Effects of aporphine and emetine alkaloids on central dopaminergic mechanisms in rats

EUROPEAN JOURNALOF PHARMACOLOGY20 (1972) 71-79. NORTH-HOLLANDPUBLISHINGCOMPANY

EFFECTS OF APORPHINE AND EMETINE ALKALOIDS ON C E N T R A L DOPAMINERGIC MECHANISMS IN RATS 1 Samarthji LAL2, Theodore L. SOURKES, Krystyna MISSALA and George BELENDIUK Department of Psychiatry, McGill University, Montreal, Quebec, Canada

Received 18 April 1972

Accepted 5 July 1972

S. LAL, T.L. SOURKES, K. MISSALA and G. BELENDIUK, EffeVts ofaporphine and emetine alkaloids on central dopaminergic mechanisms in rats, European J. Pharmacol. 20 (1972) 71-79. A series of aporphine and emetine alkaloids was studied for induction of stereotyped behaviour (SB), antagonism of reserpine sedation, and effect on the cerebral concentrations of homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA) in the rat. Apomorphine (5 mg/kg) induced SB, reversed the reserpine syndrome and lowered the concentration of cerebral HVA. Apocodeine (10-O-methylapomorphine) and methylenedioxyaporphine (both injected in a dose of 20 mg/kg) induced intermittent SB, reversed the reserpine syndrome and reduced cerebral HVA, whereas 10,11-dimethoxyaporphine had none of these effects. Methylenedioxyaporphine decreased the level of 5-HIAA slightly. The results suggest that apocodeine and methylenedioxyaporphine have similar sites of action to those affected by apomorphine, but that the first 2 alkaloids are less potent agonists than apomorphine. 6 other analogues of apomorphine, including 1,2,10,11-tetrahydroxyaporphine (the N-methyl derivative of a compound theoretically derivable from tetrahydropapaveroline), were tested at doses of 20 mg/kg, but none induced SB. Cephaeline and emetine did not influence the concentrations of HVA or 5-HIAA in the brain, nor did they induce

SB. Aporphine alkaloids

Cephaeline

Dopamine metabolism

Emetine

1. INTRODUCTION Since 1874 apomorphine has been known to cause stereotyped behaviour in rodents, characterized by gnawing, sniffing, biting and licking (Harnack, 1874). Amsler (1923)later described the stereotypy in greater detail. More recently evidence has been gathered that indicates similar sites of action of dopa, dopamine and apomorphine in the brain (Sourkes, 197 l b). This evidence comes from experiments in which these compounds have been implanted in specific regions of the brain (Smelik and Ernst, 1966), from pharmacol1

The abbreviations used are: HVA, homovanillic acid; 5-HIAA, 5-hydroxyindoleacetic acid; SB, stereotyped behaviour; DMSO, dimethylsulfoxide. 2 Fellow of the Medical Research Council (Canada) during initial part of this work.

Reserpine antagonism Stereotyped behaviour

ogical study of the chemoceptor zone of the emetic centre (Peng, 1963) and from biochemical-pharmacological studies with drugs that interfere in the biosynthesis or metabolism of specific brain monoamines (Fekete et al., 1970; Goldstein et al., 1970; Larochelle et al., 1971; Sourkes, 1971a). Several authors (Cohen and Collins, 1970; Davis and Walsh, 1970; Oster, 1942, Scheckel et al., 1969; Sourkes, 1971a) have directed attention to the variety of alkaloidal structures that could be derived in animal tissues from dopamine, taking as the paradigm the formation of tetrahydropapaveroline in vitro from dopamine in the presence of monoamine oxidase (Holtz et al., 1964). Furthermore, the structural resemblance of portions of the apomorphine molecule to dopamine renders apomorphine and related compounds as worthwhile candidates for investiga-

72

S. Lal et aL, Aporphine alkaloids

tion for possible action on dopamine-sensitive receptors. Such compounds would be of therapeutic interest in view of the known improvement of Parkinsonian symptoms produced by apomorphine (Braham et al., 1970; Cotzias et al., 1970; Schwab et al., 1951; Struppler and Von Uexktill, 1953; Von Uexkiill, 1953). Whereas the apomorphine-induced stereotypy is thought to be induced by specific stimulation of dopamine-sensitive receptors in the striatum (Roos, 1969; And6n et al., 1967; Ernst, 1967) emetic action is induced by stimulation of similar receptors in the chemoceptor zone of the emetic centre (Peng, 1963). This suggested that tests of alkaloids of the emetine series be undertaken also. The measures of action on dopaminergic mechanisms which we have studied are the effect of various aporphine alkaloids, emetine and cephaeline on their ability to (a) induce stereotyped behaviour, (b) antagonise the effects of reserpine, and (c) alter levels of homovanillic acid (HVA) in rat

HO

O~~N

CH30

1.

brain. In addition, we have studied the effect of these agents on 5-hydroxyindoleacetic acid (5-HIAA) to assess possible action on serotoninergic brain mechanisms.

2. MATERIALS AND METHODS

2.1. Drugs The following compounds were tested: apomorphine* hydrochloride; apocodeine* ( 10-methoxy- 11-hydroxy-aporphine hydrogen oxalate, no. 614817); 10, l l-dimethoxyaporphine* hydrogen-d-tartrate (no. 619373); 10,11-methylenedioxyaporphine* hydrochloride (no. 615644); morphothebaine* hydrochloride (no. 616027); 5,6-dihydroxy-6-methyl-4H-dibenzo-[d,e,g]-quinoline-lO,11-dione (no. 616520); 2,10,11-trihydroxyaporphine** hydrobromide (no. 615642); 1,2,1 O,11-tetrahydroxyap orphine** hydrobromide hemihydrate (no. 614822); tetrahydro-

O~

CH3

2.

CH30~

OC,~"~ Hs

CH3

3.

CH3

cH3o OH

i 4.

u

CH3

5.

OH

l

CH3

6.

OH

OH

OH

HO

OH

HO

HO

I

7.

CH3

CH3

I

8.

CH3

Br Br

9.

CH3

Fig. 1. Aporphine derivatives. 1, apomorphine; 2, apocodeine; 3, 10,11-dimethoxyaporphine; 4, 10,11-methylenedioxyaporphine; 5, (-)morphothebaine; 6, 5,6-dihydro-6-methyl-4H-dibenzo [d,e,g] quinoline-10,11-dione; 7, 2,10,11-trihydroxyaporphine; 8, 1,2,10,11 -tetrahydroxyaporphine; 9, 8,9-dibromo-1O,11-dihydroxyaporphine.

73

S. Lal et al., Aporphine alkaloids

CH3O~~ HO""v~"v~"~ OCH3 T IT I o.c. OcH3 C H 3 0 ~ OCH3 C H 3 0 ~ HsCf

Y

H5C2~ v v CEPHAELINE

v

EMETINE

Fig. 2. Emetine,and cephaeline. papaveroline hydrochloride (no. 712823); 8,9-dibromo- 10,11-dihydroxyaporphine* hydrobromide (no. 614821); emetine; and cephaeline. The last two compounds were purchased from K and K Laboratories, Plainview, New York. All the other compounds were gifts of Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486. The starred alkaloids in the aporphine series have an asymmetric carbon atom in the 6a-position. Those with one asterisk belong to the R-series; those with two asterisks to the S-series. The structures are illustrated in figs. 1 and 2. These compounds were administered in aqueous solution (controls received water), with the following two exceptions. The quinoline-dione, compound no. 616520, was first dissolved in a small volume of ethanol and was then diluted with water. Control rats received an equal volume of the ethanol solution alone. In the biochemical studies the methylenedioxy derivative of apomorphine, compound no. 615644, was injected as a suspension in saline in one experiment and as a solution in dimethylsulfoxide (DMSO) in a second. Again, control animals received the corresponding diluent by injection. Drug solutions were prepared immediately before use. All drugs were given intraperitoneally in a volume of 1.0 ml. Administration of drugs and observation of the animals were carried out in a small inner room artificially lit, and of steady temperature 24°C. Doses of drugs are expressed as mg of the base per kg body weight. Apomorphine was given in a dose of 5 mg/kg. 2.2. A n i m a l s Male Sprague-Dawley rats were used in this work; they were housed in pairs. In most experiments animals weighing approximately 150 g were used, but in view of the small quantities of some of the test com-

pounds that were available additional experiments were carried out on smaller animals, as indicated in text and tables. The number of rats used in given experiments is indicated in the appropriate place. Animals were used only once. 2.3. Behavioral tests Rats were injected with the test compound or control solution and were observed continuously for 2 hr. The development of sniffing, licking, biting and ptosis was noted. Ptosis was graded according to Lapin's scale (Lapin, 1967): complete ptosis, 4; more than half-closed, 3; half-closed, 2; less than halfclosed, 1 ; absence of ptosis, 0. In studying antagonism to the effects of reserpine, groups of animals were injected with reserpine, 2.5 mg/kg, and 3 hr later they were tested for evidence of reserpinization. Criteria for complete reserpinization consisted of: diarrhoea, suppressed locomotion, hunched rigid posture, complete ptosis, and positive tests of catalepsy. Those rats which were completely reserpinized were selected for the next part of the experiment, and others were discarded. The selected rats were injected with either a test compound, 20 mg/kg, apomorphine, 5 mg/kg, or the control solution. Then continuous observations were made on posture, motor behaviour and eye aperture. Tests of catalepsy were performed at 15, 40, 60 and 90 min after injection of the test compound. Additional control animals received distilled water instead of reserpine, followed by a second injection of distilled water, apomorphine, or test compound. Two tests of catalepsy were used. The rubber stopper method of Weissman (1969) was used except that the scale of the apparatus was modified as necessary for the sizes of rats used in this work. Catalepsy was judged to be present (positive) if the animal maintain-

74

S. Lal et aL, Aporphine alkaloids

ed its awkward position on all four stoppers for at least 30 sec. Failure to maintain this position for this time period was judged as a negative response. The second test for catalepsy was the 'vertical wire test'. A vertical wire screen, 30 cm high and 14.5 cm wide, with grid dimensions of 0.7 cm 2 was used. Animals were placed on the centre of the screen and were observed. The test was judged positive if the rat remained in position on the wire for at least 30 sec. 2.4. Chemical determinations Rats were given drug treatments as indicated in tables 2 and 3, and were killed 75 rain later by guillotine. Brains were immediately removed, and were frozen and stored at - 2 0 ° until analysis was to be performed. Because of the low concentration of HVA in the rat brain, two brains were pooled for a single determination. Extraction and analysis were carried out as recommended by O'Keefe et al. (1970) with the addition of the following step when the TRIS extracts were milky in appearance: a further extraction was carried out with a small volume of heptane to clear the solutions. 5-HIAA was measured in a portion of the TRIS extract by the method of Bogdanski et al. (1956).

3. RESULTS

3.1. Induction o f stereotyped behaviour Rats treated with control solutions quickly settled down after the injection and remained together, asleep for most of the test period. Occasional feeding, grooming and exploratory behaviour was noted. None of the 44 rats so tested manifested evidence of SB (stereotyped behaviour). Almost immediately after the injection of apomorphine, 5 mg/kg, animals commenced to sniff, lick and bite in a continuous manner. Initially the sniffing might be directed upwards, with the animal rearing on its hind legs, but this soon gave way to SB directed towards the floor of the cage. During the initial 5 - 1 0 min there was sometimes forward locomotion with the SB but thereafter, apart from occasional steps, locomotion was virtually absent until the last 10 min of SB. With the onset of SB normal activities of social interaction, grooming and feeding were abolished. Ptosis, grade 4, appeared within 10 min of the injection of apomorphine. In

the terminal 10 min of SB there was a gradual interspersion of normal activities, until eventually the animals grouped themselves together and fell asleep or rested quietly. 5 of 44 rats given apomorphine failed to develop SB. Of the remainder the duration of SB was 58 + 0.9 min (mean + S.E.M.) with a range of 4 0 - 6 5 min. None of the other compounds tested in a dose of 5 mg/kg (4 rats/group) developed SB. At a dose of 20 mg/kg (4 rats/group) both apocodeine and methylenedioxyaporphine caused intermittent SB. None of the other compounds tested at this dose was effective. The results with O-methylated aporphines are summarized in table 1. In a further experiment with rats weighing about 65 g, following the onset of intermittent SB, 4/4 rats receiving apocodeine and 1/4 receiving methylenedioxyaporphine (20 mg/kg each) developed convulsions; after the fit the rats resumed intermittent SB. No convulsions occurred with apomorphine itself (5 mg/kg). The pattern of SB in the 65 g animals resembled that in the adult rats. Data for both weight groups have been combined in table 1. 3.2. Antagonism of reserpine effects Control animals that had received distilled water but no reserpine (n=22) behaved as described for the controls of the previous experiment. It was impossible to place these rats on the stoppers in the Weissman test, even momentarily. On the vertical screen test the rats either climbed over the top of the screen or round the side where they alighted head down and leading with forepaws. In contrast to this, all rats that were completely reserpinised (n=22) gave positive tests of catalepsy at 15, 40, 60 and 90 min. Rats that had received apomorphine, but not reserpine, gave a negative test for catalepsy in the Weissman test, but 8 of the 22 rats tested on the vertical screen at 15 and 40 min remained on the wire, though continuing to sniff, lick and bite in this position. The others either slipped down until the hind paws touched the tabletop or else climbed up or around the screen before alighting. In the case of rats given apomorphine after reserpinization, all but one of 22 animals showed an immediate reversal of the rigid, hunched posture and the onset of some forward locomotion, together with typical features of SB. Ptosis remained unchanged. These results are summarized in table 1. Only one rat

Table 1 Effects of apomorphine and some derivatives on behaviour of the rat. Parameter observed

Apomorphine 5 mg/kg

Apocodeine 20 mg/kg

10,11-Dimethoxyaporphine 20 mg/kg

10,11-Methylenedioxyaporphine 20 mg/kg

Overt behaviour

43/48a: Immediate onset of continuous sniffing licking and biting. Ptosis, grade 4 within 10 rain 5/48: no response

8/8a: Initial alerting. Within 5 min intermittent sniffing, licking or biting for 5 - 1 0 sec and pauses of 10 sec, occasionally up to 60 sec Ptosis, grade 2 - 4 within 10 min

4/4: Resting or asleep, with occasional grooming and exploratory activity

8/8a: Similar to apocodeine, except that pauses were more variable within the 10-60 sec range

Duration (min)

40-65

35-50

45 - 5 0

25/26a: Immediate reversal of hunched, rigid posture, and onset of SB. Some forward locomotion. No change in ptosis

7/7a: Immediate reversal 4/4: Reserpine state unchanged of hunched, rigid posture. Within 5 min, intermittent SB as above. Some steps of forward locomotion. Ptosis unchanged

8/8a: As with apocodeine. In some animals, reversal of ptosis initially, with eyes beginning to close again at 20 rain

25/26 25/26 9/26

7/7 7/7 5/7

0/4 0/4 0/4

8/8 4/8 0/8

16/26 c 16/26 c 9•26

7/7 7/7 2/7

0/4 0[4 0/4

8/8 8/8 0/8

Reserpine-induced state b (1) Overt behaviour

(2) Reversal of catalepsy (i) Rubber stopper test: 15 min 40 min 60 min (ii) Vertical wire test 15 min 40 min 60 min

a 4 of these rats weighed approximately 65 g. Otherwise the rats weighed about 150 g. b 2.5 mg of reserpine per kg given i.p. 3 hr before the test compound. c Though 10 of these animals were considered positive in the wire test, 9 of these exhibited SB in this position.

Table 2 Concentrations of HVA and 5-HIAA in brains of rats injected with aporphine alkaloids. Drug treatment

None Apomorphine Apocodeine Methylenedioxyaporphine DMSO a Methylenedioxyaporphine/DMSO

Dose (mg/kg)

5 20 20 20

HVA

5-HIAA

nb

Mean -+ S.E.M.

nb

Mean + S.E.M.

6 8 5 2e 6 3e

81 -+ 5.2 40 -+ 3.7 d 48 -+ 7.4 d 53, 63 98 -+ 11.3 74 -+ 7.2

6 8 5

414 +- 10 432 +- 13 453 +- 14 c

5 3

415 +- 8 348 -+ 18

a Dimethylsulfoxide, used as solvent. b Each analysis was performed on the pooled brains (minus cerebellum) of rats. n refers to the number of such pools. c 0.05 > p > 0.01. d p < 0:01. e When the data for methylenedioxyaporphine in the two sets were estimated as percentage of control, the mean + S.E M. (n = 5) was 56% +- 5.2. This decrease is significant (p < 0.01).

76

S. Lal et al., Aporphine alkaloids

showed a positive test for catalepsy on the rubberstopper test at 15 and 40 min. In the wire-screen test 10 rats showed positive tests at these times, but 9 of these continued to display SB on the wire. By 60 min most of the rats showed a positive response on both tests and by 90 min all animals appeared to be completely reserpinized once again. The time from apomorphine injection to termination of SB and restoration of the reserpinized stance was 59 -+ 1 min. This is to be compared with 60 +- 1.1 min for the duration of SB in animals given apomorphine alone. Both apocodeine and methylenedioxyaporphine reversed the reserpine state and induced intermittent SB. The behaviour, in form and duration, was similar to that of test compound administered alone (table 1). The results were similar when 65 g rats were used (4/group, table 1); none of these rats convulsed. Other compounds tested at dosage of 20 mg/kg, were morphothebaine, tetrahydroxyaporphine, trihydroxyaporphine, dibromodihydroxyaporphine, 5,6dihydro-6-me thyl-4H-dibenz oquinoline- 10,11 -dione (3 rats for each), tetrahydropapaveroline, dimethoxyaporphine, emetine and cephaeline (4 rats for each). None of these compounds had any effect on the reserpinized rats. 3.3. Effects on cerebral H V A and 5-HIAA The effects of apomorphine and its methylated derivatives on cerebral HVA and 5-HIAA concentra-

tions are set out in table 2. As already demonstrated by other investigators, the administration of apomorphine resulted in a decrease in the concentration of HVA in the brain, with little change from control levels in respect to 5-HIAA. Apocodeine (20 mg/kg) also depressed the level of HVA. The dimethylated apomorphine, i.e. dimethoxyaporphine, had no demonstrable effect of this kind, but methylenedioxyaporphine was about as effective as apocodeine, in the same dose of 20 mg/kg. Of the compounds tested in table 2 apocodeine provoked a mean increase of less than 10% in the concentration of 5-HIAA. Methylenedioxyaporphine seemed to cause a decrease of about 20% (iv < 0.05). Emetine and cephaeline were tested in the same way, at doses that are considered emetic (Sollman, 1948). The results in table 3 show that under the conditions of our tests these alkaloids had no effect on brain HVA or 5-HIAA.

4. DISCUSSION The pharmacological properties o f apomorphine have been investigated for many years and as long ago as 1874 it was known to induce SB in rodents and other animals (Harnack, 1874). In contrast, there are only a few pharmacological studies of closely related derivatives of apomorphine (Gunn, 1941, Schwartze,

Table 3 Concentrations of HVA and 5-HIAA in brains * of rats injected with emetine and cephaeline. Expt.

Drug

Treatment schedule

HVA

5-HIAA

no.

n

Mean -+ S.E.M. (ng/g)

n

Mean +-S.E.M. (ng/g)

1

None Emetine

Control injections Injections of drug, 2 mg/kg s.c. at zero time and 1 hr; killed at 2.5 hr

4 4

124 -+ 1 157 -+ 12

4 4

484 -+ 7 488 -+ 14

2

None Emetine

Control injections Injections of drug, 2 mg/kg s.c. at zero time and 1 hr; killed at 4.5 hr

4 4

80 +-4 91 -+ 7

4 4

587 -+ 15 601 -+ 13

3

None Emetine

Control injections Injections of drug, 2 mg/kg at zero time and 1 hr; killed at 3 hr. Same schedule as for emetine but in doses of 1.0 mg/kg

3 4

75 -+ 10 73 -+ 3

2 4

291,339 340 +- 13

4

86 + 4

4

326 + 6

Cephaeline

* Whole brain was used in expts. 2 and 3, but in expt. 1 the cortex and cerebellum were removed before analysis.

S. Lal et al., Aporphine alkaloids

1931 ; Hensiak et al., 1965; Cannon et al., 1967; Koch et al., 1968). Hensiak et al. (1965) and Koch et al. (1968) found that N-alkylnorapomorphine derivatives induce compulsive gnawing in mice, but apocodeine does not do so (Koch et al., 1968). In the present study apocodeine in large doses was able to induce intermittent SB in rats. This difference is probably species-related as it is known that apomorphine induces only a weak compulsive gnawing in mice (Ther and Schramm, 1962; Scheel-Kr~iger, 1970) in contrast to rats. In regard to the extent of O-methylation, the monomethylapomorphine derivative apocodeine induced intermittent SB as did the methylenedioxy compound, but the dimethyl derivative and the dione were inactive. It has been postulated that the catecholic character of the apomorphine molecule is not likely to be essential for the pharmacological effects of this alkaloid (Kier and Truitt, 1970). The present results do not support this contention, and are in keeping with the conclusion of Pinder et al. (1971) relating the action of apomorphine, at least in part, to the catechol structure. Of course, it is conceivable that apocodeine and methylenedioxyaporphine undergo transformation to apomorphine before initiating SB. Hydroxylation at the C2 position of either apomorphine and apocodeine, or at both C1 and C2 positions of apomorphine rendered the compound inactive in inducing SB. The 1,2,10,11-tetrahydroxyaporphine is the N-methyl derivative of one of the two tetrahydroxynoraporphines theoretically derivable from dopamine via tetrahydropapaveroline (Sourkes, 1971a). According to Granchelli et al. (1971) 7hydroxyaporphine and 11-hydroxyaporphine possess apomorphine-like activity in rats. In cats and dogs (Harnack, 1874) and in man apomorphine induces vomiting, whereas in animal species which do not vomit SB is induced. Neither emetine, a central emetic agent in man nor cephaeline induced SB in the rat. In keeping with the findings of Janssen et al. (1965) and Ther and Schramm (1962), but in contrast to Fekete et al. (1970), pretreatment with reserpine did not inhibit apomorphine-induced SB. In addition, the present results show that during the SB the characteristic fixed hunched posture and positive tests of catalepsy of the reserpinised state were over-

77

come. This effect on the reserpine-state was also achieved with apocodeine and methylenedioxyaporphine. Apomorphine treatment leads to a reduction of HVA in the brain (Roos, 1969; Goldstein et al., 1970). Although tyrosine hydroxylase is inhibited by apomorphine (Goldstein et al., 1970), this occurs in vitro only with large doses of the drug, and could not be expected to occur under the conditions used in these experiments. It is conceivable that the neurons bearing dopamine-sensitive receptors relay information back to the dopaminergic fiber (e.g. through strio-nigral fibers). This inhibitory feedback would result in decreased production of dopamine and of HVA (Sharman, 1966; Sourkes and Poirier, 1969). Another possibility is transsynaptic feedback causing less dopamine to be released into the synaptic cleft (H~ggendal, 1970; Farnebo and Hamberger, 1971). Apocodeine and methylenedioxyaporphine also decreased cerebral HVA but they are not as potent as apomorphine (table 2). The short duration of action of apomorphine on behaviour noted here parallels its brief-lasting action in ameliorating Parkinsonian symptoms (Schwab et al., 1951). In previous work we have shown that the duration of apomorphine-induced SB is proportional to the logarithm of the dose (Lal and Sourkes, 1971). This would indicate the importance of searching for a long-acting dopamine analog. Such compounds could be screened by the tests mentioned in this paper. These tests show good correlation between induction of SB, antagonism to reserpine, and lowering of brain HVA. The production of these effects is consistent with action of the drug on striatal dopamine-sensitive receptor sites.

ACKNOWLEDGEMENTS This work was supported by grants of the Medical Research Council (Canada). We thank Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania, for their generous gift of apomorphine and other aporphine alkaloids.

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