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The behavioural, cardiovascular and respiratory actions of morphine-N-oxide in the dog
EUROPEAN JOURNAL OF PHARMACOLOGY 8 (1969) 261-268. NORTH-HOLLANDPUBLISHINGCOMP., AMSTERDAM
THE BEHAVIOURAL, CARDIOVASCULAR AND RESPIRATORY A C T I O N S O F M O R P H I N E - N - O X I D E IN T H E D O G M.R. FENNESSY Department of Pharmacology, University of Melbourne, Parkville, Victoria 3052, Australia
Received 16 June 1969
Accepted 17 September 1969
M.R. FENNESSY, The behavioural, cardiovascular and respiratory actions of morphine-N-oxide in the dog, European J. Pharmacol. 8 (1969) 261-268. A comparison between the actions of morphine and morphine-N-oxide (MNO) on behaviour, blood pressure, heart rate and respiration of both conscious and anaesthetised dogs has been studied. In the consciousdog morphine was more potent than MNO in producing sedation when both drugs were given subcutaneously. MNO had no antimorphine activity. Intravenously administered morphine or MNO caused sham rage, tachycardia and an increased respiratory rate in the conscious dog. Morphine was more potent than MNO in producing these actions. The effects of these drugs on the blood pressure of conscious dogs varied; all doses of morphine caused pressor responses, whereas small doses of MNO produced pressor responses while larger doses cause depressor responses. In the anaesthetised dog intravenous injections of morphine or MNO produced biphasic depressor responses, bradycardia and decreased respiratory rate. Any difference in activity between these drugs may be due to morphine having a greater ease of entry into the brain than MNO. Morphine Morphine-N-oxide
Conscious dog Anaesthetised dog
1. INTRODUCTION It has previously been suggested that morphineN-oxide (MNO) is an intermediary metabolite of morphine (Woo, Gaff and Fennessy, 1968). MNO possesses a weak analgesic action compared with morphine (Fennessy, 1968) but is considerably less toxic than the parent compound (Fennessy and Fearn, 1969). In the present studies behavioural, cardiovascular and respiratory actions of MNO have been compared with those of morphine in conscious and anaesthetised dogs.
2. METHODS 2.1. Conscious dogs Behavioural and cardiovascular responses were measured in conscious dogs ( 1 0 - 2 0 kg) using the
Behaviour Cardiovascular
method described by Fennessy and Ortiz (1968). Blood pressure was recorded from a cannulated exteriorized carotid artery using a Statham pressure transducer (P23 AA) coupled to an ink writing pen recorder (Offner Dynograph 504A). Lead II electrocardiograms were monitored using a Cardiofax MS3 electrocardiograph. Intravenous injections were made into a cannulated cephalic vein. 2.2. Anaesthetised dogs Dogs (10-15 kg) were anaesthetised by an intravenous injection of pentobarbitone sodium (30 mg/kg). Blood pressure was recorded as described above and heart rate was monitored using a Beckman Type 9851 cardiotachometer. Respiratory excursions were recorded by a resistance strain gauge tied around the thorax of the dog. Records were displayed on the pen recorder. Drugs were injected intravenously into a cannulated cephalic vein.
262
M.R.FENNESSY
2.3. Drugs The following drugs were used: morphine sulphate, morphine-N-oxide (MNO), tacrine, amiphenazole hydrochloride, nalorphine hydrobromide, atropine sulphate, mepyramine maleate, compound 48/80, chlorpromazine hydrochloride, tetrasodium ethylenediaminetetraacetate (EDTA), methysergide bimaleate, propanolol hydrochloride, hexamethonium bromide, reserpine (Serpasil) and pentobarbitone Na. The concentrations of morphine, MNO, tacrine and reserpine are expressed in terms of base while the concentrations of all other drugs are expressed in terms of their salts. MNO was prepared according to the method of Freund and Speyer (1910). Solutions of MNO for injection were freshly prepared each day by dissolving it in 0..1 M HC1 and adjusting to pH 5 with NaOH. Dogs were reserpinized with daily intraperitoneal injections of 0.5 mg/kg of reserpine for two days, and with 0.25 mg/kg on the morning of the third day. The dogs were used on the afternoon of the third day.
3. RESULTS 3.1. Behaviour in conscious dogs 3.1.1. Subcutaneous administration Subcutaneous injections of morphine (7.5 mg/kg) in conscious dogs produced sedation culminating in deep sleep 30 rain after administration. This stage of narcosis lasted from 6 to 8 hr. Within 15 rain after morphine the respiratory rate usually increased about three-fold and all dogs which had not received hyoscine (0.6 mg]kg) vomited. The respiratory rate gradually returned to normal over the next 2 hr and very seldom did it fall below control levels. MNO in doses up to 25 mg/kg when injected subcutaneously, produced little or no depression and certainly no sign of narcosis. After MNO the dogs were much quieter than normal, and unlike the effects of morphine, there was never any sign of disorientation or lack of coordination in sitting, standing and walking. Respiratory rate increased as with morphine but no vomiting was observed in the dogs after injections of MNO. The ability of drugs to reverse morphine narcosis has been used by Shaw, Gershon and Bentley (1957)
to assess antimorphine activity. Drugs to be tested for analeptic activity are injected intravenously 45 min after the onset of narcosis. A good arousal was recorded if the dog stood up and walked spontaneously; a mild arousal if the animal sat up spontaneously but walked only when stimulated; a slight arousal when the dog raised only its head when stimulated. Nalorphine, amiphenazole and tacrine aroused dogs narcotised with morphine in a dose dependent manner (table 1). Within 5 min of the administration of any of these drugs the dogs were standing and walking, respiration was stimulated and excitement was observed in most cases. MNO possessed no antimorphine activity but in doses which showed no narcotic action themselves the effects of morphine appeared to be potentiated (table 1). 3.1.2. Intravenous administration Fast intravenous injections of morphine in the conscious dog produced sham rage. This behavioural response has already been described (Domer and Josselson, 1964; Fennessy and Oritz, 1968). The duration of the rage response is dose dependent (table 2) and the characteristic effects include violent struggling, vocalization, hyperventilation, salivation, urination and defaecation. Intravenous injections of MNO also produced sham rage, the threshold being approximately 5 mg/kg (table 2). MNO had about 1/10th-1/20th the potency of morphine in producing the characteristic pattern of sham rage. As with morphine, subsequent injections of MNO given 30 min to 48 hr after the initial administration did not cause sham rage, and at least 4 days had to elapse before morphine or MNO again caused the rage response. The sham rage induced by MNO (10 mg/kg) or morphine (2 mg/kg) was abolished by intravenously pretreating dogs with doses of morphine (0.2 mg/kg) or MNO (2 mg/kg) which by themselves did not produce sham rage. Other drugs which abolished the rage response were nalorphine (1 mg/kg), amiphenazole (20 mg/kg), tacrine (2 mg/kg), chlorpromazine (2 mg/kg), mepyramine (2 mg/kg), compound 48/80 (200/~g/kg) and EDTA (5 mg/kg). These drugs were injected 1/2 hr before the administration of MNO or morphine except for compo~und 48/80 which was given 2 hr previously. As with morphine the MNO-induced rage was not abolished
MORPHINE-N-OXIDE (MNO)
263
Table 1 The effect of morphine-N-oxide, nalorphine, amiphenazole and tacrine on the arousal of dogs narcotized with subcutaneously administered morphine (7.5 mg/kg). These drugs were injected intravenously 45 min after the administration of morphine. The figures in parentheses represent the number of animals used. Dose
Drug Morphine-N-oxide
Nalorphine hydrobromide
(mg/kg)
Arousal Respiratory effects
Other observations
1.0 (2) 2.5 (3) 5.0 (4) 10.0 (3) 20.0 (3) 25.0 (3)
None None None None None None
All doses of morphine-N-oxide produced Dogs appeared to be more deeply natrespiratory stimulation 1-2 min after cotized after morphine-N-oxide injection. The rate decreased over the next 10 min and by 30 min respiration had almost ceased. (Dogs were resuscitated by injection of nalorphine.)
1 (6) 2 (4)
Good Good
Respiratory rate usually doubled within 2 min
Tremors, restlessness and slight excitement
Amiphenazole
10 20 30 40
(3) (4) (6) (5)
Slight Mild Good Good
Slight increase in respiratory rate
Little excitement
Tacrine
1 2 5 10
(4) (5) (7) (4)
None Mild Good Good
Respiratory rate increased within the dose range of 5-10 mg/kg
Once aroused dogs showed pronounced excitement, convulsions occurred with doses over 5 mg/kg
b y methysergide (0.5 mg/kg), atropine (1 mg/kg), propranolol (1 mg/kg), hexamethonium (10 mg/kg) or reserpine.
3.2. Cardiovascular responses 3.2.1. Conscious do.gs The effects of intravenously administered morTable 2 Duration of sham rage responses of conscious dogs to intravenous morphine or morphine-N-oxide. T~he figures in parentheses are the number of animals used. Morphine *
Morphine-N-oxide
Dose (mg/kg)
Mean duration (sec) + S.E.
Dose (mg/kg)
Mean duration (sec) +_S.E.
0.19 0.38 0.75 1.50 3.75 7.50
0 .4.7 11.8 25.8 44.0 49.6
0.25 0.50 1 2 5 10
0 (6) 0 (6) 0 (8) 0 (5) 6.2 +_ 1.7 (6) 15.9 + 2.4 (6)
(5) _+1.3 (6) +_ 1.9 (6) + 2.4 (5) + 3.5 (4) _+2.5 (5)
* The results for the duration of sham rage of morphine are taken from Fennessy and Ortiz (1968).
phine and MNO on the blood pressure and heart rate o f the conscious dog are shown in fig. 1 and table 3. Initial doses o f morphine, up to 7.5 mg]kg, regularly produced sharp but transient increases in blood pressure. Weaker pressor responses were elicited by the lower doses of MNO up to 2 mg/kg with maximal effect being produced by a dose o f 1 mg/kg. However, initial injections of 5 to 10mg/kg of MNO caused depressor responses. After an initial dose o f either drug had caused a pressor ,esp,~nse, the same dose repeated 15 min later t',ad no effect on the blood pressure, but when the second dose was increased, there was a sharp transient depressor response to which tolerance rapidly developed (fig. I). Fig. 1C shows that successive doses of 5 mg/kg of MNO given every 15 min produced depressor responses which gradually decreased in amplitude. The initial depressor response obtained with 5 or 10 mg/kg of MNO was not altered by atropine (1 mg/kg) but was significantly reduced by mepyramine (1 m g / k g ) o r nalorphine (1 mg/kg). Both morphine and MNO produced a dosedependent tachycardia lasting approximately 1 min in duration. Tolerance gradually developed to this effect
264
M.R.FENNESSY
Table 3 Cardiovascular responses of conscious dogs to intravenous injections of morphine and morphine-N-oxide. These responses represent the effect of initial doses of either drug. The figures in parentheses are the number of animals. Morphine *
Morphine-N-oxide
Dose (mg/kg)
Mean increase in blood pressure (ram Hg) _+S.E.
Mean increase in heart rate (beats/min) + S.E.
0.19 0.38 0.75 1.50 3.75 7.50
88.3 + 5.1 (4) 119.2_+ 12,7 (5) 125.8 + 6,4 (6) 136.3 + 10.6 (4) 134.0+15.8(3) 141.7 + 23.2 (3)
55.6 _+ 6.2 (4) 60.7 + 9.9 (5) 132.0_+ 18.5 (6) 186.1 _+16.7 (4) Sham rage too violent to record heart rate or ECG
Dose (mg/kg)
0.25 0.5 1 2 5 10
Mean increase in blood pressure (mm Hg) _+S.E.
Mean increase in heart rate (beats/rain) _+S.E.
15.6 _+ 3.3 (3) 57.3 + 6.2 (6) 21.3 _+ 5.7 (4) -78.4_+11.6(3) -89,7 _+14.4 (3)
16.4 + 3.3 (3) 52.3 + 8.6 (3) 115.6 + 9.2 (6) 150.0 + 13.8 (4) 192.1+20.7(3) Sham rage too violent to record heart rate of ECG
* The responses to morphine are taken from Fennessy and Ortiz (1968). and by about the fifth injection no further tachycardia was observed with either drug. Both drugs appeared to be equipotent in their ability to produce tachycardia (table 3), and neither affected the ECG.
BP mmH 9 lrnin ~
(a)
50,-
X
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,i
~,
0"75
0"75
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7'5
~: ":%, ~__a= MNO
M~
1
,,,
1
MNO
S
S
.
o
MNO 5
5
$
MNO 5
Fig. 1. Effects on the blood pressure of the conscious dog produced by intravenous injections of morphine (M) and morphine-N-oxide (MNO). The figure under each panel is the dose in mg/kg. Doses were given at intervals of 15 min. Traces (a), (b) and (c) are from separate experiments.
3.2.2. Anaethetised dogs The effects of morphine and MNO on the blood pressure of the anaesthetised dogs are summarized in table 4 and illustrated in fig. 2. Initially both drugs cause a biphasic depressor response consisting of a sharp immediate fall with partial recovery followed by a more prolonged fall with gradua! recovery. This effect occurred about 10 sec ai-ter intravenous administration. Morphine and MNO were equipotent in depressing blood pressure except at the lower dose levels where morphine was more potent (table 4). Pretreatment with 1 mg/kg of atropine had no effect on the depressor response produced by morphine or MNO whereas mepyramine (2 mg/kg) completely abolished the first phase of the depressor respo!ase and partially reduced the second phase (table 5). On the other hand, nalorphine (1 mg/kg) did not affect the first phase but abolished the second phase of the depressor response. When dogs were pretreated with both mepyramine and nalorphine, both phases of the depressor response were abolished. When the injections of morphine or MNO were repeated eve,ry 15 rain the depressor responses decreased and b y the fourth or fifth administration no further effect was observed (fig. 2).
MORPHINE-N-OXIDE (MNO)
265
Table 4 Cardiovascular responses of anaesthetised dogs to morphine and morphine-N-oxide. Dogs were anaesthetised with an intravenous injection of pentobarbitone (30 mg/kg). The responses represent the effect of initial intravenous injections of morphine or morphine-N-oxide. The figures in parentheses are the number of observations. Morphine
Dose (mg/kg)
Mean decrease in blood pressure (mm Hg) +_S.E.
0.19
42.3 + 11.2 (3)
0.38 0.75 1.50
68.7 + 9.4 (5) 85.0 + 13.6 (6) 89.5 + 19.4 (3)
Morphine-N-oxide Mean decrease in heart rate (beats/min) +S.E. 0
Mean decrease in blood pressure (mm Hg) +S.E.
Dose (mg/kg)
(3)
0.25
15.6 + 6.1 (4)
12.2 + 3.6 (5) 24.6 + 5.2 (6) 29.3 + 5.8 (3)
0.5 0.75 1.5
26.3 + 8.4 (4) 78.3 +__10.7 (5) 85.7 + 15.3 (4)
Both morphine and MNO produced a slight and transient bradycardia (table 4). This effect occurred within 15 sec of adminstration, and by 2 min the heart rate had geturned to the control level. At the higher dose levels morphine and MNO were equipotent in depressing heart rate. Neither drug affected the ECG. Atropine (lmg/kg) or mepyramine (2 mg/kg) did not effect the bradycardia whereas nalorphine (1 mg/kg) completely abolished it (table 5). Tolerance gradually developed to the morphine- or MNO-induced bradycardia. 3.3. Respiration in anaesthetised dogs Both morphine and MNO when injected intravenously produced an immediate increase in respiratory rate of the anaesthetised dog (fig. 3). This effect occurred about 10 sec after administration. Morphine was approximately 30 to 50 times more potent than MNO. Morphine also caused an immediate increase in the depth of respiration. The increased respiratory rate lasted about 3 min for both drugs and a gradual tolerance to this effect was seen after repeated administration. At no stage was a decrease in respiratory rate observed with either drug.
4. DISCUSSION The results presented in this paper indicate that the pharmacological actions of morphine and MNO in the dog appear to be similar and are dependent on the
Mean decrease in heart rate (beats/min) +_S.E. 0
(4)
0 (4) 17.3 + 4.3 (5) 30.0 + 6.1 (4)
route of administration and on the level of consciousness of the dog. In the conscious dog subcutaneous injections of morphine or MNO produce depression whereas rapid intravenous injections of either drug produce sham rage followed by depression. A difference was observed on the cardiovascular system of the dog. Morphine (up to 7.5 mg/kg) and 1rain mmHg
(a)
::°of
::f ~
,~m .~
•
Q
•
•
M
M
M
M
O'?S
O-~S
~S
0,7S
(b~
ISO~-
';t
•
•
MNO
MNO
MNO
MNO
0.75
0"~$
0'TS
O'TS
•
•
Fig. 2. The effects of intravenous injections of morphine (M) or morphine-N-oxide (MNO) on the blood pressure of dogs anaesthetised with pentobarbitone Na. The figure under each panel is the dose in mg/kg. Doses were given at 15 min intervals. Traces (a) and (b) are from two separate experiments.
266
M.R.FENNESSY
Table 5 Cardiovascular responses to morphine and morphine-N-oxide in anaesthetised dogs after pretreatment with atropine, mepyramine or nalorphine. Mean decrease in blood pressure (mm Hg) -+ S.E.
Mean decrease in heart rate (beats/min) _+S.E.
Morphine (0.75 mffkg)
Morphine-N-oxide (0.75 mg/kg)
Morphine (0.75 mg/kg)
Morphine-N-oxide (0.75 mg/kg)
85.0 82.9 30.5 76.2
78.3 73.3 34.6 69.0
24.6-+ 5.2 (6) 24.4 + 4.8 (4) 27.2 -+ 6.4 (5) 0 (6)
17.3 -+ 4.6 (5) 15.5 -+ 6.7 (4) 20.0 + 6.1 (4) 0 (5)
Premedication
Control Atropine (1 mg/kg) Mepyramine (2 mg/kg) Nalorphine (1 mg/kg) Mepyramine (2 mg/kg) + nalorphine (1 mg/kg)
-+ 13.6 (6) -+ 9.4 (4) -+ 5.7 (5) -+ 10.4 (6)
10.6 -+ 1.3 (3)
8.5 -+ 1.7 (4)
low doses of MNO (up to 2 mg/kg) produced pressor responses whereas doses of 5 mg]kg and higher of MNO caused depressor responses in the conscious dog. In the anaesthetised animal both drugs lowered the blood pressure. The observations that dogs narcotized with morphine were aroused with aminophenazole or tacrine confirms the findings of Shaw and Bentley (1949, 1952) that these drugs are antagonists of some of the
(a)
ln~in
038
0.38
0 38
0'38
(b)
~NO 'I
~NO I
~NO I
-+10.7(5) -+19.4 (4) -+ 4.2 (4) -+ 8.8 (5)
~NO 'I
Fig. 3. The effect of intravenous injections of morphine (M) or morphine-N-oxide (MNO) on respiratory rate and depth of the dog anaesthetised with pentobarbitone Na. The figure under each panel is the dose in mg/kg. Drugs were injected at 15 min intervals. At T the tension on the strain gauge was reduced.
0
(3)
0
(4)
central depressant actions of morphine. MNO in doses up to 25 mg/kg produced slight sedation but no narcosis, and, in contrast to morphine, MNO did not cause vomiting. MNO, which is closely related to both morphine and nalorphine, did not antagonize the morphine-induced narcosis but appeared to deepen it which suggests that MNO is not a narcotic antagonist but rather, it is a drug having similar but less potent activity than morphine. This is also evident from the sham rage experiments in which MNO was much less active than morphine. Since the sham rage reaction was abolished by drugs which competitively antagonize or prevent release of histamine it is probable that histamine release in the CNS is involved (Domar and Josselson, 1964; Fennessy and Ortiz, 1968). The low activity o f MNO compared with that of morphine may be a consequence of its decreased ease of entry into the CNS since it has been reported that N-oxides are less lipid soluble than their parent bases (Culvenor, 1953). It has been suggested that the morphine-induced pressor response in the conscious dog may be due in part to a central sympathetic stimulation and partly to a histamine-induced release o f catecholamines from the periphery (Fennessy and Ortiz, 1968). With MNO however, pressor responses occurred with low doses and depressor responses with higher doses. The pressor response was not accompanied by sham rage whereas the rage response was exhibited with a depressor response. From this and the results obtained with morphine it would seem that the effects of
MORPHINE-N-OXIDE(MNO) these drugs on the blood pressure and on behaviour of the conscious dog are independent of one another. Another explanation may be that the threshold dose of morphine needed to produce a pressor response is lower than that required to cause sham rage and that, in fact, these responses are not independent but are produced by the same mechanism. The actions of MNO on the blood pressure of conscious dogs are difficult to interpret. One explanation could be based on the ease of entry of MNO into the CNS. With low doses the small amounts of MNO passing the blood-brain barrier may be sufficient to liberate histamine in subthreshold quantities for the induction of sham rage. However this released histamine may be of sufficient concentration to stimulate central sympathetic centres and induce a pressor response. Peripherally released histamine may also produce catecholamine secretion, an effect which would summate with the central sympathetic influences. With high doses of MNO sham rage may be induced which is accompanied by a depressor response. In these cases sufficient MNO may pass the blood-brain barrier to precipitate sham rage. However, much of the increased dose of MNO may act peripherally, releasing sufficiently large amounts of histamine to overcome the sympathetically induced pressor response by its vasodilator action. The observation that mepyramine antagonized the depressor response of MNO indicates a histaminergic origin. A corollary of thes0 observations may be that morphine, when given in sufficient quantity, may cause a fall in blood pressure as was reported by Schmidt and Livingston (1933) after doses of 200 mg/kg of morphine to conscious dogs. The actions of morphine and MNO on the blood pressure of anesthetised dogs were similar. Initially, both drugs produced biphasic depressor responses which were unaffected by atropine. The first phase of the response was abolished by mepyramine whereas the second phase was antagonized by nalorphine. These results are in agreement with those of Kayaalp and Kaymakcalan (1966) who suggested that, in the anaesthetised cat, the first phase of the morphineinduced depressor response was due to histamine release whereas the second phase was due to a central depressant action of morphine. Nalorphine did not antagonize this histamine releasing action of mor¢
267
phine or MNO, whereas mepyramine did not alter the central action. In the conscious dog both morphine and MNO caused tachycardia whereas in the anaesthetised dog both drugs produced slight bradycardia. Neither atropine nor mepyramine affected the bradycardia in anaesthetised dogs whereas it was abolished by nalorphine. This suggests that these analgesics depress the heart rate not by an increase in vagal activity but maybe by a direct depressant action. In the conscious dog the morphine-induced tachycardia has been shown to be reduced by nalorphine, and by drugs affecting histaminergic and adrenergic mechanisms (Fennessy and Ortiz, 1968). These authors suggested that morphine may have a direct stimulant action on the heart. However since the actions of morphine or MNO on the heart have opposite effects at different levels of consciousness of the dog it is difficult to postulate a mechanism of action and it is obvious that further work is required. Morphine and MNO produced respiratory stimulation in both concious and anaesthetised dogs. Similar effects have been shown in conscious dogs with morphine by Martin and Eades (1961) who suggest that the respiratory rate is increased in association with maximal loss of body heat. In addition the CNS stimulant action of morphine may also be partly responsible. In anaesthetised dogs the increase in respiratory rate may be due to baroreceptor stimulation as a consequence of the depressor response.
ACKNOWLEDGEMENTS Thanks are due to Drs. C. Raper and L. Mashford for their criticisms of the manuscript and to H.W. Woods Pry. Ltd. for providing a grant for technical assistance.
REFERENCES Culvenor, C.C.J., 1963, Amine oxides, Rev.Pure Appl.Chem. 3,83. Domer, F.R. and F. Josselson, 1964, Behavioural effect of intravenous morphine administration to the unaesthetised dog, Arch. Intern. Pharmacodyn. 147,470.
268
M.R.FENNESSY
Fennessy, M.R., 1968, The analgesic action of morphineN-oxide, Brit. J. Pharmacol. 34,337. Fennessy, M.R. and H.J. Fearn, 1969, Some observations on the toxicology of morphine-N-oxide, J, Pharm. Pharmacol. 21,668. Fennessy, M.R. and A. Ortiz, 1968, The behavioural and caxdiovascular actions of intravenously administered morphine in the conscious dog, Europ. J. Pharmacol. 3, 177. Freund, M. and E. Speyer, 1910, Enwirkung von Wasserstoff superoxyd auf Thebain, Morphin und dessert Ather, Bet. Deut. Chem. Ges. 43, 3310. Kayaalp, S.O. and S. Kaymakcalan, 1966, A comparative study of the effects of morphine in unanaesthetised and anaesthetised cats, Brit. J. Pharmacol. 26,196. Martin, W.R. and C.G. Eades, 1961, Demonstration of tolerance and physical dependence in the dog following a short term infusion of morphine. J. Pharmacol. exptl. Therap. 133,262.
Schmidt, C.F. and A.E. Livingston, 1933, The action of morphine on the mammalian circulation, J. Pharmacol. Exptl. Therap. 47, 411. Shaw, F.H. and G.A. Bentley, 1959, Some aspects of the pharmacology of morphine, with special reference to its antagonism by 5-amino acridine and other chemically related compounds, Med. J. Australia 2,868. Shaw, F.H. and G.A. Bentley, 1952, Morphine antagonism, Nature 169, 712. Shaw, F.H., S. Gershon and G.A. Bentley, 1957, Morphine antagonism, J. Pharm. Pharmacol. 9,666. Woo, J.T.C., G.A. Gaff and M.R. Fennessy, 1968, A note on the effects of 2,4-diamino-5-phenylthiazole and 1,2,3,4-tetrahydro-9-aminoacridine on morphine metabolism, J. Pharm. Pharmacol. 20, 763.
THE BEHAVIOURAL, CARDIOVASCULAR AND RESPIRATORY A C T I O N S O F M O R P H I N E - N - O X I D E IN T H E D O G M.R. FENNESSY Department of Pharmacology, University of Melbourne, Parkville, Victoria 3052, Australia
Received 16 June 1969
Accepted 17 September 1969
M.R. FENNESSY, The behavioural, cardiovascular and respiratory actions of morphine-N-oxide in the dog, European J. Pharmacol. 8 (1969) 261-268. A comparison between the actions of morphine and morphine-N-oxide (MNO) on behaviour, blood pressure, heart rate and respiration of both conscious and anaesthetised dogs has been studied. In the consciousdog morphine was more potent than MNO in producing sedation when both drugs were given subcutaneously. MNO had no antimorphine activity. Intravenously administered morphine or MNO caused sham rage, tachycardia and an increased respiratory rate in the conscious dog. Morphine was more potent than MNO in producing these actions. The effects of these drugs on the blood pressure of conscious dogs varied; all doses of morphine caused pressor responses, whereas small doses of MNO produced pressor responses while larger doses cause depressor responses. In the anaesthetised dog intravenous injections of morphine or MNO produced biphasic depressor responses, bradycardia and decreased respiratory rate. Any difference in activity between these drugs may be due to morphine having a greater ease of entry into the brain than MNO. Morphine Morphine-N-oxide
Conscious dog Anaesthetised dog
1. INTRODUCTION It has previously been suggested that morphineN-oxide (MNO) is an intermediary metabolite of morphine (Woo, Gaff and Fennessy, 1968). MNO possesses a weak analgesic action compared with morphine (Fennessy, 1968) but is considerably less toxic than the parent compound (Fennessy and Fearn, 1969). In the present studies behavioural, cardiovascular and respiratory actions of MNO have been compared with those of morphine in conscious and anaesthetised dogs.
2. METHODS 2.1. Conscious dogs Behavioural and cardiovascular responses were measured in conscious dogs ( 1 0 - 2 0 kg) using the
Behaviour Cardiovascular
method described by Fennessy and Ortiz (1968). Blood pressure was recorded from a cannulated exteriorized carotid artery using a Statham pressure transducer (P23 AA) coupled to an ink writing pen recorder (Offner Dynograph 504A). Lead II electrocardiograms were monitored using a Cardiofax MS3 electrocardiograph. Intravenous injections were made into a cannulated cephalic vein. 2.2. Anaesthetised dogs Dogs (10-15 kg) were anaesthetised by an intravenous injection of pentobarbitone sodium (30 mg/kg). Blood pressure was recorded as described above and heart rate was monitored using a Beckman Type 9851 cardiotachometer. Respiratory excursions were recorded by a resistance strain gauge tied around the thorax of the dog. Records were displayed on the pen recorder. Drugs were injected intravenously into a cannulated cephalic vein.
262
M.R.FENNESSY
2.3. Drugs The following drugs were used: morphine sulphate, morphine-N-oxide (MNO), tacrine, amiphenazole hydrochloride, nalorphine hydrobromide, atropine sulphate, mepyramine maleate, compound 48/80, chlorpromazine hydrochloride, tetrasodium ethylenediaminetetraacetate (EDTA), methysergide bimaleate, propanolol hydrochloride, hexamethonium bromide, reserpine (Serpasil) and pentobarbitone Na. The concentrations of morphine, MNO, tacrine and reserpine are expressed in terms of base while the concentrations of all other drugs are expressed in terms of their salts. MNO was prepared according to the method of Freund and Speyer (1910). Solutions of MNO for injection were freshly prepared each day by dissolving it in 0..1 M HC1 and adjusting to pH 5 with NaOH. Dogs were reserpinized with daily intraperitoneal injections of 0.5 mg/kg of reserpine for two days, and with 0.25 mg/kg on the morning of the third day. The dogs were used on the afternoon of the third day.
3. RESULTS 3.1. Behaviour in conscious dogs 3.1.1. Subcutaneous administration Subcutaneous injections of morphine (7.5 mg/kg) in conscious dogs produced sedation culminating in deep sleep 30 rain after administration. This stage of narcosis lasted from 6 to 8 hr. Within 15 rain after morphine the respiratory rate usually increased about three-fold and all dogs which had not received hyoscine (0.6 mg]kg) vomited. The respiratory rate gradually returned to normal over the next 2 hr and very seldom did it fall below control levels. MNO in doses up to 25 mg/kg when injected subcutaneously, produced little or no depression and certainly no sign of narcosis. After MNO the dogs were much quieter than normal, and unlike the effects of morphine, there was never any sign of disorientation or lack of coordination in sitting, standing and walking. Respiratory rate increased as with morphine but no vomiting was observed in the dogs after injections of MNO. The ability of drugs to reverse morphine narcosis has been used by Shaw, Gershon and Bentley (1957)
to assess antimorphine activity. Drugs to be tested for analeptic activity are injected intravenously 45 min after the onset of narcosis. A good arousal was recorded if the dog stood up and walked spontaneously; a mild arousal if the animal sat up spontaneously but walked only when stimulated; a slight arousal when the dog raised only its head when stimulated. Nalorphine, amiphenazole and tacrine aroused dogs narcotised with morphine in a dose dependent manner (table 1). Within 5 min of the administration of any of these drugs the dogs were standing and walking, respiration was stimulated and excitement was observed in most cases. MNO possessed no antimorphine activity but in doses which showed no narcotic action themselves the effects of morphine appeared to be potentiated (table 1). 3.1.2. Intravenous administration Fast intravenous injections of morphine in the conscious dog produced sham rage. This behavioural response has already been described (Domer and Josselson, 1964; Fennessy and Oritz, 1968). The duration of the rage response is dose dependent (table 2) and the characteristic effects include violent struggling, vocalization, hyperventilation, salivation, urination and defaecation. Intravenous injections of MNO also produced sham rage, the threshold being approximately 5 mg/kg (table 2). MNO had about 1/10th-1/20th the potency of morphine in producing the characteristic pattern of sham rage. As with morphine, subsequent injections of MNO given 30 min to 48 hr after the initial administration did not cause sham rage, and at least 4 days had to elapse before morphine or MNO again caused the rage response. The sham rage induced by MNO (10 mg/kg) or morphine (2 mg/kg) was abolished by intravenously pretreating dogs with doses of morphine (0.2 mg/kg) or MNO (2 mg/kg) which by themselves did not produce sham rage. Other drugs which abolished the rage response were nalorphine (1 mg/kg), amiphenazole (20 mg/kg), tacrine (2 mg/kg), chlorpromazine (2 mg/kg), mepyramine (2 mg/kg), compound 48/80 (200/~g/kg) and EDTA (5 mg/kg). These drugs were injected 1/2 hr before the administration of MNO or morphine except for compo~und 48/80 which was given 2 hr previously. As with morphine the MNO-induced rage was not abolished
MORPHINE-N-OXIDE (MNO)
263
Table 1 The effect of morphine-N-oxide, nalorphine, amiphenazole and tacrine on the arousal of dogs narcotized with subcutaneously administered morphine (7.5 mg/kg). These drugs were injected intravenously 45 min after the administration of morphine. The figures in parentheses represent the number of animals used. Dose
Drug Morphine-N-oxide
Nalorphine hydrobromide
(mg/kg)
Arousal Respiratory effects
Other observations
1.0 (2) 2.5 (3) 5.0 (4) 10.0 (3) 20.0 (3) 25.0 (3)
None None None None None None
All doses of morphine-N-oxide produced Dogs appeared to be more deeply natrespiratory stimulation 1-2 min after cotized after morphine-N-oxide injection. The rate decreased over the next 10 min and by 30 min respiration had almost ceased. (Dogs were resuscitated by injection of nalorphine.)
1 (6) 2 (4)
Good Good
Respiratory rate usually doubled within 2 min
Tremors, restlessness and slight excitement
Amiphenazole
10 20 30 40
(3) (4) (6) (5)
Slight Mild Good Good
Slight increase in respiratory rate
Little excitement
Tacrine
1 2 5 10
(4) (5) (7) (4)
None Mild Good Good
Respiratory rate increased within the dose range of 5-10 mg/kg
Once aroused dogs showed pronounced excitement, convulsions occurred with doses over 5 mg/kg
b y methysergide (0.5 mg/kg), atropine (1 mg/kg), propranolol (1 mg/kg), hexamethonium (10 mg/kg) or reserpine.
3.2. Cardiovascular responses 3.2.1. Conscious do.gs The effects of intravenously administered morTable 2 Duration of sham rage responses of conscious dogs to intravenous morphine or morphine-N-oxide. T~he figures in parentheses are the number of animals used. Morphine *
Morphine-N-oxide
Dose (mg/kg)
Mean duration (sec) + S.E.
Dose (mg/kg)
Mean duration (sec) +_S.E.
0.19 0.38 0.75 1.50 3.75 7.50
0 .4.7 11.8 25.8 44.0 49.6
0.25 0.50 1 2 5 10
0 (6) 0 (6) 0 (8) 0 (5) 6.2 +_ 1.7 (6) 15.9 + 2.4 (6)
(5) _+1.3 (6) +_ 1.9 (6) + 2.4 (5) + 3.5 (4) _+2.5 (5)
* The results for the duration of sham rage of morphine are taken from Fennessy and Ortiz (1968).
phine and MNO on the blood pressure and heart rate o f the conscious dog are shown in fig. 1 and table 3. Initial doses o f morphine, up to 7.5 mg]kg, regularly produced sharp but transient increases in blood pressure. Weaker pressor responses were elicited by the lower doses of MNO up to 2 mg/kg with maximal effect being produced by a dose o f 1 mg/kg. However, initial injections of 5 to 10mg/kg of MNO caused depressor responses. After an initial dose o f either drug had caused a pressor ,esp,~nse, the same dose repeated 15 min later t',ad no effect on the blood pressure, but when the second dose was increased, there was a sharp transient depressor response to which tolerance rapidly developed (fig. I). Fig. 1C shows that successive doses of 5 mg/kg of MNO given every 15 min produced depressor responses which gradually decreased in amplitude. The initial depressor response obtained with 5 or 10 mg/kg of MNO was not altered by atropine (1 mg/kg) but was significantly reduced by mepyramine (1 m g / k g ) o r nalorphine (1 mg/kg). Both morphine and MNO produced a dosedependent tachycardia lasting approximately 1 min in duration. Tolerance gradually developed to this effect
264
M.R.FENNESSY
Table 3 Cardiovascular responses of conscious dogs to intravenous injections of morphine and morphine-N-oxide. These responses represent the effect of initial doses of either drug. The figures in parentheses are the number of animals. Morphine *
Morphine-N-oxide
Dose (mg/kg)
Mean increase in blood pressure (ram Hg) _+S.E.
Mean increase in heart rate (beats/min) + S.E.
0.19 0.38 0.75 1.50 3.75 7.50
88.3 + 5.1 (4) 119.2_+ 12,7 (5) 125.8 + 6,4 (6) 136.3 + 10.6 (4) 134.0+15.8(3) 141.7 + 23.2 (3)
55.6 _+ 6.2 (4) 60.7 + 9.9 (5) 132.0_+ 18.5 (6) 186.1 _+16.7 (4) Sham rage too violent to record heart rate or ECG
Dose (mg/kg)
0.25 0.5 1 2 5 10
Mean increase in blood pressure (mm Hg) _+S.E.
Mean increase in heart rate (beats/rain) _+S.E.
15.6 _+ 3.3 (3) 57.3 + 6.2 (6) 21.3 _+ 5.7 (4) -78.4_+11.6(3) -89,7 _+14.4 (3)
16.4 + 3.3 (3) 52.3 + 8.6 (3) 115.6 + 9.2 (6) 150.0 + 13.8 (4) 192.1+20.7(3) Sham rage too violent to record heart rate of ECG
* The responses to morphine are taken from Fennessy and Ortiz (1968). and by about the fifth injection no further tachycardia was observed with either drug. Both drugs appeared to be equipotent in their ability to produce tachycardia (table 3), and neither affected the ECG.
BP mmH 9 lrnin ~
(a)
50,-
X
. .
,:,
,i
~,
0"75
0"75
3"7~
~ M~
7'5
~: ":%, ~__a= MNO
M~
1
,,,
1
MNO
S
S
.
o
MNO 5
5
$
MNO 5
Fig. 1. Effects on the blood pressure of the conscious dog produced by intravenous injections of morphine (M) and morphine-N-oxide (MNO). The figure under each panel is the dose in mg/kg. Doses were given at intervals of 15 min. Traces (a), (b) and (c) are from separate experiments.
3.2.2. Anaethetised dogs The effects of morphine and MNO on the blood pressure of the anaesthetised dogs are summarized in table 4 and illustrated in fig. 2. Initially both drugs cause a biphasic depressor response consisting of a sharp immediate fall with partial recovery followed by a more prolonged fall with gradua! recovery. This effect occurred about 10 sec ai-ter intravenous administration. Morphine and MNO were equipotent in depressing blood pressure except at the lower dose levels where morphine was more potent (table 4). Pretreatment with 1 mg/kg of atropine had no effect on the depressor response produced by morphine or MNO whereas mepyramine (2 mg/kg) completely abolished the first phase of the depressor respo!ase and partially reduced the second phase (table 5). On the other hand, nalorphine (1 mg/kg) did not affect the first phase but abolished the second phase of the depressor response. When dogs were pretreated with both mepyramine and nalorphine, both phases of the depressor response were abolished. When the injections of morphine or MNO were repeated eve,ry 15 rain the depressor responses decreased and b y the fourth or fifth administration no further effect was observed (fig. 2).
MORPHINE-N-OXIDE (MNO)
265
Table 4 Cardiovascular responses of anaesthetised dogs to morphine and morphine-N-oxide. Dogs were anaesthetised with an intravenous injection of pentobarbitone (30 mg/kg). The responses represent the effect of initial intravenous injections of morphine or morphine-N-oxide. The figures in parentheses are the number of observations. Morphine
Dose (mg/kg)
Mean decrease in blood pressure (mm Hg) +_S.E.
0.19
42.3 + 11.2 (3)
0.38 0.75 1.50
68.7 + 9.4 (5) 85.0 + 13.6 (6) 89.5 + 19.4 (3)
Morphine-N-oxide Mean decrease in heart rate (beats/min) +S.E. 0
Mean decrease in blood pressure (mm Hg) +S.E.
Dose (mg/kg)
(3)
0.25
15.6 + 6.1 (4)
12.2 + 3.6 (5) 24.6 + 5.2 (6) 29.3 + 5.8 (3)
0.5 0.75 1.5
26.3 + 8.4 (4) 78.3 +__10.7 (5) 85.7 + 15.3 (4)
Both morphine and MNO produced a slight and transient bradycardia (table 4). This effect occurred within 15 sec of adminstration, and by 2 min the heart rate had geturned to the control level. At the higher dose levels morphine and MNO were equipotent in depressing heart rate. Neither drug affected the ECG. Atropine (lmg/kg) or mepyramine (2 mg/kg) did not effect the bradycardia whereas nalorphine (1 mg/kg) completely abolished it (table 5). Tolerance gradually developed to the morphine- or MNO-induced bradycardia. 3.3. Respiration in anaesthetised dogs Both morphine and MNO when injected intravenously produced an immediate increase in respiratory rate of the anaesthetised dog (fig. 3). This effect occurred about 10 sec after administration. Morphine was approximately 30 to 50 times more potent than MNO. Morphine also caused an immediate increase in the depth of respiration. The increased respiratory rate lasted about 3 min for both drugs and a gradual tolerance to this effect was seen after repeated administration. At no stage was a decrease in respiratory rate observed with either drug.
4. DISCUSSION The results presented in this paper indicate that the pharmacological actions of morphine and MNO in the dog appear to be similar and are dependent on the
Mean decrease in heart rate (beats/min) +_S.E. 0
(4)
0 (4) 17.3 + 4.3 (5) 30.0 + 6.1 (4)
route of administration and on the level of consciousness of the dog. In the conscious dog subcutaneous injections of morphine or MNO produce depression whereas rapid intravenous injections of either drug produce sham rage followed by depression. A difference was observed on the cardiovascular system of the dog. Morphine (up to 7.5 mg/kg) and 1rain mmHg
(a)
::°of
::f ~
,~m .~
•
Q
•
•
M
M
M
M
O'?S
O-~S
~S
0,7S
(b~
ISO~-
';t
•
•
MNO
MNO
MNO
MNO
0.75
0"~$
0'TS
O'TS
•
•
Fig. 2. The effects of intravenous injections of morphine (M) or morphine-N-oxide (MNO) on the blood pressure of dogs anaesthetised with pentobarbitone Na. The figure under each panel is the dose in mg/kg. Doses were given at 15 min intervals. Traces (a) and (b) are from two separate experiments.
266
M.R.FENNESSY
Table 5 Cardiovascular responses to morphine and morphine-N-oxide in anaesthetised dogs after pretreatment with atropine, mepyramine or nalorphine. Mean decrease in blood pressure (mm Hg) -+ S.E.
Mean decrease in heart rate (beats/min) _+S.E.
Morphine (0.75 mffkg)
Morphine-N-oxide (0.75 mg/kg)
Morphine (0.75 mg/kg)
Morphine-N-oxide (0.75 mg/kg)
85.0 82.9 30.5 76.2
78.3 73.3 34.6 69.0
24.6-+ 5.2 (6) 24.4 + 4.8 (4) 27.2 -+ 6.4 (5) 0 (6)
17.3 -+ 4.6 (5) 15.5 -+ 6.7 (4) 20.0 + 6.1 (4) 0 (5)
Premedication
Control Atropine (1 mg/kg) Mepyramine (2 mg/kg) Nalorphine (1 mg/kg) Mepyramine (2 mg/kg) + nalorphine (1 mg/kg)
-+ 13.6 (6) -+ 9.4 (4) -+ 5.7 (5) -+ 10.4 (6)
10.6 -+ 1.3 (3)
8.5 -+ 1.7 (4)
low doses of MNO (up to 2 mg/kg) produced pressor responses whereas doses of 5 mg]kg and higher of MNO caused depressor responses in the conscious dog. In the anaesthetised animal both drugs lowered the blood pressure. The observations that dogs narcotized with morphine were aroused with aminophenazole or tacrine confirms the findings of Shaw and Bentley (1949, 1952) that these drugs are antagonists of some of the
(a)
ln~in
038
0.38
0 38
0'38
(b)
~NO 'I
~NO I
~NO I
-+10.7(5) -+19.4 (4) -+ 4.2 (4) -+ 8.8 (5)
~NO 'I
Fig. 3. The effect of intravenous injections of morphine (M) or morphine-N-oxide (MNO) on respiratory rate and depth of the dog anaesthetised with pentobarbitone Na. The figure under each panel is the dose in mg/kg. Drugs were injected at 15 min intervals. At T the tension on the strain gauge was reduced.
0
(3)
0
(4)
central depressant actions of morphine. MNO in doses up to 25 mg/kg produced slight sedation but no narcosis, and, in contrast to morphine, MNO did not cause vomiting. MNO, which is closely related to both morphine and nalorphine, did not antagonize the morphine-induced narcosis but appeared to deepen it which suggests that MNO is not a narcotic antagonist but rather, it is a drug having similar but less potent activity than morphine. This is also evident from the sham rage experiments in which MNO was much less active than morphine. Since the sham rage reaction was abolished by drugs which competitively antagonize or prevent release of histamine it is probable that histamine release in the CNS is involved (Domar and Josselson, 1964; Fennessy and Ortiz, 1968). The low activity o f MNO compared with that of morphine may be a consequence of its decreased ease of entry into the CNS since it has been reported that N-oxides are less lipid soluble than their parent bases (Culvenor, 1953). It has been suggested that the morphine-induced pressor response in the conscious dog may be due in part to a central sympathetic stimulation and partly to a histamine-induced release o f catecholamines from the periphery (Fennessy and Ortiz, 1968). With MNO however, pressor responses occurred with low doses and depressor responses with higher doses. The pressor response was not accompanied by sham rage whereas the rage response was exhibited with a depressor response. From this and the results obtained with morphine it would seem that the effects of
MORPHINE-N-OXIDE(MNO) these drugs on the blood pressure and on behaviour of the conscious dog are independent of one another. Another explanation may be that the threshold dose of morphine needed to produce a pressor response is lower than that required to cause sham rage and that, in fact, these responses are not independent but are produced by the same mechanism. The actions of MNO on the blood pressure of conscious dogs are difficult to interpret. One explanation could be based on the ease of entry of MNO into the CNS. With low doses the small amounts of MNO passing the blood-brain barrier may be sufficient to liberate histamine in subthreshold quantities for the induction of sham rage. However this released histamine may be of sufficient concentration to stimulate central sympathetic centres and induce a pressor response. Peripherally released histamine may also produce catecholamine secretion, an effect which would summate with the central sympathetic influences. With high doses of MNO sham rage may be induced which is accompanied by a depressor response. In these cases sufficient MNO may pass the blood-brain barrier to precipitate sham rage. However, much of the increased dose of MNO may act peripherally, releasing sufficiently large amounts of histamine to overcome the sympathetically induced pressor response by its vasodilator action. The observation that mepyramine antagonized the depressor response of MNO indicates a histaminergic origin. A corollary of thes0 observations may be that morphine, when given in sufficient quantity, may cause a fall in blood pressure as was reported by Schmidt and Livingston (1933) after doses of 200 mg/kg of morphine to conscious dogs. The actions of morphine and MNO on the blood pressure of anesthetised dogs were similar. Initially, both drugs produced biphasic depressor responses which were unaffected by atropine. The first phase of the response was abolished by mepyramine whereas the second phase was antagonized by nalorphine. These results are in agreement with those of Kayaalp and Kaymakcalan (1966) who suggested that, in the anaesthetised cat, the first phase of the morphineinduced depressor response was due to histamine release whereas the second phase was due to a central depressant action of morphine. Nalorphine did not antagonize this histamine releasing action of mor¢
267
phine or MNO, whereas mepyramine did not alter the central action. In the conscious dog both morphine and MNO caused tachycardia whereas in the anaesthetised dog both drugs produced slight bradycardia. Neither atropine nor mepyramine affected the bradycardia in anaesthetised dogs whereas it was abolished by nalorphine. This suggests that these analgesics depress the heart rate not by an increase in vagal activity but maybe by a direct depressant action. In the conscious dog the morphine-induced tachycardia has been shown to be reduced by nalorphine, and by drugs affecting histaminergic and adrenergic mechanisms (Fennessy and Ortiz, 1968). These authors suggested that morphine may have a direct stimulant action on the heart. However since the actions of morphine or MNO on the heart have opposite effects at different levels of consciousness of the dog it is difficult to postulate a mechanism of action and it is obvious that further work is required. Morphine and MNO produced respiratory stimulation in both concious and anaesthetised dogs. Similar effects have been shown in conscious dogs with morphine by Martin and Eades (1961) who suggest that the respiratory rate is increased in association with maximal loss of body heat. In addition the CNS stimulant action of morphine may also be partly responsible. In anaesthetised dogs the increase in respiratory rate may be due to baroreceptor stimulation as a consequence of the depressor response.
ACKNOWLEDGEMENTS Thanks are due to Drs. C. Raper and L. Mashford for their criticisms of the manuscript and to H.W. Woods Pry. Ltd. for providing a grant for technical assistance.
REFERENCES Culvenor, C.C.J., 1963, Amine oxides, Rev.Pure Appl.Chem. 3,83. Domer, F.R. and F. Josselson, 1964, Behavioural effect of intravenous morphine administration to the unaesthetised dog, Arch. Intern. Pharmacodyn. 147,470.
268
M.R.FENNESSY
Fennessy, M.R., 1968, The analgesic action of morphineN-oxide, Brit. J. Pharmacol. 34,337. Fennessy, M.R. and H.J. Fearn, 1969, Some observations on the toxicology of morphine-N-oxide, J, Pharm. Pharmacol. 21,668. Fennessy, M.R. and A. Ortiz, 1968, The behavioural and caxdiovascular actions of intravenously administered morphine in the conscious dog, Europ. J. Pharmacol. 3, 177. Freund, M. and E. Speyer, 1910, Enwirkung von Wasserstoff superoxyd auf Thebain, Morphin und dessert Ather, Bet. Deut. Chem. Ges. 43, 3310. Kayaalp, S.O. and S. Kaymakcalan, 1966, A comparative study of the effects of morphine in unanaesthetised and anaesthetised cats, Brit. J. Pharmacol. 26,196. Martin, W.R. and C.G. Eades, 1961, Demonstration of tolerance and physical dependence in the dog following a short term infusion of morphine. J. Pharmacol. exptl. Therap. 133,262.
Schmidt, C.F. and A.E. Livingston, 1933, The action of morphine on the mammalian circulation, J. Pharmacol. Exptl. Therap. 47, 411. Shaw, F.H. and G.A. Bentley, 1959, Some aspects of the pharmacology of morphine, with special reference to its antagonism by 5-amino acridine and other chemically related compounds, Med. J. Australia 2,868. Shaw, F.H. and G.A. Bentley, 1952, Morphine antagonism, Nature 169, 712. Shaw, F.H., S. Gershon and G.A. Bentley, 1957, Morphine antagonism, J. Pharm. Pharmacol. 9,666. Woo, J.T.C., G.A. Gaff and M.R. Fennessy, 1968, A note on the effects of 2,4-diamino-5-phenylthiazole and 1,2,3,4-tetrahydro-9-aminoacridine on morphine metabolism, J. Pharm. Pharmacol. 20, 763.