 of the acetylcholine (Ach)-induced contractions of the frog rectus abdominis muscle suspended in a 25 ml bath containing frog Ringer solution. Number of observations is given in the brackets. All values differ significantly (p ,(0.01) when compared to the control responses to acetylcholine in the absence of OG 30. 3.3. E f f e c t on heart
3.3.1. Open chest preparation of dog Both OG 30 and DMPP increased the blood pressure and the force of myocardial contraction in the open chest preparation of dog (fig. 8). Hexamethonium abolished the effects of both compounds.
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Fig. 8. Effect of OG 30, 0.5 mg/kg (O) and DMPP (D) on myocardial contractile force and blood pressure. Dog, 7.5 kg, d. Upper trace, time mark 5 sec; middle trace, myocardial contractile force; lower trace, blood pressure. OG 30 and DMPP increased the blood pressure and myocardial force; these effects were blocked after 5 mg/kg hexamethonium (at arrow); compare the two panels.
P.R.Saxena, Actions ofOG 30
81
Table 1 Acute toxicity of OG 30 in mice. Route of administration i.v.
i.p.
oral
Dose* (mg/kg) 7.5 11.25 15.0 30.0
% Mortality 20 80 100 100
25.0 50.0 75.0 100.0
0 30 70 100
50.0 300.0 1000.0 2000.0
0 0 0 0
LD~ff (mg/kg) 10.25
Toxicity symptoms clonic convulsion, loss of righting reflex, respixatory failure and cyanosis before stopping of the heart As above
57.75
NIL
NIL
* The number of mice injected at each dose level was 10. ** Calculated using the method of Miller and Tainter (1944).
3.3.2. Perfused guinea-pig heart On the isolated perfused guinea-pig heart, OG 30 (up to 5 mg) had no appreciable effect except for a small transient increase in coronary outflow or an occasional slight decrease in heart contraction. DMPP (10-20/ag) always increased the heart contractions after a slight initial decrease. 3.4. Acute toxicity The results presented in table 1 establish the LDso of OG 30 as 10.25 and 57.75 mg/kg by the i.v. and i.p. routes respectively. However, the compound was non-toxic orally, even in a dose of 2 g/kg. Under similar circumstances, the LDso of DMPP was 1.6 and 18.75 mg/kg by the i.v. and i.p. routes respectively. The toxic symptoms were the same as in the case of OG 30. 3.5. Effect of OG 30 after intra-intestinal injection Since OG 30 was non-toxic orally in mice it was injected into stomach, duodenum or small intestine of anaesthetised cats to see, ff it was ineffective by the enteral route in the cat. Injections of OG 30 (up to 60 mg/kg) did not raise the blood pressure when observed for up to four hours; the effective intravenous dose was 0.2 to 0.5 mg/kg.
3.6. Comparison of the effects of OG 30 by systemic or portal venous injection in cat To rule out the possibility that breakdown of OG 30 in the liver was responsible for its lack of activity after oral or enteral administration, the effects of injections into the femoral vein and into a tributary of the superior mesenteric vein were compared in two cats. OG 30 (1 mg/kg) produced a similar pressor response when injected by either route.
4. DISCUSSION Study of the ganglion-blocking action of OG 30 was prompted because its actions generally resembled those of DMPP which, in higher amounts, can block ganglia (Chen and Portman, 1954; Leach, 1957). It is evident from the results that OG 30 could also depress the ganglion-cells following an initial stimulant action. This ganglion-blockade by OG 30 was shortlived presumably due to rapid elimination of the compound from the site of action as is likely with DMPP (Chen and Portman, 1954). The impairment of ganglionic-transmission resulting from high doses of OG 30 does not appear to be due to depolarization
82
P.R.Saxena, Actions of OG 30
since responsiveness to KCI was unaffected (Trendelenburg, 1957). However, an initial depolarizing block, as has been conclusively demonstrated for nicotine (Paton and Perry, 1953; Trendelenburg, 1966a, 1966b) and suggested for DMPP (Leach, 1957), may have passed off even before KC1 could be injected, but, pending an electrophysiological study, it was concluded that OG 30 produced a "nondepolarizing" block. Suppression of McN-A-343 response by OG 30 may be similar to that observed between DMPP and McN-A-343 (Smith, 1966). The neuromuscular-blocking action of OG 30 was clearly demonstrated in both in vivo and in vitro experiments. This effect was mainly due to a depolarizing action, because of: (i) quick contraction of the non-stimulated tibialis anterior muscle on close i.a. injection, (ii) short period of fasciculation and potentiation of the maximal twitch before the block of neuromuscular transmission (see fig. 6), (iii) sustained tetanus and lack of post-tetanic twitch potentiation during neuromuscular block, (iv) slight potentiation (and not antagonism) of neuromuscular blocking action of OG 30 by anti-cholinesterases in cat gastrocnemius preparation, (v) a potentiation by OG 30 of acetvlcholine-induced frog rectus abdominis muscle contractions, (vi) a contracture of the rectus muscle by OG 30 itself and (vii) blockade by d-tubocurarine of the OG 30-induced rectus contractions. Furthermore, it has been observed (Saxena et al., 1970) that OG 30 resembled the actions of McN-A-343 on a new in vitro sciatic nerve-tibialis anterior muscle preparation (Van Riezen, 1968a, 1968b) in blocking the stimulant action of the depolarizing drugs in a lower concentration; higher amounts of OG 30 did increase the chick muscle tension and blocked the nervestimulated muscle contractions. The latter effect also points to a depolarizing action of OG 30. The other ganglionic stimulants, nicotine (Thesleff, 1955) and DMPP (Kaller, 1956; Ling, 1959) also cause neuromuscular blockade by depolarization. Like DMPP, OG 30 stimulated the dog heart, in vivo by an action at ganglia. However, unlike DMPP and other ganglion stimulants, OG 30 did not have a stimulant effect on isolated guinea pig heart. The in vitro cardiostimulant action of nicotine, DMPP and McN-A-343 is known but the existence of sympa-
thetic ganglia in the heart has been denied (Lee and Shideman, 1959; Leaders and Long, 1962; Bhagat, 1966). Bhagat (1966) concluded that these ganglionstimulants produce cardiac stimulation by liberating catecholamines directly from the stores associated with postganglionic sympathetic nerve endings. DMPP has further been shown to have a tyramine-like effect on isolated atria (Lindmar and Muscholl, 1961; Bhagat et al., 1967). OG 30 thus seems to act more specifically on the ganglia and lacks this extraganglionic action. An inhibitory effect on the heart associated with parasympathetic ganglia was also not consistently seen, again indicating a weaker parasympathetic ganglion stimulation with OG 30 (see Saxena, 1969). In view of the similar toxicity symptoms of OG 30 and DMPP (loss of righting reflex, respiratory arrest before cardiac arrest and cyanosis) and also because of the observation of Chen et al. (1951) that artificial respiration could prevent the toxicity of DMPP in rabbits, it is possible that death due to parenteral administration of these compounds is mainly, if not entirely, the result of respiratory failure due to neuromuscular block. Observations in spontaneously breathing anaesthetised cats, where a very high dose of OG 30 caused cessation of respiration before hypotension and stoppage of heart (fig. 3), also support the above statement. It was interesting to observe that even very large amounts of OG 30, a ganglionstimulant which lacks quaternary-ammonium character, were ineffective orally in mice or when injected in the alimentary canal of anaesthetised cats. Whether OG 30 is destroyed in the gut, is not absorbed orally, or is broken down in the liver after absorption, are points for consideration. OG 30 was inactive after administration at three different sites (stomach, duodenum and intestine), but since it was stable at acid pH, it was unlikely to be destroyed in the gut. Its destruction during passage through the liver after oral absorption was also unlikely since i.p. injections following which absorption into the blood takes place through the visceral rather than the parietal peritoneum (Goodman and Gilman, 1965), were effective. Moreover, administration of the same quantities of OG 30 through a systemic vein and a portal vein had similar effects. Thus, ineffectiveness of oral doses of OG 30 were probably due to poor absorption.
P.R.Saxena, Actions o f OG 30 ACKNOWLEDGEMENTS Grateful acknowledgement is made to Dr. H.J.Panneman for making available sufficient OG 30 for the study, and to Mr. P.van Houwelingen and Mr. H.Spierenburg for technical assistance in some of the experiments.
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