The use of quaternary narcotic antagonists in opiate research

NeuropharmacologyVol. 24, No. 3, pp. 181-191, 1985 Printed in Great Britain. All rights reserved

0028-3908/85 $3.00+ 0.00 Copyright c 1985Pergamon Press Ltd

REVIEW THE USE OF QUATERNARY NARCOTIC ANTAGONISTS IN OPIATE RESEARCH D. R. BROWN* and L. I. GOLDBERG? *Department of Veterinary Biology, University of Minnesota-Twin Cities, College of Veterinary Medicine, 295 Animal Science/Veterinary Medicine Building, 1988 Fitch Avenue, St Paul, MN 55108 and tDepartments of Pharmacological and Physiological Sciences, and of Medicine, and the Committee on Clinical Pharmacology, The University of Chicago, 947 East 58th Street, Chicago, IL 60637 U.S.A. (Accepted 12 June 1984)

Summary-Quatemary ammonium derivatives of narcotic antagonists are commonly used in determining sites of action of opiates in the central nervous system and the periphery because it is widely assumed that they do not readily cross the blood-brain barrier, in contrast to their relatively non-polar tertiary counterparts. However, these compounds possess several unique pharmacological properties which have not been taken into consideration in the design of numerous investigations. This article reviews the current state of knowledge concerning the pharmacology of the quatemary narcotic antagonists, examines their use in physiological and behavioral studies of action of opiates, and proposes guidelines for the design of experiments involving these compounds. Key words: quaternary opiates, quaternary opiate antagonists, N-methylnaloxone, Nmethylnalorphine, N-methylnaltrexone, N,N-diallyl nalorphine, N-methyllevallorphan, central nervous system, sites of opiate action.

A common method of ascertaining the central or peripheral action of opiates involves the use of pure opiate narcotic antagonists, such as naloxone- or naltrexone, or the mixed opiate agonist-antagonist nalorphine, to antagonize the effects of exogenously administered opiates or to interrupt activity in endogenous opioid systems. These antagonists are highly lipid-soluble and rapidly diffuse across biological membranes, including the blood-brain barrier (BBB), and this property has consistently frustrated attempts to confine these compounds to either the central nervous system (CNS) or to peripheral tissues (Kaufman, Semo and Koski, 1975; Berkowitz, Spector and Lee, 1976; Herz and Teschemacher, 1971, 1978). One approach to this problem has been to develop congeners of these antagonists bearing an additional alkyl substituent on the nitrogen atom in their ring structures (Fig. 1). These quaternary amine derivatives (Goldberg, Merz and Stockhaus, 1979), which retain some antagonist activity, were designed to traverse the blood-brain barrier less readily than their tertiary counterparts as a consequence of their relatively greater polarity and reduced lipid solubility. It has been known for over a century that quaternary forms of opiate agonists, such as Nmethylmorphine and N-methylcodeine, display little or no analgesic activity and do not reduce body temperature when given by peripheral routes, although quaternary morphine decreases the gut motility in a manner similar to morphine (CrumN.P. 24,SA

181

Brown and Fraser, 1868; Dott and Stockman, 1890; Eddy, 1933; Foster, Jenden and Lomax, 1967; Herz and Teschemacher, 1971). On the other hand, Nmethylmorphine is effective in producing analgesia and hypothermia when administered into the cerebral ventricles, suggesting that these latter effects are produced through interactions with opiate receptors in the CNS (Foster et al., 1967; Herz and Teschemacher, 1971). The use of quaternary opiates is complicated however by their low affinity for opiate receptors compared to the tertiary forms (Eddy, 1933; Kosterlitz, Lord and Watt, 1973; Kosterlitz and Waterfield, 1975; Opheim and Cox, 1976) and their ability, similar to other quaternary ammonium compounds including the quaternary narcotic antagonists, to block autonomic ganglia and the neuromuscular junction in large doses (Crum-Brown and Fraser, 1968; Dott and Stockman, 1890; Eddy, 1933; Foster et al., 1967). The quaternary narcotic antagonists have assumed increasing importance as pharmacological tools in the determination of the sites at which opiates act within the brain and periphery, The interest in this field of research has resulted in a steadily growing body of literature and it is the purpose of the present review briefly to survey and critically to evaluate these findings. PHARMACOLOGICAL

CONSIDERATIONS

Quaternary narcotic antagonists have been used with increasing frequency to localize the action of

182

D. R. BROWNand L. I. GOLDBERG

@_$y”

d2d HO

0 NALTREXONE

HO 0 0 N-METHYLNALTREXONE

0

&‘“”

HO

&2-CH=CH2

0

0

HO 0 0 N-METHYLNALOXONE

NALOXONE

Hk==(O% NALORPHINE

HO N.N

0

OH

DIALLYLNALORPHINE

(DANM)

FH3

H6 METHYLLEVALLORPHAN

Hb LEVALLORPHAN

N-ALLYL

LEVORPHANOL

HO N-ALLYL

LEVALLORPHAN

Fig. 1. Chemical structures of some commonly-used narcotic antagonists and their quatemary forms. Table

1. Activity

opiates. However, there has been frequent lack of adherence to the methodological detail necessary to prevent error. Before reviewing this literature, it might be helpful to consider some safeguards which are frequently omitted. First, dose-response relationships of the quaternary antagonist must be determined for the experiments in which the antagonist is used. Quaternary narcotic antagonists require larger doses than their tertiary congeners for equivalent opiate antagonism in several different biological systems. These apparent differences in potency in uivo may be related to a number of pharmacological variables: these include membrane permeability, distribution of the drug across the blood-brain barrier and within CNS and peripheral tissues, biological half-life and metabolic conversion to other active and inactive forms, and affinity for opiate receptors. Most of these determinants have received little attention or are unknown. Differences in receptor affinity and pharmacological potency are best evaluated in bioassays in uitro where distribution and metabolic variables do not assume the importance they do in whole animal preparations (Kosterlitz and Waterfield, 1975). Quaterinization of the opiate antagonists generally greatly diminishes their affinity for opiate receptors. For example, methylnaloxone has generally been found to possess only 2 to 4% of the opiate antagonist activity of naloxone in vitro and potency differences between methylnaltrexone and naltrexone are of the same order (Table 1). On the other hand, methylnalorphine has been shown to vary widely in potency, relative to nalorphine, in similar assays from different laboratories. It has been reported to possess only 2% of the activity of nalorphine in displacing [3H]etorphine from membranes from the rat brain but nearly 30% in displacing (Table l), [3Hldiprenorphine (Kobylecki, Lane, Smith, Wakelin, Cruse, Egert and Kennard, 1982). Moreover, methylnalorphine antagonizes the acute effects of morphine on the assay using the field-stimulated longitudinal muscle of the guinea-pig, with a potency only 12% that of nalorphine, although one study

of quaternary

in vitro*

opiate antagonists

Guinea-pig

Compound Methylnaloxone Methylnaltrexone Methylnalorphine N-Methyllevallorphan

Rat brain, opiate receptor afinityt (%) 3.6 1.9 1.9 1.3 2.1 -

(1)ll (2) (I) (3) (1)

Morphine antagonism $ (%) 2.3 (1) 3.6 (4) 2.6 (1) 12.1 (5) 112.6 (6)

ileum Precipitate abstinence $ (%) 2.6 (1) 10.2 (4) 3.9 (1) -

*Activity expressed as a percentage of antagonist potency relative to the tertiary form. tAbility to displace 50% of stereospecifically-bound [‘Hletorphine from membranes from rat brain in the presence of 150mM sodium. iBased on K, value for antagonizing the action of morphine in reducing electrically-induced contractions of longitudinal muscle. §Based on concentrations which are 50% effective in producing withdrawal contractions in longitudinal muscle from morphine-dependent animals. 11Data obtained from the following references: (1) Valentino el al. (1983); (2) Manara ef al. (1982); (3) Valentino ef al. (1981); (4) Killian, Schuster, Wainer and Merz (1981); (5) Kosterlitz and Waterfield (1975); (6) Bianchetti et al. (1983).

Quatemary narcotic antagonists reported that it is 5-fold more potent than nalorphine in antagonizing the effects of normorphine (Smith, Buchan, Parsons and Wilkinson, 1982). Quaternary nalorphine exhibits only 6% of the potency of nalorphine in antagonizing the effects of methionine enkephalin in the assay using the isolated vas deferens of the mouse, but is almost 40% as potent as nalorphine in reversing the actions of normorphine in this preparation (Table 1; Smith et al., 1982). Finally, N-methyllevallorphan has only 4% of the potency of levallorphan in displacing [3Hlnaltrexone from membranes of the rat brain, but appears to be slightly more potent than its tertiary form in antagonizing the actions of morphine in the guinea-pig ileum (Table 1; Bianchetti, Nisato, Sacilotto, Dragonetti, Picerno, Tarantino, Manara, Angel and Simon, 1983). These various experimental differences could arise from the actions of drugs at different subtypes of opiate receptors, the partial agonist character of nalorphine and levallorphan, or the wide variability between laboratories in ligand binding assays. These many possibilities remain to be investigated. The relative potency difference between the tertiary and quaternary forms of an opiate antagonist has been an often neglected factor in the design and interpretation of in vivo experiments involving these compounds. Indeed, experimental results based on the administration of single doses of drugs offer little information and are not useful in comparisons of pharmacological potency. Second, dose-effect curves obtained after the peripheral administration of a quaternary opiate antagonist should be compared when possible to those obtained after its administration into the CNS. A major assumption in the use of these compounds is that they do not readily penetrate the blood-brain barrier. Thus, for example, the absence of an effect by a quaternary antagonist after peripheral administration is taken to imply the existence of an opiate site of action in the CNS. However, these compounds may pass intact into the brain across the blood-brain barrier or several more permeable membranes, such as those located near the area postrema, for instance, at some finite rate of diffusion. A seeming lack of pharmacological effect may be due to the relatively low affinity of these quaternary compounds for opiate receptors. Indeed, when [methyl-‘4C]nalorphine was subcutaneously administered to mice, a small amount of radiolabel was present in brain tissue as soon as 15 min after injection (Smith et al., 1982). In other experiments, quaternary naloxone, administered by peripheral routes, had only l-2% of the potency of tertiary naloxone in reversing catalepsy in rats induced by morphine and in precipitating a withdrawal syndrome in monkeys dependent on morphine (Brown, Robertson and Goldberg, 1983; Valentino, Katz, Medzihradsky and Woods, 1983). These differences in potency are similar to those found in vitro, indicating that quaternary naloxone gains entry to the CNS from the peripheral circu-

183

lation. In contrast, quaternary naltrexone was less than 0.1% as potent as naltrexone in reversing catalepsy in rats and completely ineffective in precipitating withdrawal in dependent monkeys even at doses up to 32 mg/kg. Moreover, methylnaltrexone was lO,OOO-fold more potent in reversing catalepsy when given intracerebroventricularly (i.c.v.) than it was upon subcutaneous administration (Brown et al., 1983). As quaternary naltrexone possesses l-4% of the affinity of naltrexone for opiate receptors, these differences in potency between the quaternary and tertiary forms are greater than could be explained by differences in affinity for opiate receptors alone; this consideration and the high potency of centrallyadministered methylnaltrexone indicate that it probably does not readily traverse the blood-brain barrier, at least during the times and in the species examined. Thus, in this case, these potency differences observed between quaternary and tertiary antagonists were probably due to distributional variables in vivo. Third, the time intervals during which the effects of quaternary antagonists are studied should be chosen with caution. Little is known about the pharmacokinetics and metabolism of these compounds. The antagonist may be readily metabolized to either more active, lipid-soluble forms or to products devoid of opiate antagonist activity. Moreover, they may slowly diffuse across the blood-brain barrier and achieve pharmacologically effective concentrations in the CNS. Injected into the CNS, they might also diffuse into the peripheral circulation with time. The rate of penetration of drug across the blood-brain barrier may also vary according to species of animal, route of administration, and the experimental procedure; the latter variable may produce changes in the permeability of the blood-brain barrier. Some quaternary antagonists may penetrate biological membranes more readily than others as a function of their lipid solubility, rate of metabolism and other factors. Fourth, it is important to assess the chemical purity of the compounds under investigation. A small amount of contamination by tertiary congeners, for example, could lead to erroneous results. Finally, the quaternary narcotic antagonists produce effects unrelated to the blockade of opiate receptors. This has been documented for tertiary opiate antagonists such as naloxone (Sawynok, Pinsky and LaBella, 1979). In large doses, quaternary antagonists may block the neuromuscular junction or autonomic ganglia (Argentieri and McArdle, 1983). Quaternary naloxone produces tremors and convulsions at doses of approx. 1&3Opg (i.c.v.) in rats (Brown and Holtzman, 1981; Hemmer, Olson, Kastin, McLean and Olson, 1982; Ramabadran, Suaudeau and Jacob, 1982). Quaternary naltrexone, at a concentration of 160 PM, has recently been shown to alter the amplitude and time course of end-plate currents at the neuro-muscular junction of the rat, suggesting that the compound blocks ionconductance channels associated with cholinergic re-

D. R. BROWN and

184

Table 2. Summary

L. I. GOLDBERG

of experiments on the ability of quaternary opiate antagonists induced inhibition of gastrointestinal transit

Route of administration

of quaternary

antagonist

Peripheral N-methylnaloxone N-methylnaltrexone N-methylnalorphine DANM N-methyllevallorphan N-allyllevallorphan CennYzl (i.c.v.) DANM

to reverse morphine-

Route of administration Peripheral l(R); I(M) l(R); 44 l(R); -(W 1(R); -(M) -(R); l(M) -(R); l(M)

of morphine Central

(i.c.v.)

O(R); l(M) O(R); -(M)

-(R)%4 -(R); O(M)

l(R)

Key: 0 = no change; I_= antagonism; - = not tested; R = rat; M = mouse. References to specific experiments are found in the text. DANM = N,N-diallylnalorphine.

ceptors (Argentieri and McArdle, 1983). Clearly, the opiate antagonist activity of these compounds must be distinguished from their non-specific effects. With these experimental considerations taken into account, the use of the quaternary antagonists in a variety of physiological and behavioral systems is discussed below. GASTROINTESTINAL MOTILITY

Opiates inhibit gastric emptying and the propulsive motor activity of the intestine, thereby decreasing the rate of intestinal transit and producing constipation. These antimotility and antitransit actions can be produced by the administration of opiate agonists into both the systemic circulation and the brain. Quaternary narcotic antagonists have been employed in many experiments designed to examine this dual localization of action. These compounds are quite effective in antagonizing the inhibitory actions of opiates on the intestinal smooth muscle in vitro. In many cases, they are also effective in inhibiting the antimotility effects of opiates in vivo, particularly when both the agonist and quaternary antagonist are administered by similar routes. The degree of which peripherally-administered quaternary antagonists inhibit the actions of opiate agonists, injected into the CNS, may reflect their ability to enter the brain from the periphery. The effects of the quaternary antagonists on the antitransit actions of morphine are summarized in Table 2 and in the discussion below. In the rat, the subcutaneous or intraperitoneal (i.p.) administration of methylnaloxone or methylnaltrexone, in doses up to 4 and 30mg/kg respectively, antagonized the inhibitory effects of peripherally-administered morphine on intestinal transit (Ferritti, Bianchi, Tavani and Manara, 1981; Russell, Bass, Goldberg, Schuster and Merz, 1982; Bianchi, Fiocchi, Tavani and Manara, 1982; Manara, Bianchi, Fiocchi, Notarnicola, Peracchia and Tavani, 1982). The N,N-diallyl derivative of nalorphine (DANM; Fig. 1) was also effective in large doses (8 mg/kg), when given by peripheral routes, although its effect was short-lived (Tavani, Bianchi and Manara, 1979; Burleigh, 1981; Burleigh, Galligan and Burks, 1981). Methylnalorphine, like DANM, is relatively weak and its effects are of short duration

(Bianchi et al., 1982). A relatively large dose of DANM (100 pg) was found to reverse the antitransit actions of morphine injected peripherally when injected into the cerebral ventricles, but it did not inhibit the effects of loperamide, an opiate-like antidiarrheal agent which does not appreciably enter the CNS after peripheral administration (Burleigh et al., 1981). On the other hand, methylnaloxone and methylnaltrexone, at doses up to 8 mg/kg (i.p.), had no effect on the antitransit actions of morphine (36-2OOpg, i.c.v.) in the rat (Manara et nl., 1982; Parolaro, Sala, Crema, Spazzi, Cesana and Gori, 1983). Moreover, methylnaloxone injected peripherally was ineffective in antagonizing the antitransit effects of the opioid peptide dermorphin; however, it did block the actions of this peptide upon intracerebroventricular administration (Parolaro et al., 1983). The inhibition of transit induced by the intracerebroventricular administration of the other opioid peptides, /?-endorphin or p-Ala*-methionine enkephalinamide, was not affected by DANM (10 mg/kg) given by peripheral routes to rats (Galligan and Burks, 1982). In mice, large intravenous or oral doses of methylnaltrexone (up to 30 and 720 mg/kg, respectively) did not alter the actions of subcutaneously administered morphine on diarrhea induced by prostaglandin (Russell et al., 1982). On the other hand, moderate doses of methylnaloxone (5-10 mg/kg, s.c.) have been reported to reverse the antitransit actions of morphine (Schulz, Wuster and Herz, 1979; J. Rick and L. I. Goldberg, unpublished). The N-methyl and N-ally1 quaternary derivatives of levallorphan (Fig. 1) were effective in antagonizing the antitransit actions of 12 mg/kg of morphine (s.c.) with doses of 4 and 16 mg/kg (s.c.) respectively inhibiting 50% of the effect of the opiate (Bianchetti et al., 1983; Dragonetti, Bianchetti, Sacilotto, Giudice, Ferrarese, Cattaneo and Manara, 1983). In contrast, the diastereoisomer of N-methyllevallorphan, N-allyllevorphanol (Fig. l), was completely ineffective in subcutaneous doses in excess of 50 mg/kg (Dragonetti et al., 1983). Methylnaloxone administered peripherally appears to cross the blood-brain barrier in the mouse. The antagonist, at a dose of 10 mg/kg (i.p.), was found to inhibit the antimotility effects of morphine administered in the CNS (Schulz et al., 1979). Indeed, the

Quatemary

narcotic

subcutaneous dose of methylnaloxone required to reduce the centrally-induced antitransit effects of morphine by 50% has been estimated at 2 mg/kg (Bianchetti, Giudice, Picemo and Carminati, 1982). N-Methyl- and N-allyllevallorphan, however, at doses up to 60mg/kg (s.c.) did not inhibit the antimotility effects of 2 pg morphine (i.c.v.) (Dragonetti et al., 1983). In the dog, peripheral doses of both methylnaloxone and methylnaltrexone as small as 0.5 mg/kg antagonized the inhibitory effects of morphine on the electrical concomitants of intestinal motor activity (Russell et al., 1982). ANTINOCICEPTION

The analgesic or antinociceptive actions of the opiates are thought to be mediated at sites within the CNS and are reversed by systemically-administered narcotic antagonists. Quaternary antagonists produce variable effects on opiate-induced antinociception when given by peripheral routes, depending upon the species, the analgesia assay system, and the pretreatment intervals for drugs under examination. No investigation has yet examined the antagonist effects of these compounds after their administration into the CNS. In the mouse, morphine decreases the writhing induced by intraperitoneal injections of acetic acid, an effect which was not altered by up to 16mg/kg (i.p.) of methylnalorphine (Smith et al., 1982). On the other hand, methylnaloxone, and probably to a lesser extent methylnaltrexone appear to inhibit antinociception produced in mice by morphine, suggesting that they cross the blood-brain barrier in this species. Methylnaloxone at doses of 5-30 mg/kg (i.p.) reduced increases in tail-flick latency produced by morphine (Schulz et al., 1979). It was found to be only 1.6 and 4% as potent as tertiary naloxone in blocking antinociception induced by morphine, as determined in a shock-vocalization (Nilsen test) and hot-plate tests (Bianchetti et al., 1982; Dragonetti et al., 1983). Because methylnaloxone inhibits these effects of morphine at doses comparable to those of naloxone when differences in potency are taken into account, this data is additional evidence that the quaternary form, or an active metabolite, readily crosses the blood-brain barrier of mice. Methylnaltrexone also decreased the effects of morphine on the latency of hot-plate escape in mice when given at doses exceeding 10 mg/kg (i.p.) (Russell et al., 1982). Mice, defeated in attacks by other mice, have been reported to display an increase in the latency of tail-flick, an antinociceptive response believed to involve an endogenous opioid system. This analgesic state was eliminated by 1 mg/kg (i.p.) of naltrexone, but remained unaffected by 40 mg/kg of methylnaltrexone (Miczek, Thompson and Shuster, 1982). Peripherally-administered methylnaloxone or methylnaltrexone have no immediate effects on nor-

antagonists

185

mal sensitivity to pain even in large doses, although methylnaltrexone has been reported to increase slightly the sensitivity of mice 2 hr after treatment. This was thought to be due to diffusion of the compound across the blood-brain barrier and subsequent inhibition of endogenous opioid pathways involved in responsivity to pain (Ramabadran, 1982). However, methylnaltrexone, administered into the cerebral ventricles in large doses (lO&lOOO pg/kg), actually increased the latency of hot-plate escape (Ramabadran et al., 1982). Finally, N-allyllevallorphan inhibits the antinociceptive effects of morphine (24mg/kg, s.c.) in mice as assessed in the hot-plate paradigm with a dose of 15 mg/kg (s.c.) reducing the action or morphine by 50%. On the other hand, N-methyllevallorphan does not affect antinociception induced by morphine at doses exceeding 30mg/kg (s.c.) (Dragonetti et al., 1983). In the rat, studies with quaternary antagonists are often contradictory. For example, DANM was reported to inhibit the antinociceptive actions of morphine by 66% at a dose of 8 mg/kg (i.p.) in a hot-date test (Tavani et al., 1979). However, recent studies from the same laboratory detected no appreciable antagonist activity at doses of DANM up to 60 mg/kg (s.c.) in a similar paradigm (Bianchi et al., 1982). This discrepancy might have arisen by administration of DANM through different routes (intraperitoneal versus subcutaneous) but the importance of this variable was not determined. Furthermore, although 0.1 mg/kg (s.c.) of methylnaloxone was reported to antagonize the actions of morphine on the hot-plate test (Rios and Jacob, 1982), other studies have shown significant antagonist activity only at doses exceeding 16 mg/kg (Ferritti et al., 1981; Bianchi et al., 1982). In the absence of opiates, small doses (l-10 pg) of methylnaloxone, injected into the inflamed paws of rats, actually decreased responses to pain (Rios and Jacob, 1983). Methylnaltrexone was found to have no appreciable morphine antagonist activity in a hot-plate paradigm even at toxic doses of 10&300mg/kg (i.p.) (Russell et al., 1982). Using the same technique but longer pretreatment intervals, others have detected significant antagonist activity with doses of methylnaltrexone as small as 8mg/kg (s.c.), suggesting that the drug had entered the CNS of the rat at the later time periods (Bianchi et al., 1982). Due to a lack of data on the antagonist activity of these compounds after administration into the CNS, the use of single doses of drug, instead of determinations of dose-effect relationships, and widely varying doses of morphine, the ability of quaternary antagonists to block antinociception induced by opiates has yet to be resolved. PRECIPITATED OPIATE-WITHDRAWAL

A well-recognized and sensitive index of opiate antagonist activity is the ability of a drug to precip-

186

D. R. BROWN and L. I. GOLDBERG

itate the withdrawal syndrome in animals dependent on opiates. As discussed above, quaternary narcotic antagonists can precipitate abstinence-related contractions of intestinal smooth muscle from dependent animals in vitro. In such isolated tissue preparations, the differences in potency between the tertiary and quaternary forms in precipitating withdrawal are similar to those obtained for binding to opiate receptors and antagonizing the acute effects of opiates (Table 1). In rats dependent on morphine, DANM was found to be approximately three orders of magnitude less potent than nalorphine in precipitating withdrawal after peripheral administration, but nearly equipotent when administered centrally (Laschka, Herz and methylBlasig, 1976). Peripherally-administered naltrexone was found to be completely ineffective in precipitating withdrawal in dependent rhesus monkeys even at doses as large as 32 mg/kg; in contrast, naltrexone was effective at 4pgg/kg (Valentino, Herling, Woods, Medzihradsky and Merz, 1981; Valentino et al., 1983). Mice made acutely dependent upon morphine displayed only slight withdrawalrelated reactions after the administration of methylnaloxone or methylnaltexone at 3-30 mg/kg (s.c.) (Ramabadran, 1982). Although this latter finding might imply that these antagonists did not cross the blood-brain barrier in mice, it is also quite likely that the degree of dependence achieved after the administration of a single dose of morphine and the magnitude of the resulting abstinence syndrome would be quite small. On the other hand, the relatively narrow loo-fold separation in the potencies of DANM-nalorphine and methylnaloxone-naloxone in precipitating withdrawal in dependent rhesus monkeys approximates the differences in potency obtained in in vitro bioassay systems (Valentino et al., 1983). Using precipitated withdrawal as an index of penetration of the blood-brain barrier, methylnaloxone and DANM or their active metabolites enter the brain readily after peripheral administration in a number of species. However, the differing degrees of dependence on opiates obtained in the investigations discussed, the possibility that the intensity of dependence on morphine may affect the integrity of the blood-brain barrier, and obvious species-differences, are some variables that may complicate this interpretation. Diarrhea is one common manifestation of the opiate-withdrawal syndrome. It presumably occurs as the combined result of enhanced intestinal motor activity and diminished absorption of fluid from the intestinal lumen. Although tertiary narcotic antagonists are quite potent in eliciting withdrawal diarrhea in dependent rats, methylnaltrexone and DANM were recently found to be ineffective in producing either overt signs of withdrawal or, specifically, diarrhea after peripheral administration at doses up to 50mg/kg (i.p.) (Schreier and Burks, 1982). Methylnaltrexone, in doses up to 50 mg/kg (s.c.), failed to

produce diarrhea in dogs dependent on morphine (Russell et al., 1982). However, when injected into the cerebral ventricles of dependent rats in large doses of 20 and 50 p g respectively, both methylnaltrexone and DANM produced the withdrawal syndrome, including diarrhea (Schreier and Burks, 1982). Methylnaltrexone has been found to decrease absorption of fluid from closed loops of the jejunum and colon of rats dependent on morphine, when administered at 0.01-3 mg/kg, intravenously. This antiabsorptive action also occurs after intracerebroventricular injections in doses up to 10 pg (Chang, Brown, Field and Miller, 1984). Thus, the disturbances in intestinal motility and transport of fluid occurring in precipitated opiate withdrawal may have CNS, as well as peripherally-localized, components. OTHER PHYSIOLOGICAL SYSTEMS

Cardiovascular

effects

Opiates have pronounced effects on the cardiovascular system and endogenous opioids may be involved physiologically in some aspects of cardiorespiratory regulation (Holaday, 1983). In conscious dogs, both methylnaloxone and methylnaltrexone produce hypotension and tachycardia after intravenous administration. The effects of methylnaloxone are dose-related, with doses as small as 0.25 and 0.5 mg/kg producing significant changes in blood pressure and heart rate, respectively (Giles, Sander and Merz, 1983). In contrast, naloxone (1 mg/kg, i.v.) produces hypertension and tachycardia (Sander, Kastin and Giles, 1982). Both quaternary antagonists given by intravenous bolus also inhibit the vasopressor and tachycardic responses to leucine enkephalin in small intravenous doses, although the opiate antagonist activity of methylnaloxone is of short (< 4 min) duration (Giles et al., 1983). It has not been determined whether these actions of the quaternary antagonists are due to an effect at opiate receptors or are the result of ganglionic blockade (see below). Endogenous opioids may be involved in the decompensatory adjustments observed in circulatory shock (Holaday, 1983). Naloxone and naltrexone, at 1-2.5mg/kg (s.c.) significantly increased the survival of mice undergoing anaphylactic shock. Equivalent doses of methylnaltrexone did not increase survival (Amir, 1982, 1983). In a related study, methylnaltrexone in doses of 1 or 5 mg/kg (s.c.) did not alter hyperglycemia in mice in response to the administration of Escherichia coli endotoxin. However, subcutaneous injections of either naloxone or naltrexone, in the same dose range, were effective in decreasing the magnitude of the hyperglycemia induced by endotoxin (Amir and Harel, 1983). Based on these data, these investigators suggested that central opioid pathways were involved in some aspects of circulatory shock. Since the doses of quaternary naltrexone used

Quatemary narcotic antagonists

in these studies were considerably smaller than those of the tertiary antagonist when differences in potency are recognized, and were not proved to be effective upon administration into the CNS, it is difficult to make conclusions regarding a role for endogenous opioids in the pathophysiology of shock from these data. Finally, methylnaltrexone has recently been found to dose-dependently reduce mean arterial blood pressure and increase heart rate in rats when given intravenously in doses ranging from 0.005 to lOmg/kg, due to an anticholinergic action on the heart (Willette, Krieger and Sapru, 1983). Moreover, the quaternary antagonist at 5 mg/kg (i.v.) markedly effects of l,lattenuated the cardiovascular dimethyl-4-phenylpiperazinium (DMPP), a ganglion stimulant, the bradycardic actions of methacholine, and the reflex bradycardia associated with the administration of phenylephrine. Methylnaltrexone also abolished the depressant effects of enkephalins on respiration acting at pulmonary sensory receptors. Therefore the compound, in moderate to large doses, appears to block nicotinic ganglionic and cardiac muscarinic receptors as well as opiate receptors (Willette et al., 1983). Endocrinological

effects

Opiates alter the release of certain pituitary hormones. Two reports have been published in which methylnaloxone was used to determine the site(s) at which morphine stimulates the release of growth hormone and prolactin and inhibits the secretion of luteinizing hormone in the rat. When given intraperitoneally at a dose of 2.5 mg/kg, it had no effect on the release of growth hormone or prolactin, induced by morphine. However, methylnaloxone was quite effective in inhibiting these effects at 5pg (i.c.v.). Neither naloxone nor its quaternary form had any effect on the basal secretion of growth hormone, although naloxone decreased the basal release of prolactin at intraperitoneal doses, greater than 5 mg/kg (Panerai, Casanueva, Martini, Mantegazza and DiGiulio, 1981). On the other hand, both naloxone and methylnaloxone increased the basal release of luteinizing hormone and reversed the inhibitory actions of morphine (Panerai, Martini, Casanueva, Petraglia, DiGiulio and Mantegazza, 1983). The peripheral administration of a quaternary opiate agonist, N-methylmorphine, decreased the release of luteinizing hormone but did not alter that of growth hormone or prolactin. However, when injected intracerebroventricularly it increased secretion of both growth hormone and prolactin (Panerai et al., 1981, 1983). These findings tentatively suggest that opiates act centrally to modulate the release of growth hormone and prolactin, but outside the blood-brain barrier to influence that of luteinizing hormone. Large doses of narcotic antagonists have been shown to elevate levels of corticosterone in the

187

plasma of rats. For example, a lOmg/kg dose of naltrexone significantly raised the level of corticosterone in plasma within 15 min of intravenous administration; in contrast, doses of methylnaloxone or methylnaltrexone up to 20 mg/kg (i.v.) had no appreciable effect on hormonal level. However, when either quaternary antagonist was administered at a dose of 50 pg (i.c.v.), significant increases in the levels of corticosterone were observed, lasting up to 60 min after the injection (Eisenberg, 1984). On the basis of relative differences in potency, the intravenous doses of quaternary antagonists employed were considerably smaller than those of naltrexone necessary to promote hormonal secretion and therefore would not be expected to elevate the level of corticosterone in plasma. Moreover, large intracerebroventricular doses of the quaternary analogs produce, as discussed above, convulsive episodes in rats and these could certainly have contributed to the hormonal alterations occurring after central administration of drugs in this study. Thus, it remains unclear whether quaternary narcotic antagonists increase the level of corticosterone in plasma through a direct interaction with opiate receptors or by non-specific means, and whether the site(s) of this effect predominately lie in the CNS or the periphery. Renal effects

Opiates acting at putative kappa-receptors, such as ethylketocyclazocine and bremazocine, produce diuresis in rats. Urination induced by bremazocine was inhibited by the subcutaneous administration of a number of narcotic antagonists. However, methylnaltrexone, in doses up to 20 mg/kg, was completely ineffective in reducing this diuretic action (Leander, 1983). It was therefore concluded that diuresis induced by opiates may be mediated at sites in the CNS. Unfortunately, the effects of quaternary naltrexone were assessed at 2 and 5 hr after administration of drug, time intervals during which it is likely that the antagonist activity had subsided. Furthermore, no analysis of the action in the CNS of the quaternary analog was attempted. Behavioral studies

Quaternary narcotic antagonists have been used in several studies of the action of opiates on animal behavior. Although most of these investigations have examined models of spontaneous and operant behavior, one report has been published in which quaternary naloxone was used in a classical conditioning paradigm. Peripherally-administered morphine was shown to inhibit a learned response in rabbits motivated aversively, an effect abolished by small intravenous doses of naloxone. Methylnaloxone, at doses up to 100 times the effective antagonist dose of naloxone, failed to alter the action of morphine. Taking into account the differences in potency between tertiary and quaternary naloxone, it was concluded that these actions of morphine were central in

188

D. R. BROWN and L. I. GOLDBERG

origin (Mauk, Madden, Barchas and Thompson, 1982). Other studies in which quaternary antagonists have been employed, included those of selfadministration of narcotics, discriminative stimulus effects and ingestive behavior. These are discussed in separate subsections below. Blockade of self-administration of opiates

It is well recognized that animals dependent on narcotics will perform operant tasks to receive infusions of opiates. Not surprisingly, opiate antagonists have been shown to alter the pattern of operant responding for injections of opiates. Naloxone, given by peripheral or central routes, increases the rate of self-administration of morphine by dependent rhesus monkeys and the self-administration of heroin in rats (Sanchez-Ramos and Schuster, 1976; Britt and Wise, 1983). N,N-Diallylnalorphine only slightly increased the self-administration of morphine by monkeys when administered intravenously at doses up to 10 mg/kg, yet it produced large increases in responding when injected intracerebroventricularly at doses up to lOOpgg/kg. Moreover, DANM was approximately three orders of magnitude more potent in precipitating abstinence signs in monkeys after administration into the CNS than it was after peripheral treatment (Sanchez-Ramos and Schuster, 1976). In the rat, DANM, injected into the ventral tegmental area, but not other brain areas, increased operant responding for intravenous infusions of heroin (Britt and Wise, 1983). It appears from these results that specific sites in the brain may mediate some aspects of behavior involved in the selfadministration of opiates. Discriminative stimulus effects

Pure narcotic antagonists inhibit the discriminative stimulus effects of opiates in non-dependent animals and act as discriminative stimuli in small doses in animals which are tolerant and dependent on opiates (Young and Woods, 1982). Naltrexone blocks the discriminative stimulus effects of morphine in nondependent pigeons at intramuscular (i.m.) doses of less than 0.1 mg/kg and those of etorphine and ethylketocyclazocine in non-dependent rhesus monkeys at doses of less than 32pg/kg (i.m.). In nondependent and morphine-dependent pigeons, operantly trained to discriminate between naltrexone and saline and drugs of other classes, naltrexone produced naltrexone-appropriate responding in doses ranging from 3.2 to 32 and 0.1 to 0.32 mg/kg, respectively. In contrast, methylnaltrexone, at doses up to 32 mg/kg, failed to alter the discriminativestimulus effects of opiates in pigeons and monkeys and generally possessed little or no ability to produce naltrexone-appropriate responding in dependent and non-dependent pigeons (Hein, Young, Herling and Woods, 1981; Valentino et al., 1981). It did, like naltrexone, depress the rates of responding at relatively large doses (Valentino et al., 1981; Young and

Woods, 1982). Although antagonist activity of methylnaltrexone administered centrally has never been evaluated in this procedure, the greater than 300-fold separation between the potencies of tertiary and quaternary naltrexone demonstrated in these studies indicates that the discriminative stimulus effects of opiates and opiate antagonists are mediated at sites within the CNS. Ingestive behavior

Endogenous opioids have been implicated in the regulation of the intake of food and water, an hypothesis supported by two principal lines of evidence (Sanger, 1981; Morley, Levine, Yim and Lowy, 1983). First, a number of opiate agonists have been shown to induce feeding and drinking in otherwise satiated animals, when administered by either perinhera1 or central routes. Second, in numerous species of animal, narcotic antagonists markedly suppress the intake of food and water stimulated by deprivation, physiological challenges and many other factors, presumably through an interruption of the activity of endogenous opioids. The effects of quaternary narcotic antagonists have been compared with those of their tertiary forms in a few studies of ingestive behavior. Nalorphine was found to reduce consumption of water in rats deprived of water at a dose of lOmg/kg (s.c.); an identical dose of DANM was without effect; an expected result in view of the large differences in potency between nalorphine and DANM (Ostrowski, Rowland, Foley, Nelson and Reid, 198 1). Several investigators, using methylnaloxone, have found it to have no effect on the fluid intake of rats deprived of water when administered by peripheral routes, at doses up to lOmg/kg; in comparison, tertiary naloxone suppresses intake at doses of less than 1 mg/kg (Brown and Holtzman, 1981; Hemmer et al., 1982; Cooper and Turkish, 1983). In one study, methylnaloxone was found to reduce fluid intake at 50 mg/kg (ip.) (Hemmer et al., 1982). Although this effect might have been due to penetration of the blood-brain barrier by the drug in large doses, it is equally possible that the phenomenon resulted from a reduction in motor activity caused by ganglionic blockade. Like quaternary naloxone, methylnaltrexone was found to be completely ineffective in suppressing intake of water of deprived rats at peripherally administered doses up to 1 mg/kg (Brown and Holtzman, 1981). Nevertheless, both quaternary antagonists attenuate intake of water when injected intracerebroventricularly at doses as small as 1Opg in the rat (Brown and Holtzman, 1981). While these findings suggest that the narcotic antagonists act within the brain to produce their effects on ingestive behavior, the large intracerebroventricular doses of these compounds necessary to suppress this behavior are close to those producing seizures (see above). Detailed behavioral analyses will be required to establish whether the effects of these compounds, administered by the

Quatemary narcotic antagonists intracerebroventricular route, are specific to ingestive behavior or are maliifestations of toxicity. One recent study examined the effects of naloxone and methylnaloxone on feeding induced by electrical stimulation of the lateral hvDothalamus of rats. Subcutaneous injections of naioxone (0.2 and 1 mg/kg) increased the frequency threshold required to induce feeding behavior. Quaternary naloxone, at doses up to 10 mg/kg (s.c.), had no effect on the stimulation parameters (Carr and Simon, 1983).

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CONCLUSIONS Since the end of the last decade, a growing number of investigations into sites of action of opiates have utilized the quaternary narcotic antagonists. These compounds have been assumed to possess a low permeability to biological membranes, such as the blood-brain barrier, relative to their parent tertiary forms. From the evidence considered above, however, it is apparent that many of these quaternary antagonists, or their active metabolites, penetrate the blood-brain barrier quite readily and that the degree to which they cross is-dependent-upon many variables including species differences, route of administration, pretreatment intervals and dose of drug. Too often, these factors have not been taken into account in the design of many experiments and hence the results obtained are at best difficult to interpret. Fortunately, efforts are being made to develop novel peripherally-acting opiate antagonists having a greater ratio of peripheral to central selectivity and a lower margin of toxicity. The N-methyl quaternary derivative of levallorphan represents one such compound (Bianchetti et al., 1983). This and other compounds currently in development offer promise as future pharmacological probes into the function of opiates. Finally, quaternary narcotic antagonists may have considerable potential in clinical settings as well. The parenteral administration of these compounds may be useful in inhibiting undesirable side-effects of opiate analgesics, such as constipation (Goldberg et al., 1979) and respiratory depression (Willette and Sapru, _. 1982). Aside from their ability to reverse the effects of exogenously-administered opiates, narcotic antagonists and their quaternary derivatives may also possess a number of as yet unexplored therapeutic actions relating to the interruption of peripherallylocalized endoeenous ouioid activitv. These intriauing possibilities await investigation. I

Acknowlednements-The

authors would like to thank Dr H. N. Sapru (New Jersey Medical School), Dr Thomas Giles and Dr Gary Sander (Tulane University), Dr James H. Woods (University of Michigan) and Dr Luciano Manara (Sanofi-Midy, S.p.A., Milan, Italy) for graciously supplying manuscripts of their research work prior to publication, and Dr Charis R. Schuster (Universit; of Chicago) for helpful discussions on this manuscript. This work was supported in part by NIMH Postdoctoral Training Fellowship MH 14274 to D.R.B. and NIH grant GM-22220.

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