Drug and Alcohol Dependence, 18 (1986) 87-96 87 Elsevier Scientific Publishers Ireland Ltd.

BIDIRECTIONAL CROSS-TOLERANCE BETWEEN METHADONE ( m u ) - A N D ETHYLKETOCYCLAZOCINE ( k a p p a ) - TOLERANT RATS*

OKSOON HONG, GERALD A. YOUNG and NAIM KHAZAN

Department of Pharmacology and Toxicology, School of Pharmacy, University of Maryland at Baltimore, 20 North Pine Street, Baltimore, MD 21201 (U.S.A.)

(Received February 10th, 1986)

SUMMARY

Our laboratory previously reported on unidirectional cross-tolerance between morphine and methadone, both mu opioid agonists, and between morphine and ethylketocyclazocine (EKC), the lat ter being a relatively selective kappa opioid agonist. Morphine-tolerant rats were found to be non-cross-tolerant to methadone and EKC, but methadone- and EKC- tolerant rats were cross tolerant to morphine. In the present study, we characterized the cross-tolerance between methadone and EKC. A group of female adult Sprague-Dawley rats was made tolerant to methadone by a series of automatic i.v. injections ranging from 0.25 mg/kg per 2 h on the first day to 2.0 mg/kg per 1.5 h on the ninth day. Another group of rats was similarly made tolerant to EKC with doses ranging from 0.5 mg/kg per 2 h on the first day to 4 mg/kg per h on the ninth day. Relatively similar degrees of tolerance development to the EEG and behavioral effects of methadone and EKC were reflected by decreases in durations of action and decreases in opioid-induced EEG power spectral changes. Methadone- tolerant rats were found to be cross-tolerant to the EEG and behavioral effects of EKC, and, similarly, EKC-tolerant rats were found to be cross- tolerant to those of methadone. Thus, a bidirectional cross-tolerance be- tween a mu and a kappa agonist was demonstrated. The present results together with those reported earlier indicate that cross-tolerance may not be directly related to the receptor selectivity of the opioids. It is possible that differential physicochemical properties of these opioids may play a more decisive role in the phenomenon of cross-tolerance.

*This work was supported by National Institute on Drugs Abuse Grant DA-01050. Prelimi- nary data were reported at the meeting of the Federation of American Societies for Experimental Biology, Anaheim, California, April 21-26, 1985 and at the 1985 meeting of the Committee on Problems of Drug Dependence, Inc.

0376-8716/86/$03.50 (~) Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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Key words: Methadone - Ethylketocyclazocine -Cross-tolerance - Opioids - EEG

INTRODUCTION

Tolerance to morphine and methadone has been previously demon- strated in the rat using electroencephalographic (EEG) and behavioral correlates [1,2]. Furthermore, it w~as shown that morphine-tolerant rats were not cross-tolerant to methadone or ethylketocyclazocine (EKC), but methadone- and EKC-tolerant rats were cross-tolerant to morphine [3,4]. Thus, a unidirectional cross-tolerance was noted between morphine and methadone and between morphine and EKC. Using mouse locomotor activity, Gwynn and Domino [5] have recently demonstrated the existence of unidirectional cross-tolerance between morphine and EKC. Also, uni- directional cross-tolerance between morphine and methadone has been demonstrated in other studies [6-8]. Thus, in spite of the fact that both morphine and methadone are mu agonists, there is a lack of bidirectional cross-tolerance between them. On the other hand, differential analgesic cross-tolerance to morphine between lipophilic and hydrophilic opioid agon- ists has been reported [8,9]. Methadone is reported to be significantly more lipophilic than morphine [10]. Similar to methadone, EKC (unpublished data from our laboratory) is highly lipophilic. The purpose of the present s tudy was, therefore, to characterize the phenomenon of cross-tolerance between methadone, a selective mu agonist, and EKC, a selective kappa agonist [11,12], both of which have a similarly high lipid solubility.

METHODS

Female adult Sprague-Dawley rats (250-300 g) were used. They were anesthetized with ketamine hydrochloride (100 mg/kg, i.p.) and prepared with chronic cerebrocortical and temporalis muscle electrodes to record EEGs and electromyograms (EMGs), respectively [13]. Bipolar EEG elec- trodes were placed epidurally over the cortex, 2 mm anterior to bregma and 2 mm lateral to the midline, and 3 mm posterior to bregma and 2 mm lateral to the midline. Each rat was also prepared with a chronic silicone rubber cannula inserted into the jugular vein for drug administration [14]. After recovery from surgery, rats were housed in individual cages with food and water available ad libitum. They were connected to a Grass Model 7 polygraph by a flexible EEG cable [2], where a mercury pool swivel [15] provided a noise-free contact between the recording cable and the polygraph, which permitted relatively unrestrained movement of the rats. Saline and drug injections were administered through the swivel via a feed-through cannula using a Harvard infusion pump controlled by solid state programming equipment (BRS/LVE, Beltsville, MD). An alternate light-dark cycle was maintained with illumination from 0600 to 2200.

EEG and EMG activities were collected 24 h /day on a Grass Model 7

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polygraph from each rat. Using a Hewlett-Packard 3960 tape recorder, the EEG was stored on FM magnetic tape for subsequent computer analysis. The drug-induced changes in the overt behavior of the rats were observed and noted below the corresponding EEG tracings. The direct EEG, integ- rated EMG and behavior of the rats were used to distinguish the different behavioral states of stupor or catalepsy, arousal, quiet awake and slow- wave sleep (SWS) [2]. The durations of SWS suppression after drug administration were determined and the average values were calculated.

Spectral analysis of EEG samples was accomplished with a Nicolet MED-80 minicomputer utilizing the Disk Frequency Analysis Package. EEG power spectra were derived from EEG epochs at 5 min after each injection that were digitized at a sampling rate of 100/s [16,17]. Average EEG power spectra were obtained by averaging spectra derived from six successive 10-s epochs. The average EEG power spectra were smoothed by using a geometric algorithm and plotted on a Hewlet t Packard X-Y plotter. The percent increase of total EEG spectral power after drug administration over that during the quiet awake state was studied and compared.

Methadone hydrochloride (Merck and Co., Inc., Rahway, NJ) was dissol- ved in physiological saline and EKC methanesulfonate (Sterling-Winthrop Research Institute, Rensselaer, NY) was dissolved in physiological saline with emulsifying agent (Emulphor) and ethanol (18:1:1). All injections were administered i.v. in a volume of between 0.05 and 0.4 ml/rat .

Intravenous challenge doses of methadone (2 mg/kg, i.v.) or EKC (4 mg/ kg, i.v.) were administered in a randomized fashion to two groups of six naive rats each. An additional two groups of six rats each were made tolerant to and physically dependent on either methadone or EKC by a series of automatic i.v. injections. Methadone was initially administered at 0.25 mg/kg per 2 h for 2 days, then increased to 0.5, 1.0 and 2.0 mg/kg per 2 h every other day, and, finally administered at a dose of 2.0 mg/kg per 1.5 h for I day. EKC was initially administered at 0.5 mg/kg per 2 h for 2 days, then increased to 1.0, 2.0 and 4.0 mg/kg per 2 h every other day, and, finally administered at a dose of 4.0 mg/kg h for i day. These chronic dose schedules of methadone and EKC were empirically deter- mined on the basis of being able to produce approximately similar degrees of tolerance. After chronic administration of methadone and EKC, toler- ance and cross-tolerance to methadone (2 mg/kg, i.v.) and EKC (4 mg/kg, i.v.) were determined. Cross-tolerance was assessed by substi tuting one opioid for the other in place of a regularly scheduled automatic i.v. injection. Data were analyzed by analyses of variance followed by Least Significant Difference tests for significant differences among means.

RESULTS

Since the administration of opioids disrupts the sleep-awake cycle and suppresses SWS, the latency to first episode of EEG and behavioral SWS was used to reflect the degree of tolerance development. The mean laten-

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cies to SWS in the different groups are shown in Table I. After me thadone (2 mg/kg , i.v.) injections in non- to le ran t rats, the first occurrence of SWS emerged af ter 144.5 -+ 13.8 min (mean -+ S.E.M.). However, in methadone- to le ran t and EKC-to le ran t rats, SWS appeared at 49.9 -+ 3.8 and 69.0 -+ 5.3 min, respectively. These values in the to le ran t ra t s are signif icantly different f rom those in the non- to le ran t ra t s (P < 0.001). Also, the m e a n la tency to the first episode of SWS af ter EKC (4 mg/kg , i.v.) admin i s t ra t ion in EKC-to le ran t (36.8 -+ 6.5 min) and me thadone - to l e ran t (37.4 -+ 2.5 min) ra t s decreased in compar ison to t h a t in non- to le ran t ra t s (115.0-+ 6.9 min), indica t ing significant tolerance and cross-tolerance.

The admin i s t ra t ion of m e t h a d o n e (2 mg/kg , i.v.) in non- to le ran t ra t s produced dense high-vol tage slow-wave EEGs and increases in associated E E G power spect ra in the lower frequencies as a p r edominan t peak. However, the same dose of m e t h a d o n e produced m u c h smal ler changes in cortical E E G t rac ings and associated E E G power spect ra in metha- done- to lerant ra t s (Fig. 1). Increases in total E E G spectral power ( 0 - 2 5 Hz) af ter d rug admin i s t ra t ion were compared to those dur ing the control awake s ta te (Table II). The admin i s t ra t ion of me thadone in non-

TABLE I

LATENCIES TO ONSET OF SWS IN NON-TOLERANT, METHADONE-TOLERANT AND EKC-TOLERANT RATS GIVEN METHADONE AND EKC i.v. CHALLENGES

Pre-treatment Latency a (min.)

Methadone EKC

Non-tolerant 144.5 ± 13.8 115.0 ± 6.9 Methadone-tolerant 49.9 ± 3.8*** 37.4 ± 2.5*** EKC-tolerant 69.0 ± 5.3*** 36.8 ± 6.5***

aMean -+ S.E.M. ***P < 0.001, when compared to non-tolerant group; Least Significant Difference Test.

TABLE II

INCREASES IN SPECTRAL POWER IN NON-TOLERANT, METHADONE-TOLERANT AND EKC-TOLERANT RATS GIVEN METHADONE AND EKC i.v. CHALLENGES

Pre-treatment EEG spectral power a (% of control awake)

Methadone EKC

Non-tolerant 509 ± 112 852 ± 163 Methadone-tolerant 160 ± 19"* 208 ± 32*** EKC-tolerant 275 ± 44* 217 ± 33***

aMean ± S.E.M. *P < 0.05, **P < 0.01, and ***P < 0.001, when compared to non-tolerant group; Least Significant Difference Test.

TOLERANCE DEVELOPMENT T O EEG AND ~EEG POWER SPECTRAL EFFECTS OF METHADONE

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A N

W

CONTROL AWAKE

1 sec

mmmmI

0 15 Hz

METHADONE (2 mg/kg, iv) a. NON- TOLERANT b. TOLERANT

0 15 Hz 0 15 Ha

Fig. 1. Direct cortical EEG recordings and related EEG power spectra during control wakeful- ness (upper), and after methadone adminis t ra t ion (2 mg/kg, i.v.) in (a) a non-tolerant and (b) a methadone- tolerant rat.

tolerant and methadone-tolerant rats increased total EEG spectral power by 509 -+ 112% (mean -+ S.E.M.) and 160 -+ 19% compared to control awake spectral power, respectively; these increases are significantly different from one another at the 0.01 level. Furthermore, rats chronically treated with EKC displayed cross-tolerance to methadone. The average increase in EEG spectral power in these rats was 275 +- 44% of control awake values which is significantly different from the value obtained in non-tolerant rats (P < 0.05).

The administration of EKC in non-tolerant rats produced dense high-voltage EEG bursts and increased total EEG spectral power (852 -+ 163% of control). Administration of EKC produced smaller increases in total EEG spectral power in both EKC-tolerant (217 + 33% of control) and

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methadone-tolerant (208-+ 32% of control) rats; these values are signifi. cantly different from that in non-tolerant rats (P < 0.001).

Representative EEG and behavioral effects produced by the adminis- tration of methadone (2 mg/kg, i.v.) in non-tolerant, methadone tolerant and EKC-tolerant rats are shown in Fig. 2. In non-tolerant rats, methadone produced behavioral stupor accompanied by high-voltage slow- frequency EEG bursts for about 30-60 min which was followed by be- havioral arousal associated with low-voltage desynchronized EEG for about 30-60 min (Fig. 2; top). In methadone-tolerant rats, methadone produced less severe stupor lasting for a much shorter period of time which was then followed by a shorter period or arousal (Fig. 2; middle). Also,

A R O U S A L

A W A K E

STUPOR

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NON-TOLERANT M E T H A D O N E

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I I I

METHADONE TOLERANT M E T H A D O N E

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HOURS Fig. 2. Graphic representation of EEG and behavioral responses of representative rats to a 2 mg/kg i.v. dose of methadone during the non-tolerant, methadone-tolerant and EKC-to|erant states.

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EKC-tolerant rats showed cross-tolerance to methadone as reflected by a reduced duration of stupor and, to a relatively lesser extent, of arousal (Fig. 2; bottom).

Representative EEG and behavioral effects produced by the adminis- tration of EKC (4 mg/kg, i.v.) on EEG and behavior in non-tolerant, EKC- tolerant and methadone-tolerant rats are depicted in Fig. 3. In non- tolerant rats, similar to methadone, EKC produced a biphasic EEG and behavioral response consisting of 30-60 min of behavioral stupor with high-voltage EEG bursts followed by up to 60 min of arousal associated with low-voltage desynchronized EEG (Fig. 3; top). After chronic treatment with EKC, a significant degree of tolerance developed to the behavioral

AROUSAL

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HOURS Fig, 3. Graphic representat ion of EEG and behaviora l responses of representat ive rats to a 4 mg /kg i.v. dose of EKC during the non-tolerant, EKC-tolerant, and methadone- tolerant states.

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effects of EKC, as reflected in a reduced duration of the stuporous and arousal phases (Fig. 3; middle). Methadone-tolerant rats also demonstrated a high degree of cross-tolerance to the EEG and behavioral effects of EKC (Fig. 3; bottom).

DISCUSSION

We found earlier that morphine-tolerant rats were not cross-tolerant to the EEG and behavioral effects of methadone, but, on the other hand, methadone-tolerant rats were cross-tolerant to morphine [3]. Also, mor- phine-tolerant rats were not cross-tolerant to the effects of EKC on EEG and behavior, but EKC-tolerant rats were cross-tolerant to the effects of morphine [4]. Unidirectional cross-tolerance between morphine and methadone and between morphine and EKC has also been reported by others using different experimental protocols [5,7,8]. The present s tudy demonstrates bidirectional cross-tolerance between methadone and EKC. The development of tolerance and cross-tolerance to the EEG and behavior- al effects of morphine, methadone and EKC in rats is summarized in Table III.

It was shown that in vitro tolerance developed to the inhibitory effects of selective mu and kappa opioid agonists on electrically-induced muscle twitches in the guinea-pig ileum preparation [18]. In this study, cross- tolerance among the selective mu agonists was found as well as among the selective kappa agonists; however, cross-tolerance between the mu and kappa agonists was not found. This led the authors to conclude that separate populations of mu and kappa receptors exist in the guinea-pig ileum. It was also shown that selective mu, kappa and delta opioid agonists differentially protect receptor binding sites from deactivation by N-ethylmaleimide [19]. Based upon cross-tolerance data with mu and delta agonists, Yaksh [20] suggested that once animals are made tolerant to one

TABLE III

CROSS-TOLERANCE AMONG MORPHINE, METHADONE AND EKC

Rats Given: Cross-tolerance tolerant to:

Morphine Methadone ( - ) Methadone Morphine ( + )

Morphine EKC (- ) EKC Morphine ( + )

Methadone EKC ( + ) EKC Methadone ( + )

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opioid agonist, selective cross-tolerance develops such that they are cross- tolerant only to other opioids which activate the same opioid receptor subtype. However, our present data suggest that the phenomenon of cross-tolerance among opioid agonists may not be a suitable index for defining multiple opioid receptor populations. Other experimental parame- ters are better suited to define opioid receptor subtypes; e.g., protection studies using receptor binding techniques.

Many investigators have tried to explain differential cross-tolerance of opioids on the basis of lipid solubility [8,9,21]. For example, relative lipid solubility may influence the abilities of agonists to cross the blood-brain barrier [9] or the accessibilities of different opioids to receptors at the cellular level, especially to internalized receptors [21,22]. Interestingly, morphine has a very low partition coefficient (1.42 at pH 7.4 and 37°C) [10], while methadone [10] and EKC (unpublished data from our labora- tory) have very high partition coefficients; 116.33 and 112.4, respectively.

Altered pharmacological characteristics of the opioid receptors upon tolerance development may be involved in differential cross-tolerance. It was found that the endogenous opioid peptide dynorphin, a kappa opioid agonist [23-27], antagonizes morphine analgesia in non-tolerent mice [28]. In contrast, dynorphin was found to augment morphine-induced analgesia in morphine-tolerant mice [28]. Also, dynorphin has been reported to suppress withdrawal symptoms in heroin-dependent humans [29] as well as in morphine dependent monkeys [30] and rats [31]. Furthermore, in self-administration studies, it has been shown that dynorphin, ketocycla- zocine and EKC substituted for morphine in dependent rats self-adminis- tering morphine and sustained a state of dependence [32,33]. Thus, kappa agonists appear to antagonize morphine effects in naive subjects; however, they substitute for morphine or heroin in dependent subjects. These data suggest that critical changes may have occurred in receptor properties during the development of tolerance. These changes in receptor properties may also play an important role in defining emerging uni- or bidirectional cross-tolerance.

In conclusion, the phenomena of opioid tolerance and cross-tolerance appear to involve multidimensional factors which may include receptor selectivity, lipid solubility and changes in receptor characteristics as- sociated with the development of tolerance.

ACKNOWLEDGEMENT

We thank Mrs. Patricia Tretter for the professional preparation of this manuscript.

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