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Depression by morphine-6-glucuronide of nociceptive activity in rat thalamus neurons: comparison with morphine
BRAIN RESEARCH ELSEVIER
Brain Research 722 (1996) 132-138
Research report
Depression by morphine-6-glucuronide of nociceptive activity in rat thalamus neurons: comparison with morphine Ilmar Jurna a, *, Joseph Baldauf ", Wolfgang Fleischer b a
Institutfiir Pharmakologie und Toxikologie der Universitiit des Saarlandes, D-66424 Homburg/Saar, Germany b Mundipharma GmbH, D-65549 Limburg/Lahn, Germany
Accepted 30 January 1996
Abstract
To assess the contribution of the active metabolite of morphine, morphine-6-glucuronide (M6G), to the analgesic effect of systemically administered morphine, experiments were carried out on rats under urethane anesthesia in which nociceptive activity was evoked by electrical stimulation of afferent C fibers in the sural nerve and recorded from single neurons in the ventrobasal complex of the thalamus. Intravenous (i.v.) injections of morphine completely blocked the activity at doses of 500 and 1000 /xg/kg, the EDso being 44/xg/kg. M6G administered by i.v. injection reduced the evoked nociceptive activity only by about 40% at 80 and 160 /xg/kg, the EDso being 6 /xg/kg. After intrathecal (i.t.) injection, morphine produced maximum depression of 55% of the control activity at 20/xg; the ED5o is 18 /xg. M6G injected i.t. produced maximum depression of 40% at doses ranging from 0.2 to 10/xg. The EDs0 of M6G i.t. is below 0.2 /xg. The effects of morphine and M6G were reversed by naloxone (200 /~g/kg i.v.). The results show that M6G is more potent than morphine, regardless of the route of administration, while morphine is more effective when injected i.v. Due to the low efficacy of M6G, it seems unlikely that this glucuronide contributes substantially to the analgesic effect of morphine when renal function is normal. The results also make evident that the maximum effect of morphine results from an action at spinal and supraspinal sites. Keywords: Analgesia; Thalamus neuron; Morphine; Morphine-6-glucuronide; Intravenous; Intrathecal
1. Introduction
Morphine is largely metabolized by glucuronidation in the liver, and the glucuronides have generally been considered as pharmacologically inactive [15]. Therefore it was surprising that relative overdosing occurred in patients with renal failure but not in those with impaired hepatic function [11,37]. This suggested an important role of the kidneys in the elimination of morphine. However, the excretion of morphine by the urine in patients with renal failure is not reduced, while that of morphine glucuronides is markedly delayed [25,29,31,36,38,39,41]. It seems now to be established that increased levels and prolonged elimination of M6G are responsible for relative overdosing of morphine in impaired renal function [30]. Already in 1950, a powerful antinociceptive action of M6G in the animal experiment has been reported [7]. Actually, M6G was about three times more potent than
* Corresponding author. Fax: (49) (6841) 16-6411. 0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0006-8993(96)00205- 3
morphine, and the duration of its action was twice as long as that of morphine, while morphine-3-glucuronide (M3G) proved to be ineffective. Later experiments confirmed these observations and, in addition, demonstrated that M6G is more potent when administered by intracerebroventricular injection [1,14,34,42], microinjection into the periaqueductal gray [33] or i.t. injection [34] than by systemic routes. M3G was reported to be devoid of an antinociceptive action [28,33], but it either antagonized the antinociceptive effect of morphine or M6G [16,43] or potentiated that of morphine [28]. M6G is more selective than morphine for opioid receptors of t h e / z type [1,8,9,14,33,34] and penetrates the blood brain barrier without previous hydrolysis [51], which is possibly due to the high lipophilicity of the folded form of the molecule [6]. The two glucuronides, M6G and M3G, were detected in the cerebrospinal fluid after systemic administration [17,25,38,46,48] which, when considering the low capacity of the CNS to produce glucuronides from morphine [47], indicates that glucuronides formed in the liver have passed the blood brain barrier. It has conse-
I. Jurna et al. / Brain Research 722 (1996) 132-138
quently been proposed that the formation of M6G contributes to the pain relief caused by morphine [17,25,31,32]. In fact, an open study showed that M6G administered i.v. caused marked analgesia that lasted about 7 h [31]. Moreover, a single-blind crossover study carried out with i.t. injections showed thall M6G was more effective in producing analgesia than morphine [18]. It is in agreement with these clinical observations that local application of M6G to the spinal cord as well as i.v. injection of M6G was more potent than morphine in depressing the activity of dorsal horn neurons that was elicited by electrical stimulation of afferent A and C fibers, C fiber-evoked activity being more suscepl:ible to the two drugs [44]. Because of the importance of the question to what extent M6G contributes to the analgesic action of morphine, experiments were carried out on rats in which the effect of i.v. and i.t. injections of this glucuronide was determined on nociceptive activity evoked in single neurons of the ventrobasal complex of the thalamus and compared with that of morphine (see also [5]). In this study, it will be strictly distinguished if a drug is more effective (i.e. producing a larger effect) or more potent (i.e. producing a given effect at a lower dose) than the other.
2. Materials and methods The experiments were carded out on 174 rats of either sex (Wistar/Charles River; 250-300 g body weight). The animals were housed in macrolon cages (six animals to a cage) and offered standard diet (Altromin) and tap water ad libitum. They received an i.p. injection of urethane (1.2 g / k g ) to induce and mainl:ain anesthesia for surgery and the experiment proper. At the end of surgery, an additional subcutaneous injection of urethane (120 m g / k g ) was made. In separate experiments performed on the righting reflex it was found that sleeping ti:me achieved by this treatment was about 6 h. Preparing the animals, searching for neurons and recording activity before and after injection of drugs lasted no longer than 3 h. The animals breathed spontaneously. Body temperature was monitored in the rectum and kept between 3'7.5 and 38°C by radiant heat. The procedure to prepare the animals for the experiment and to elicit and record C fiber-evoked activity from thalamus neurons has been described previously [5]. A cannula was inserted into a tail-vein for i.v. injections. For i.t. injections, a laminectomy was performed at the level of T 8 to T10 and a polyethyle:ae catheter (outer diameter 0.4 mm, length inserted into th,e spinal canal 14-20 mm) was introduced into the subarachnoid space of the lumbosacral spinal cord, its outer end being fitted by a 20 gauge injection needle to a microinjection syringe [23]. The exposed cord was covered with warm agar which, when cooling, sealed the spinal canal and fixed the i.t. catheter. The left sural nerve was prepared for electrical stimulation with a pair of platinum wire electrodes and cut distal to the
133
electrodes. The nerve was stimulated employing single rectangular impulses delivered from a Grass stimulator (Model $4) with stimulus isolation unit at a frequency of 0.1 Hz and an impulse duration of 0.5 ms. The stimulation strength was 2-2.5 times higher than that producing maximum responses and supramaximal for afferent C fibers (42 to 68 V) in the sural nerve. These stimulation parameters have been employed in a previous investigation [22] in which activity in single axons of the rat spinal cord was elicited by electrical stimulation of the sural nerve. Tungsten microelectrodes (tip diameter 1 /zm; resistance 10 Mg2) attached to a micromanipulator with a stereotaxic device were used to record activity from single neurons in the ventrobasal complex of the thalamus. For ipsi- and contralateral recording, a hole was drilled into the skull on both sides for introducing the microelectrode. The co-ordinates for recording activity in the ventral posterolateral nucleus (VPL) and the ventral posteromedial nucleus (VPM) were AP: 2.3 to 2.6 mm, L: 2.8 mm, and V: 5.2 to 6.8 mm [35]. The activity of single neurons was amplified (WPI preamplifier Model DAM-5A) and displayed on a cathode ray oscilloscope (Kikusui model 5516 ST). The signals were passed through a window discriminator (WPI Model 121) and evaluated with a Cambridge electronic design computer interface (Model 1401 A) together with a personal computer (Tandon XT 10) and MRATE software. The number of addresses used was 256, the duration of each address being 4 or 8 ms. Peristimulus histograms consisting of 10 consecutive responses were summed each time and electronically integrated. Spontaneous and evoked activities were treated separately. The integrations of activity were pooled for statistical evaluation. Four to six determinations were made before drug administration and these served as controls when they were stable. If evoked activity changed by more that 10% of the mean value before drug administration, the neuron was abandoned and another one searched for. Only one neuron was tested in one animal. Therefore, the number of neurons or axons, experiments and rats used are identical. Significant differences were established by applying Student's t-test for unpaired samples. In addition, the Friedman test was employed in combination with the Wilcoxon-Wilcox test. Finally, the times were determined at which the values after drug application differed from the control values by using a MANOVA-controlled analysis of variance. These values are presented by open symbols in Figs. 2, 3, 5 and 6. This was done to avoid unreadable curves when the time course of the dose-dependent depression of evoked activity is presented with S.E.M. After the end of the experiments carried out on thalamus neurons, the position of the microelectrode tip was marked by passing current of approximately 4 mA for 30 s (Grass Lesion Maker). The animals were killed with an overdose of pentobarbital and the brain was perfused by an intraarterial injection of a 10% formaldehyde solution. The
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brain was removed, fixed in Bouin's solution and embedded in paraplast. Serial sections (10/xm) were stained with gallocyanin-chromalum [13] and counterstained with phloxin. The drugs used were morphine hydrochloride (Merck), morphine-6-glucuronide (Tasmanian Alcaloids), naloxone hydrochloride (Sigma), and urethane (Riedel-De Ha5n). Physiological NaC1 solution was used as solvent. Intrathecal injections were made with a volume of 10 /xl at a rate of 20 /xl/min. All doses are indicated as the salts.
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3. Results In total, 174 neurons in the VPL or VPM nuclei of the rat thalamus responding to supramaximal electrical stimulation of the ipsi- or contralateral sural nerve were studied. No differences in the responses of VPL and VPM neurons to stimulation or to M6G or morphine could be detected so that all neurons were treated as belonging to one group. Ninety-nine neurons were activated from the ipsilateral and 75 from the contralateral sural nerve. Fig. 1 shows recordings of the spontaneous and evoked impulse discharges recorded from a thalamus neuron and the corresponding peristimulus histograms. No ' wind up' of spontaneous activity occurred when supramaximal stimulation of the sural nerve was employed at the rate chosen (0.1 Hz). When the stimulation strength was reduced below that which generally caused activation of ascending axons in the spinal cord [22,23], i.e. below 20 V, no neuron tested continued to respond to stimulation. Moreover, all neurons activated by supramaximal stimulation of the sural nerve also responded to squeezing more than one paw and pinching the skin of various parts of the body. However, none of these neurons responded to touching or gentle stroking of the skin or air puffs applied to it. The interval between the stimulus and the maximum of evoked activity in the peristimulus histograms of the controls varied between 120 and 320 ms (see also [5]). In Fig. 1, the interval was 310 ms, and the distance between the proximal stimulation electrode (cathode) and the dorsal root entry zone containing afferents from the sural nerve was approximately 120 mm. This yields an apparent conduction velocity of 0.38 m / s which is in accord with an activation by afferent C fibers. Control injections with saline have previously been shown not to change nociceptive activity evoked in single neurons of the thalamus when made by the i.v. [5] or the i.t. [23] route. Moreover, i.v. injection of the lowest dose of M6G tested in these experiments (2.5 /xg/kg; Fig. 3) and an i.t. injection of the lowest dose of morphine tested (10 /~g; Fig. 5) caused no significant depression of evoked activity. Therefore, and in view of the strict regulations limiting the number of animals used in experiments, no additional control experiments were conducted. Morphine administered by i.v. injection reduced the
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Fig. 1. Depression by morphine of activity in a single neuron of the thalamus evoked by sural nerve stimulation. Left hand column of recordings: impulses discharged from the neuron before and after activation by supramaximal electrical stimulation (56 V) of the ipsilateral sural nerve. Stimulus artifacts are indicated by dots under the recordings which were taken before and after i.v. injection of morphine 0.5 mg/kg. Right hand column of recordings: peristimulus histograms (ten trials each) of the activity determined before and after i.v. injection of morphine corresponding to that shown in the left hand column. Stimulation is indicated by dots under the recordings.
activity in thalamus neurons evoked by afferent C fiber stimulation (Fig. 1). The effect was dose-dependent and its maximum was reached between 5 and 20 min (Fig. 2). The threshold dose was 0.025 m g / k g , and the dose producing the maximum effect was 0.5 m g / k g (Figs. 2 and 4). The EDs0 of morphine i.v. established graphically is 0.044 m g / k g or 44 /xg/kg (Fig. 4) This value agrees with that obtained in previous experiments, i.e. 0.05 m g / k g [5]. The effect of morphine was reversed by an i.v. injection of naloxone (0.2 m g / k g ; Figs. 1 and 2). M6G injected i.v. reduced the nociceptive activity evoked in thalamus neurons in a dose-dependent manner (Fig. 3). As in the case of morphine, the effect reached its maximum between 5 and 20 min. The threshold dose lies between a dose of 2.5 /xg/kg, which was ineffective, and a dose of 5 ~ g / k g , which significantly reduced the activity by about 18% of the controls from 5 to 30 min. Thus, on the basis of threshold doses, M6G was about 8 times
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I. Jurna et al.// Brain Research 722 (1996) 132-138
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Fig. 2. Time course of the depression of C fiber-evoked activity in single neurons of the thalamus caused by i.v. injection of morphine. Ordinate: change in number of impulses discharged in response to C fiber stimulation (evoked activity) in per ceat of the controls. Abscissa: time in minutes before and after injection. The points on the curves are the mean values of the determinations whose number is indicated in brackets for each dose. Open symbols indicate that the values differ significantly from the controls (P < 0.05).
more potent than morphine. The m a x i m u m depression was caused b y M 6 G at a dose of 40 / x g / k g and a m o u n t e d to about 40% o f the control activity (Figs. 3 and 4). Increasing the dose to 80 or even 160 / x g / k g failed to increase the effect. The ED50 o f M 6 G derived from the d o s e - r e sponse curve is about 6 / x g / k g and, hence, 7 times lower than that of m o r p h i n e (Fig. 4). Therefore M 6 G is more potent than m o r p h i n e but less effective w h e n the two drugs are administered b y i.v. injection. The depression of evoked
Fig. 4. Relationship between the depression of C fiber-evoked activity in single neurons of the thalamus caused by i.v. injection of morphine (open circles) and M6G (closed circles). Ordinate: amount of depression in per cent of control activity. Abscissa: doses on a logarithmic scale. The values presented are the mean values determined 10 min after the injection and correspond to those of Figs. 2 and 3.
activity persisted until 60 m i n after i.v. injection of M 6 G 80 and 160 / ~ g / k g and was abolished by an i.v. injection of naloxone 200 / x g / k g (Fig. 4). W h e n m o r p h i n e was applied directly to the spinal cord by i.t. injection, it produced a d o s e - d e p e n d e n t depression of the activity in thalamus n e u r o n s e v o k e d by afferent C fiber stimulation (Fig. 5). A dose of 1 0 / x g was ineffective, and a dose of 2 0 / x g produced a depression lasting from 5 to 30 m i n after the injection. The m a x i m u m depression was produced by 8 0 / x g and a m o u n t e d to about 55% of the control activity. At a dose of 160 ~ g , m o r p h i n e appeared
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L Jurna et al./Brain Research 722 (1996) 132-138
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more potent than morphine, but there is no remarkable difference in the effectiveness of the two drugs.
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to be rather less effective than at a dose of 80 /xg which means that no complete block of evoked nociceptive activity could be achieved by i.t. injections of morphine. The EDs0 derived from the dose-response curve is 18 /zg (Fig. 7). M6G administered by i.t. injection reduced the evoked nociceptive activity in a dose-dependent fashion (Fig. 6). The effect of all doses was significant till 60 rain after injection when naloxone (200 /zg/kg) was injected i.v. which abolished M6G-induced depression. The lowest dose tested was 0.2 /zg and this caused a maximum reduction of 23% of the control activity at 10 rain after the injection. It is difficult to decide which dose of M6G between 0.2 and 10 /zg produced maximum depression of evoked activity (Fig. 7). In any case it amounted to no more than 40% of the controls. The EDs0 can only roughly be estimated as being less than 0.2 /xg. There is no significant difference between the values determined 10 rain after i.t. injection of M6G 0.5, 1 and 10/xg and that after morphine. The values after M6G 3/xg and morphine 80/zg differed significantly ( P < 0.05). It appears that, at the spinal level, M6G is depression
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Fig. 7. Relationship between the depression of C fiber-evoked activity in single neurons of the thalamus caused by i.t. injection of morphine (open circles) and M6G (closed circles). Details as in Fig. 4.
The results of the present study confirm those of other authors according to which M6G is more potent than morphine in producing an antinociceptive effect (for literature see Introduction). Nociceptive activity evoked in thalamus neurons by afferent C fiber stimulation was reduced by M6G at doses 7 to 8 times lower than those of morphine when using the i.v. route of administration. The difference is even more marked when the two drugs were administered by i.t. injection. In that case, the dose-response curves of M6G and morphine are clearly separated (Fig. 7), while after i.v. injection the dose-response curves of M6G and morphine overlap (Fig. 4). The effective doses of M6G appear to be at least 10 times lower than those of morphine after i.t. injection. However, morphine was more effective than M6G when the two drugs were injected i.v. Morphine was capable to completely block the nociceptive activity evoked in thalamus neurons, while M6G reduced it by no more than 50%. The results obtained with i.v. injections of morphine correspond to those of a previous series of experiments carried out some years ago in which the EDs0 of morphine was found to be 0.05 m g / k g and doses of 0.5 and 1 m g / k g reduced the evoked activity to zero as in the present study [5]. A similarly low EDs0 had been determined by Guilbaud and coworkers when studying the effect of i.v. injections of morphine on nociceptive activity elicited in thalamus neurons by noxious stimuli applied to peripheral tissue [3]. Both M6G as well as morphine failed to suppress altogether the nociceptive activity when administered by i.t. injection. This is in agreement with results in the literature which show that morphine injected i.v. reduced nociceptive activity evoked in neurons of the dorsal horn of the spinal cord by a maximum of about 70% [24,26,27,45] or in axons ascending from the spinal cord to the brain by 50 to 60% [21,22] of the control activity in spinal animals. Moreover, microiontophoretic application of morphine to dorsal horn neurons was found to reduce evoked nociceptive activity not in all but only in part of these neurons [4,12]. Similarly, it was observed in a study in which M6G was applied locally to the spinal cord that it reduced nociceptive activity only in part of the neurons tested and that, in the neurons affected by M6G, nociceptive activity was reduced not completely but only by about 90% of the controls [44]. In addition, it was found in that study that systemically administered M6G produced a mean inhibition of C fiber-evoked activity of about 80%. According to Yaksh and Rudy [49], morphine-induced analgesia or antinociception is not simply the sum of the effects exerted by morphine at spinal and supraspinal sites,
L Jurna et al./Brain Research 722 (1996) 132-138
but instead the effect o f morphine at one or the other site predominates. However, it has been demonstrated that the antinociceptive effect of irttracerebroventricular injections of morphine was potential!ed by i.t. injections and vice versa, and it was therefore concluded that both spinal and supraspinal sites are essent~ial to produce the full antinociceptive or analgesic effect caused by systemic administration of morphine [50]. This fully agrees with the observation that morphine was more effective in depressing nociceptive reflex responses in intact rats than in rats with their Spinal cords transected [10,19,20] or after lesioning the dorsolateral funiculus [2]. These findings obviously indicate that morphine activates a descending inhibitory control operating from the brain stem and enhancing the spinal effect of morphine. Therefore, the depressant effect of morphine at the spinal level seems to add to the effects produced at supraspinal sites so that the strong antinociceptive effect observed in l:halamus neurons results. It is, however, an open question why such summing up of spinal and supraspinal effects wa,; not detectable in the case of M6G. Higher lipophilicity of the folded molecule might account for the higher potency of M6G, but it cannot explain why it is less effective than morphine following i.v. administration to the animal with an intact neuraxis. However, the higher efficacy in this condition might result from different affinities of the two compounds to opioid receptor subtypes. The results obtained following i.v. injections of morphine and M 6 G show that morphine is more effective than M 6 G in depressing nociceptive activity in the thalamus, while M 6 G is more potent in this respect. Because of the relatively poor effectiveness of M 6 G it is doubtful that the glucuronide contributes in a substantial way to the analgesic effect of systemicalily administered morphine. A similar conclusion has been reached when evaluating the low concentrations of M 6 G established in the cerebrospinal fluid after epiduml injections of morphine to patients suffering from cmacer pain [40]. However, the situation may be different when the excretion of M 6 G is markedly reduced in renal failure leading to relative overdosing of morphine.
Acknowledgements The authors are indebted to Mrs. Karen Wolske, Mrs. Birgit Spohrer and Mrs. Gabriele Ulrich for valuable and skilful technical assistance.
References [1] Abbot, F.V. and Palmour, R.M., Morphine-6-glucuronide:analgesic effects and receptor binding profile in rats, Life Sci., 43 (1988) 1685-1695. [2] Barton, C., Basbaum, A.I. and Fields, H.L., Dissociation of
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supraspinal and spinal actions of morphine: a quantitative evaluation, Brain Res., 188 (1980) 487-498. [3] Benoist, J.M., Kayser, V., Gautron, M. and Guilbaud, G., Low doses of morphine strongly depresses responses of specific nociceptive neurones in the ventrobasal complex of the rat, Pain, 15 (1983) 333-344. [4] Calvillo, O., Henry, J.L. and Neuman, R.S., Effects of morphine and naloxone on dorsal horn neurones in the cat, Can. J. Physiol. Pharmacol., 52 (1974) 1207-1211. [5] Carlsson, K.-H., Monzel, W. and Jurna, I., Depression by morphine and the non-opioid analgesic agents, metamizol (dipyrone), lysine acetylsalicylate and paracetamol, of activity in rat thalamus neurones evoked by electrical stimulation of nociceptive afferents, Pain, 32 (1988) 313-326. [6] Carrupt, P.-A., Testa, B., Bechalany, A., E1 Tayar, N., Descas, P. and Perrissoud, D., Morphine-6-glucuronide and morphine-3glucuronide as molecular chameleons with unexpected lipophilicity, J. Med. Chem., 34 (1991) 1272-1275. [7] Casparis, P., Su alcuni derivati della morfina e loro azione analgesica, Boll. Chim. Farm., 89 (1950) 309-318. [8] Chen, Z.R., Irvine, R.J., Somogyi, A.A. and Bochner, F., Mu receptor binding of some commonly used opioides and their metabolites, Life Sci., 48 (1991) 2165-2171. [9] Christensen, C.B. and Jorgensen, L.N., Morphine-6-glucuronidehas high affinity for the opioid receptor, Pharmacol. Toxicol., 60 (1987) 75-76. [10] Dewey, W.L., Snyder, J.W., Harris, L.S. and Howes, J.F., The effect of narcotics and narcotic antagonists on the tall-flick response in spinal mice, J. Pharm. Pharmacol., 21 (1969) 548-550. [11] Don, H.F., Dieppa, R.A. and Taylor, P., Narcotic analgesics in anuric patients, Anesthesiology, 42 (1975) 745-747. [12] Duggan, A.W., Hall, J.G. and Headley, P.M., Suppression of transmission of nociceptive impulses by morphine: selective effects of morphine administered in the region of the substantia gelatinosa. Br. J. Pharmacol., 61 (1977) 65-76. [13] Einarson, L., On the theory of gallocyanin-chromalum staining and its application for quantitative estimation of basophilia: a selective staining of exquisite progressivity, Acta Pathol. Microbiol. Scand., 28 (1951) 82-102. [14] Franc6s. B., Gout, R., Campistron, G. Pancon, E. and Cros, J., Morphine-6-glucuronide is more mu-selective and potent in analgesic tests than morphine, Progr. Clin. Biol. Res., 328 (1990) 477-480. [15] Glare, P.A. and Walsh, T.D., Clinical pharmacokinetics of morphine, Ther. Drug Monit., 13 (1991) 1-23. [16] Gong, Q.-L., Hedner, J., Bj6rkman, R. and Hedner, T., Morphine-3glucuronide may functionally antagonize morphine-6-glucuronide induced antinociception and ventilatory depression in the rat, Pain, 48 (1992) 249-255. [17] Hand, C.W., Blunnie, W.P., Claffey, L.P., McShane, A.J., McQuay, H.J. and Moore, R.A., Potential analgesic contribution from morphine-6-glucuronide in CSF, Lancet, II (1987) 1207-1208. [18] Hanna, M.H., Peat, S.J., Woodham, A., Knibb, A. and Fung, C., Analgesic efficacy and CSF pharmacokinetics of intrathecal morphine-6-glucuronide:comparison with morphine, Br. J. Anaesth., 64 (1990) 547-550. [19] Irwin, S., Houde, R.W., Bennett, D.R., Hendershot, L.C. and Seevers, M.H., The effect of morphine, methadone and meperidine on some reflex responses of some spinal animals to nociceptive stimulation, J. Pharmacol. Exp. Ther., 101 (1951) 132-143. [20] Juma, I., D~npfung repetitiver AktivierungsvorgS.ngean der spinalen Motorik durch Morphin. In R. Janzen, W.D. Keidel, A. Herz and C. Steichele (Eds.), Schmerz, Grundlagen, Pharmakologie, Therapie, Georg Thieme Verlag, Stuttgart, 1972, pp. 267-269. [21] Juma, I. and Grossmann, W., The effect of morphine on the activity evoked in ventrolateral tract axons of the cat spinal cord, Exp. Brain Res., 24 (1976) 473-484.
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[22] Jurna, I. and Heinz, G., Differential effects of morphine and opioid analgesics on A and C fibre-evoked activity in ascending axons of the rat spinal cord, Brain Res., 171 (1979) 573-576. [23] Jurna, I., Spohrer, B. and Bock, R., lntrathecal injection of acetylsalicylic acid, salicylic acid and indometacin depresses C fibre-evoked activity in the rat thalamus and spinal cord, Pain, 49 (1992) 249-256. [24] Kitahata, L.M., Kosaka, Y., Taub, A., Bonikos, K and Hoffert, M., Lamina-specific suppression of dorsal-horn unit activity by morphine sulfate, Anesthesiology, 41 (1974) 39-48. [25] Laizure, S.C., Miller, J.H., Stevens, R.C., Donahue. D.J., Laster, R.E. and Brown, D., The disposition and cerebrospinal fluid penetration of morphine and its two major glucuronided metabolites in adults undergoing lumbar myelogram, Pharmacotherapy, 13 (1993) 471-475. [26] LeBars, D., Men6trey, D., Conseiller, C. and Besson, J.M., Depressive effects of morphine upon lamina V cells activities in the dorsal horn of the spinal cat, Brain Res., 98 (1975) 261-277. [27] LeBars, D., Guilbaud, G., Jurna, I. and Besson, J.M., Differential effects of morphine on responses of dorsal horn lamina V type cells elicited by A and C fibre stimulation in the spinal cat, Brain Res., 115 (1976) 518-524. [28] Lipkowski, A.W., Can', D.B., Langlade, A., Osgood, P.F. and Szyfelbein, S.J., Morphine-3-glucuronide: silent regulator of morphine actions, Life Sci., 55 (1994) 149-154. [29] Mc Quay, H.J., Moore, R.A., Hand, C.W. and Sear, J.W., Potency of oral morphine, Lancet, 1I (1987) 1458-1459. [30] Osborne, R.J., Joel, S.P. and Slevin, M.L., Morphine intoxication in renal failure: the role of morphine-6-glucuronide, Br. Med. J., 292 (1986) 1548-1549. [31] Osborne, R., Joel, S., Trew, D. and Slevin, M., Analgesic activity of morphine-6-glucuronide, Lancet, I (1988) 828. [32] Osborne, R., Joel, S., Trew, D. and Slevin, M., Morphine and metabolite behaviour after different routes of morphine administration: demonstration of the importance of the active metabolite morphine-6-glucuronide, Clin. Pharmacol. Ther., 47 (1990) 12-19. [33] Pasternak, G.W., Bodnar, R.J., Clark, J.A. and Inturrisi, C.E., Morphine-6-glucuronide, a potent mu agonist, Life Sci., 41 (1987) 2845-2849. [34] Paul, D., Standifer, K.M., Inturrisi, C.E. and Pasternak, G.W., Pharmacological characterization of morphine-6/3-glucuronide, a very potent morphine metabolite, J. Pharmacol. Exp. Ther., 251 (1989) 477-483. [35] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, San Diego, 1986. [36] Peterson, G.M., Randall, C.T.C. and Paterson, J., Plasma levels of morphine and morphine glucuronides in the treatment of cancer apin: relationship to renal function and route of administration, Eur. J. Clin. Pharmacol., 38 (1990) 121-124.
[37] Regnard, C.F.B. and Twycross, R.G., Metabolism of narcotics, Br. Med. J., 288 (1984) 860. [38] S~iwe, J. and Odar-CederlSf, I., Kinetics of morphine in patients with renal failure, Eur. J. Clin. Pharmacol., 32 (1987) 377-382. [39] S~iwe, J., Svensson, J.O. and Odar-Cederl~Sf, I., Kinetics of morphine in patients with renal failure, Lancet, II (1985) 211. [40] Samuelsson, H., Hedner, T., Venn, R. and Michaikiewicz, A., CSF and plasma concentrations of morphine and morphine glucuronides in cancer patients receiving epidural morphine, Pain, 52 (1993) 179-185. [41] Sear, J.W., Hand, C.W., Moore, R.A. and McQuay, H.J., Studies on morphine disposition: influence of renal failure on the kinetics of morphine and its metabolites, Br. J. Anaesth., 62 (1989) 28-32. [42] Shimomura, K., Kamata, O., Ueki, S., Ida, S., Oguri, K., Yoshimura, H. and Tsukamoto, H., Analgesic effect of morphine glucuronides, Tohoku J. Exp. Med., 105 (1971) 45-52. [43] Smith, M.T., Watt, J.A. and Cramond, T., Morphine-3-glucuronide - a potent antagonist of morphine analgesia, Life Sci., 47 (1990) 579-585. [44] Sullivan, A.F., McQuay, H.J., Bailey, D. and Dickenson, A.H., The spinal antinociceptive actions of morphine metabolites morphine-6glucuronide and normorphine in the rat, Brain Res., 482 (1989) 219-224. [45] Toyooka, H., Kitahata, L.M., Dohi, S., Ohtani, M., Hanaoka, K. and Taub, A., Effects of morphine on the rexed lamina. VII. Spinal neuronal responses to graded noxious radiant heat stimulation, Exp. Neurol., 62 (1978) 146-158. [46] Van Dongen, R.T.M., Crul, B.J.P., Koopman-Kimenai, P.M. and Vree, T.B., Morphine and morphine-glucuronide concentrations in plasma and CSF during long-term administration of oral morphine, Br. J. Clin. Pharmacol., 38 (1994) 271-273. [47] Wahlstr~Sm, A., Winblad, B., Bixo, M. and Rane, A, Human brain metabolism of morphine and naloxone, Pain, 35 (1988) 121-127. [48] Wolff, T., Samuelsson, H. and Hedner, T., Morphine and morphine metabolite concentrations in cerebrospinal fluid and plasma in cancer patients after slow-release oral morphine administration, Pain, 62 (1995) 147-154. [49] Yaksh, T.L. and Rudy, T.A., Narcotic anaigetics: CNS sites and mechanisms of action as revealed by intracerebral injection techniques, Pain, 4 (1978) 299-359. [50] Yeung, J.C. and Rudy, T.A., Multiplicative interaction between narcotic agonisms expressed at spinal and supraspinal sites of antinociceptive action as revealed by concurrent intrathecal and intracerebroventricular injections of morphine, J. Pharmacol. Exp. Ther., 215 (1980) 633-642. [51] Yoshimura, H., Ida, S., Oguri, K. and Tsukamoto, H., Biochemical basis for analgesic activity of morphine-6-glucuronide. I. Penetration of morphine-6-glucuronide in the brain of rats, Biochem. Pharmacol., 22 (1973) 1423-1430.
Brain Research 722 (1996) 132-138
Research report
Depression by morphine-6-glucuronide of nociceptive activity in rat thalamus neurons: comparison with morphine Ilmar Jurna a, *, Joseph Baldauf ", Wolfgang Fleischer b a
Institutfiir Pharmakologie und Toxikologie der Universitiit des Saarlandes, D-66424 Homburg/Saar, Germany b Mundipharma GmbH, D-65549 Limburg/Lahn, Germany
Accepted 30 January 1996
Abstract
To assess the contribution of the active metabolite of morphine, morphine-6-glucuronide (M6G), to the analgesic effect of systemically administered morphine, experiments were carried out on rats under urethane anesthesia in which nociceptive activity was evoked by electrical stimulation of afferent C fibers in the sural nerve and recorded from single neurons in the ventrobasal complex of the thalamus. Intravenous (i.v.) injections of morphine completely blocked the activity at doses of 500 and 1000 /xg/kg, the EDso being 44/xg/kg. M6G administered by i.v. injection reduced the evoked nociceptive activity only by about 40% at 80 and 160 /xg/kg, the EDso being 6 /xg/kg. After intrathecal (i.t.) injection, morphine produced maximum depression of 55% of the control activity at 20/xg; the ED5o is 18 /xg. M6G injected i.t. produced maximum depression of 40% at doses ranging from 0.2 to 10/xg. The EDs0 of M6G i.t. is below 0.2 /xg. The effects of morphine and M6G were reversed by naloxone (200 /~g/kg i.v.). The results show that M6G is more potent than morphine, regardless of the route of administration, while morphine is more effective when injected i.v. Due to the low efficacy of M6G, it seems unlikely that this glucuronide contributes substantially to the analgesic effect of morphine when renal function is normal. The results also make evident that the maximum effect of morphine results from an action at spinal and supraspinal sites. Keywords: Analgesia; Thalamus neuron; Morphine; Morphine-6-glucuronide; Intravenous; Intrathecal
1. Introduction
Morphine is largely metabolized by glucuronidation in the liver, and the glucuronides have generally been considered as pharmacologically inactive [15]. Therefore it was surprising that relative overdosing occurred in patients with renal failure but not in those with impaired hepatic function [11,37]. This suggested an important role of the kidneys in the elimination of morphine. However, the excretion of morphine by the urine in patients with renal failure is not reduced, while that of morphine glucuronides is markedly delayed [25,29,31,36,38,39,41]. It seems now to be established that increased levels and prolonged elimination of M6G are responsible for relative overdosing of morphine in impaired renal function [30]. Already in 1950, a powerful antinociceptive action of M6G in the animal experiment has been reported [7]. Actually, M6G was about three times more potent than
* Corresponding author. Fax: (49) (6841) 16-6411. 0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0006-8993(96)00205- 3
morphine, and the duration of its action was twice as long as that of morphine, while morphine-3-glucuronide (M3G) proved to be ineffective. Later experiments confirmed these observations and, in addition, demonstrated that M6G is more potent when administered by intracerebroventricular injection [1,14,34,42], microinjection into the periaqueductal gray [33] or i.t. injection [34] than by systemic routes. M3G was reported to be devoid of an antinociceptive action [28,33], but it either antagonized the antinociceptive effect of morphine or M6G [16,43] or potentiated that of morphine [28]. M6G is more selective than morphine for opioid receptors of t h e / z type [1,8,9,14,33,34] and penetrates the blood brain barrier without previous hydrolysis [51], which is possibly due to the high lipophilicity of the folded form of the molecule [6]. The two glucuronides, M6G and M3G, were detected in the cerebrospinal fluid after systemic administration [17,25,38,46,48] which, when considering the low capacity of the CNS to produce glucuronides from morphine [47], indicates that glucuronides formed in the liver have passed the blood brain barrier. It has conse-
I. Jurna et al. / Brain Research 722 (1996) 132-138
quently been proposed that the formation of M6G contributes to the pain relief caused by morphine [17,25,31,32]. In fact, an open study showed that M6G administered i.v. caused marked analgesia that lasted about 7 h [31]. Moreover, a single-blind crossover study carried out with i.t. injections showed thall M6G was more effective in producing analgesia than morphine [18]. It is in agreement with these clinical observations that local application of M6G to the spinal cord as well as i.v. injection of M6G was more potent than morphine in depressing the activity of dorsal horn neurons that was elicited by electrical stimulation of afferent A and C fibers, C fiber-evoked activity being more suscepl:ible to the two drugs [44]. Because of the importance of the question to what extent M6G contributes to the analgesic action of morphine, experiments were carried out on rats in which the effect of i.v. and i.t. injections of this glucuronide was determined on nociceptive activity evoked in single neurons of the ventrobasal complex of the thalamus and compared with that of morphine (see also [5]). In this study, it will be strictly distinguished if a drug is more effective (i.e. producing a larger effect) or more potent (i.e. producing a given effect at a lower dose) than the other.
2. Materials and methods The experiments were carded out on 174 rats of either sex (Wistar/Charles River; 250-300 g body weight). The animals were housed in macrolon cages (six animals to a cage) and offered standard diet (Altromin) and tap water ad libitum. They received an i.p. injection of urethane (1.2 g / k g ) to induce and mainl:ain anesthesia for surgery and the experiment proper. At the end of surgery, an additional subcutaneous injection of urethane (120 m g / k g ) was made. In separate experiments performed on the righting reflex it was found that sleeping ti:me achieved by this treatment was about 6 h. Preparing the animals, searching for neurons and recording activity before and after injection of drugs lasted no longer than 3 h. The animals breathed spontaneously. Body temperature was monitored in the rectum and kept between 3'7.5 and 38°C by radiant heat. The procedure to prepare the animals for the experiment and to elicit and record C fiber-evoked activity from thalamus neurons has been described previously [5]. A cannula was inserted into a tail-vein for i.v. injections. For i.t. injections, a laminectomy was performed at the level of T 8 to T10 and a polyethyle:ae catheter (outer diameter 0.4 mm, length inserted into th,e spinal canal 14-20 mm) was introduced into the subarachnoid space of the lumbosacral spinal cord, its outer end being fitted by a 20 gauge injection needle to a microinjection syringe [23]. The exposed cord was covered with warm agar which, when cooling, sealed the spinal canal and fixed the i.t. catheter. The left sural nerve was prepared for electrical stimulation with a pair of platinum wire electrodes and cut distal to the
133
electrodes. The nerve was stimulated employing single rectangular impulses delivered from a Grass stimulator (Model $4) with stimulus isolation unit at a frequency of 0.1 Hz and an impulse duration of 0.5 ms. The stimulation strength was 2-2.5 times higher than that producing maximum responses and supramaximal for afferent C fibers (42 to 68 V) in the sural nerve. These stimulation parameters have been employed in a previous investigation [22] in which activity in single axons of the rat spinal cord was elicited by electrical stimulation of the sural nerve. Tungsten microelectrodes (tip diameter 1 /zm; resistance 10 Mg2) attached to a micromanipulator with a stereotaxic device were used to record activity from single neurons in the ventrobasal complex of the thalamus. For ipsi- and contralateral recording, a hole was drilled into the skull on both sides for introducing the microelectrode. The co-ordinates for recording activity in the ventral posterolateral nucleus (VPL) and the ventral posteromedial nucleus (VPM) were AP: 2.3 to 2.6 mm, L: 2.8 mm, and V: 5.2 to 6.8 mm [35]. The activity of single neurons was amplified (WPI preamplifier Model DAM-5A) and displayed on a cathode ray oscilloscope (Kikusui model 5516 ST). The signals were passed through a window discriminator (WPI Model 121) and evaluated with a Cambridge electronic design computer interface (Model 1401 A) together with a personal computer (Tandon XT 10) and MRATE software. The number of addresses used was 256, the duration of each address being 4 or 8 ms. Peristimulus histograms consisting of 10 consecutive responses were summed each time and electronically integrated. Spontaneous and evoked activities were treated separately. The integrations of activity were pooled for statistical evaluation. Four to six determinations were made before drug administration and these served as controls when they were stable. If evoked activity changed by more that 10% of the mean value before drug administration, the neuron was abandoned and another one searched for. Only one neuron was tested in one animal. Therefore, the number of neurons or axons, experiments and rats used are identical. Significant differences were established by applying Student's t-test for unpaired samples. In addition, the Friedman test was employed in combination with the Wilcoxon-Wilcox test. Finally, the times were determined at which the values after drug application differed from the control values by using a MANOVA-controlled analysis of variance. These values are presented by open symbols in Figs. 2, 3, 5 and 6. This was done to avoid unreadable curves when the time course of the dose-dependent depression of evoked activity is presented with S.E.M. After the end of the experiments carried out on thalamus neurons, the position of the microelectrode tip was marked by passing current of approximately 4 mA for 30 s (Grass Lesion Maker). The animals were killed with an overdose of pentobarbital and the brain was perfused by an intraarterial injection of a 10% formaldehyde solution. The
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brain was removed, fixed in Bouin's solution and embedded in paraplast. Serial sections (10/xm) were stained with gallocyanin-chromalum [13] and counterstained with phloxin. The drugs used were morphine hydrochloride (Merck), morphine-6-glucuronide (Tasmanian Alcaloids), naloxone hydrochloride (Sigma), and urethane (Riedel-De Ha5n). Physiological NaC1 solution was used as solvent. Intrathecal injections were made with a volume of 10 /xl at a rate of 20 /xl/min. All doses are indicated as the salts.
control control
MORPHINE 0.5mg/kg iv 5rain
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3. Results In total, 174 neurons in the VPL or VPM nuclei of the rat thalamus responding to supramaximal electrical stimulation of the ipsi- or contralateral sural nerve were studied. No differences in the responses of VPL and VPM neurons to stimulation or to M6G or morphine could be detected so that all neurons were treated as belonging to one group. Ninety-nine neurons were activated from the ipsilateral and 75 from the contralateral sural nerve. Fig. 1 shows recordings of the spontaneous and evoked impulse discharges recorded from a thalamus neuron and the corresponding peristimulus histograms. No ' wind up' of spontaneous activity occurred when supramaximal stimulation of the sural nerve was employed at the rate chosen (0.1 Hz). When the stimulation strength was reduced below that which generally caused activation of ascending axons in the spinal cord [22,23], i.e. below 20 V, no neuron tested continued to respond to stimulation. Moreover, all neurons activated by supramaximal stimulation of the sural nerve also responded to squeezing more than one paw and pinching the skin of various parts of the body. However, none of these neurons responded to touching or gentle stroking of the skin or air puffs applied to it. The interval between the stimulus and the maximum of evoked activity in the peristimulus histograms of the controls varied between 120 and 320 ms (see also [5]). In Fig. 1, the interval was 310 ms, and the distance between the proximal stimulation electrode (cathode) and the dorsal root entry zone containing afferents from the sural nerve was approximately 120 mm. This yields an apparent conduction velocity of 0.38 m / s which is in accord with an activation by afferent C fibers. Control injections with saline have previously been shown not to change nociceptive activity evoked in single neurons of the thalamus when made by the i.v. [5] or the i.t. [23] route. Moreover, i.v. injection of the lowest dose of M6G tested in these experiments (2.5 /xg/kg; Fig. 3) and an i.t. injection of the lowest dose of morphine tested (10 /~g; Fig. 5) caused no significant depression of evoked activity. Therefore, and in view of the strict regulations limiting the number of animals used in experiments, no additional control experiments were conducted. Morphine administered by i.v. injection reduced the
30rain
60rnin
i
60rain
NALOXONE 0.2mg/kg i.v. Jnts )
5min
o Is
,
.
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Fig. 1. Depression by morphine of activity in a single neuron of the thalamus evoked by sural nerve stimulation. Left hand column of recordings: impulses discharged from the neuron before and after activation by supramaximal electrical stimulation (56 V) of the ipsilateral sural nerve. Stimulus artifacts are indicated by dots under the recordings which were taken before and after i.v. injection of morphine 0.5 mg/kg. Right hand column of recordings: peristimulus histograms (ten trials each) of the activity determined before and after i.v. injection of morphine corresponding to that shown in the left hand column. Stimulation is indicated by dots under the recordings.
activity in thalamus neurons evoked by afferent C fiber stimulation (Fig. 1). The effect was dose-dependent and its maximum was reached between 5 and 20 min (Fig. 2). The threshold dose was 0.025 m g / k g , and the dose producing the maximum effect was 0.5 m g / k g (Figs. 2 and 4). The EDs0 of morphine i.v. established graphically is 0.044 m g / k g or 44 /xg/kg (Fig. 4) This value agrees with that obtained in previous experiments, i.e. 0.05 m g / k g [5]. The effect of morphine was reversed by an i.v. injection of naloxone (0.2 m g / k g ; Figs. 1 and 2). M6G injected i.v. reduced the nociceptive activity evoked in thalamus neurons in a dose-dependent manner (Fig. 3). As in the case of morphine, the effect reached its maximum between 5 and 20 min. The threshold dose lies between a dose of 2.5 /xg/kg, which was ineffective, and a dose of 5 ~ g / k g , which significantly reduced the activity by about 18% of the controls from 5 to 30 min. Thus, on the basis of threshold doses, M6G was about 8 times
135
I. Jurna et al.// Brain Research 722 (1996) 132-138
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Fig. 2. Time course of the depression of C fiber-evoked activity in single neurons of the thalamus caused by i.v. injection of morphine. Ordinate: change in number of impulses discharged in response to C fiber stimulation (evoked activity) in per ceat of the controls. Abscissa: time in minutes before and after injection. The points on the curves are the mean values of the determinations whose number is indicated in brackets for each dose. Open symbols indicate that the values differ significantly from the controls (P < 0.05).
more potent than morphine. The m a x i m u m depression was caused b y M 6 G at a dose of 40 / x g / k g and a m o u n t e d to about 40% o f the control activity (Figs. 3 and 4). Increasing the dose to 80 or even 160 / x g / k g failed to increase the effect. The ED50 o f M 6 G derived from the d o s e - r e sponse curve is about 6 / x g / k g and, hence, 7 times lower than that of m o r p h i n e (Fig. 4). Therefore M 6 G is more potent than m o r p h i n e but less effective w h e n the two drugs are administered b y i.v. injection. The depression of evoked
Fig. 4. Relationship between the depression of C fiber-evoked activity in single neurons of the thalamus caused by i.v. injection of morphine (open circles) and M6G (closed circles). Ordinate: amount of depression in per cent of control activity. Abscissa: doses on a logarithmic scale. The values presented are the mean values determined 10 min after the injection and correspond to those of Figs. 2 and 3.
activity persisted until 60 m i n after i.v. injection of M 6 G 80 and 160 / ~ g / k g and was abolished by an i.v. injection of naloxone 200 / x g / k g (Fig. 4). W h e n m o r p h i n e was applied directly to the spinal cord by i.t. injection, it produced a d o s e - d e p e n d e n t depression of the activity in thalamus n e u r o n s e v o k e d by afferent C fiber stimulation (Fig. 5). A dose of 1 0 / x g was ineffective, and a dose of 2 0 / x g produced a depression lasting from 5 to 30 m i n after the injection. The m a x i m u m depression was produced by 8 0 / x g and a m o u n t e d to about 55% of the control activity. At a dose of 160 ~ g , m o r p h i n e appeared
120
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80 80
60
60
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-20
-20
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20
l
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60 min 80
aorphine-E;-Glucuronidei.v.l Naloxone i.v. O~ 2.S/Jg/kg (N=8) | 200.ug/kg a • 5.0ug/kg (N=8) [] • 10/Jg/kg (N=7) ¢C= ~1= 20,ug/kg (N=7) ~ 1 ~ 80/Jg/kg (N=8) <>• 401Jg/kg (N=8) V • 160/Jg/kg (N=7)
Fig. 3. Time course of the depression of C fiber-evoked activity in single neurons of the thalamus caused by i.v. injection of M6G. Details as in Fig. 2.
I
0
20
40 min 60
Morphine i.t. 0 • 10/Jg <> • 20pg [] • 30 pg ~7 • 40/Jg ~= '1' 80pg Z~ • 160pg
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Fig. 5. Time course of the depression of C fiber-evoked activity in single neurons of the thalamus caused by i.t. injection of morphine. Details as in Fig. 2.
136
L Jurna et al./Brain Research 722 (1996) 132-138
120
more potent than morphine, but there is no remarkable difference in the effectiveness of the two drugs.
o~ 100
4. Discussion 80
-20
0
20
40
60
min
80
Morphine-6-Glucuronide i.t. T Naloxone i.v. 0.2pg
o.sug 1.o,o 3.0,ug loug
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Fig. 6. Time course of the depression of C fiber-evoked activity in single neurons of the thalamus caused by i.t. injection of M6G. Details as in Fig. 2.
to be rather less effective than at a dose of 80 /xg which means that no complete block of evoked nociceptive activity could be achieved by i.t. injections of morphine. The EDs0 derived from the dose-response curve is 18 /zg (Fig. 7). M6G administered by i.t. injection reduced the evoked nociceptive activity in a dose-dependent fashion (Fig. 6). The effect of all doses was significant till 60 rain after injection when naloxone (200 /zg/kg) was injected i.v. which abolished M6G-induced depression. The lowest dose tested was 0.2 /zg and this caused a maximum reduction of 23% of the control activity at 10 rain after the injection. It is difficult to decide which dose of M6G between 0.2 and 10 /zg produced maximum depression of evoked activity (Fig. 7). In any case it amounted to no more than 40% of the controls. The EDs0 can only roughly be estimated as being less than 0.2 /xg. There is no significant difference between the values determined 10 rain after i.t. injection of M6G 0.5, 1 and 10/xg and that after morphine. The values after M6G 3/xg and morphine 80/zg differed significantly ( P < 0.05). It appears that, at the spinal level, M6G is depression
80 %- eM-6-G EDso < 0.2,,ug
OMORPHINE
EDso,, 18jag
60-
40-_ f 20-
o o12
o15
;
3
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160jug
i.t.
Fig. 7. Relationship between the depression of C fiber-evoked activity in single neurons of the thalamus caused by i.t. injection of morphine (open circles) and M6G (closed circles). Details as in Fig. 4.
The results of the present study confirm those of other authors according to which M6G is more potent than morphine in producing an antinociceptive effect (for literature see Introduction). Nociceptive activity evoked in thalamus neurons by afferent C fiber stimulation was reduced by M6G at doses 7 to 8 times lower than those of morphine when using the i.v. route of administration. The difference is even more marked when the two drugs were administered by i.t. injection. In that case, the dose-response curves of M6G and morphine are clearly separated (Fig. 7), while after i.v. injection the dose-response curves of M6G and morphine overlap (Fig. 4). The effective doses of M6G appear to be at least 10 times lower than those of morphine after i.t. injection. However, morphine was more effective than M6G when the two drugs were injected i.v. Morphine was capable to completely block the nociceptive activity evoked in thalamus neurons, while M6G reduced it by no more than 50%. The results obtained with i.v. injections of morphine correspond to those of a previous series of experiments carried out some years ago in which the EDs0 of morphine was found to be 0.05 m g / k g and doses of 0.5 and 1 m g / k g reduced the evoked activity to zero as in the present study [5]. A similarly low EDs0 had been determined by Guilbaud and coworkers when studying the effect of i.v. injections of morphine on nociceptive activity elicited in thalamus neurons by noxious stimuli applied to peripheral tissue [3]. Both M6G as well as morphine failed to suppress altogether the nociceptive activity when administered by i.t. injection. This is in agreement with results in the literature which show that morphine injected i.v. reduced nociceptive activity evoked in neurons of the dorsal horn of the spinal cord by a maximum of about 70% [24,26,27,45] or in axons ascending from the spinal cord to the brain by 50 to 60% [21,22] of the control activity in spinal animals. Moreover, microiontophoretic application of morphine to dorsal horn neurons was found to reduce evoked nociceptive activity not in all but only in part of these neurons [4,12]. Similarly, it was observed in a study in which M6G was applied locally to the spinal cord that it reduced nociceptive activity only in part of the neurons tested and that, in the neurons affected by M6G, nociceptive activity was reduced not completely but only by about 90% of the controls [44]. In addition, it was found in that study that systemically administered M6G produced a mean inhibition of C fiber-evoked activity of about 80%. According to Yaksh and Rudy [49], morphine-induced analgesia or antinociception is not simply the sum of the effects exerted by morphine at spinal and supraspinal sites,
L Jurna et al./Brain Research 722 (1996) 132-138
but instead the effect o f morphine at one or the other site predominates. However, it has been demonstrated that the antinociceptive effect of irttracerebroventricular injections of morphine was potential!ed by i.t. injections and vice versa, and it was therefore concluded that both spinal and supraspinal sites are essent~ial to produce the full antinociceptive or analgesic effect caused by systemic administration of morphine [50]. This fully agrees with the observation that morphine was more effective in depressing nociceptive reflex responses in intact rats than in rats with their Spinal cords transected [10,19,20] or after lesioning the dorsolateral funiculus [2]. These findings obviously indicate that morphine activates a descending inhibitory control operating from the brain stem and enhancing the spinal effect of morphine. Therefore, the depressant effect of morphine at the spinal level seems to add to the effects produced at supraspinal sites so that the strong antinociceptive effect observed in l:halamus neurons results. It is, however, an open question why such summing up of spinal and supraspinal effects wa,; not detectable in the case of M6G. Higher lipophilicity of the folded molecule might account for the higher potency of M6G, but it cannot explain why it is less effective than morphine following i.v. administration to the animal with an intact neuraxis. However, the higher efficacy in this condition might result from different affinities of the two compounds to opioid receptor subtypes. The results obtained following i.v. injections of morphine and M 6 G show that morphine is more effective than M 6 G in depressing nociceptive activity in the thalamus, while M 6 G is more potent in this respect. Because of the relatively poor effectiveness of M 6 G it is doubtful that the glucuronide contributes in a substantial way to the analgesic effect of systemicalily administered morphine. A similar conclusion has been reached when evaluating the low concentrations of M 6 G established in the cerebrospinal fluid after epiduml injections of morphine to patients suffering from cmacer pain [40]. However, the situation may be different when the excretion of M 6 G is markedly reduced in renal failure leading to relative overdosing of morphine.
Acknowledgements The authors are indebted to Mrs. Karen Wolske, Mrs. Birgit Spohrer and Mrs. Gabriele Ulrich for valuable and skilful technical assistance.
References [1] Abbot, F.V. and Palmour, R.M., Morphine-6-glucuronide:analgesic effects and receptor binding profile in rats, Life Sci., 43 (1988) 1685-1695. [2] Barton, C., Basbaum, A.I. and Fields, H.L., Dissociation of
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supraspinal and spinal actions of morphine: a quantitative evaluation, Brain Res., 188 (1980) 487-498. [3] Benoist, J.M., Kayser, V., Gautron, M. and Guilbaud, G., Low doses of morphine strongly depresses responses of specific nociceptive neurones in the ventrobasal complex of the rat, Pain, 15 (1983) 333-344. [4] Calvillo, O., Henry, J.L. and Neuman, R.S., Effects of morphine and naloxone on dorsal horn neurones in the cat, Can. J. Physiol. Pharmacol., 52 (1974) 1207-1211. [5] Carlsson, K.-H., Monzel, W. and Jurna, I., Depression by morphine and the non-opioid analgesic agents, metamizol (dipyrone), lysine acetylsalicylate and paracetamol, of activity in rat thalamus neurones evoked by electrical stimulation of nociceptive afferents, Pain, 32 (1988) 313-326. [6] Carrupt, P.-A., Testa, B., Bechalany, A., E1 Tayar, N., Descas, P. and Perrissoud, D., Morphine-6-glucuronide and morphine-3glucuronide as molecular chameleons with unexpected lipophilicity, J. Med. Chem., 34 (1991) 1272-1275. [7] Casparis, P., Su alcuni derivati della morfina e loro azione analgesica, Boll. Chim. Farm., 89 (1950) 309-318. [8] Chen, Z.R., Irvine, R.J., Somogyi, A.A. and Bochner, F., Mu receptor binding of some commonly used opioides and their metabolites, Life Sci., 48 (1991) 2165-2171. [9] Christensen, C.B. and Jorgensen, L.N., Morphine-6-glucuronidehas high affinity for the opioid receptor, Pharmacol. Toxicol., 60 (1987) 75-76. [10] Dewey, W.L., Snyder, J.W., Harris, L.S. and Howes, J.F., The effect of narcotics and narcotic antagonists on the tall-flick response in spinal mice, J. Pharm. Pharmacol., 21 (1969) 548-550. [11] Don, H.F., Dieppa, R.A. and Taylor, P., Narcotic analgesics in anuric patients, Anesthesiology, 42 (1975) 745-747. [12] Duggan, A.W., Hall, J.G. and Headley, P.M., Suppression of transmission of nociceptive impulses by morphine: selective effects of morphine administered in the region of the substantia gelatinosa. Br. J. Pharmacol., 61 (1977) 65-76. [13] Einarson, L., On the theory of gallocyanin-chromalum staining and its application for quantitative estimation of basophilia: a selective staining of exquisite progressivity, Acta Pathol. Microbiol. Scand., 28 (1951) 82-102. [14] Franc6s. B., Gout, R., Campistron, G. Pancon, E. and Cros, J., Morphine-6-glucuronide is more mu-selective and potent in analgesic tests than morphine, Progr. Clin. Biol. Res., 328 (1990) 477-480. [15] Glare, P.A. and Walsh, T.D., Clinical pharmacokinetics of morphine, Ther. Drug Monit., 13 (1991) 1-23. [16] Gong, Q.-L., Hedner, J., Bj6rkman, R. and Hedner, T., Morphine-3glucuronide may functionally antagonize morphine-6-glucuronide induced antinociception and ventilatory depression in the rat, Pain, 48 (1992) 249-255. [17] Hand, C.W., Blunnie, W.P., Claffey, L.P., McShane, A.J., McQuay, H.J. and Moore, R.A., Potential analgesic contribution from morphine-6-glucuronide in CSF, Lancet, II (1987) 1207-1208. [18] Hanna, M.H., Peat, S.J., Woodham, A., Knibb, A. and Fung, C., Analgesic efficacy and CSF pharmacokinetics of intrathecal morphine-6-glucuronide:comparison with morphine, Br. J. Anaesth., 64 (1990) 547-550. [19] Irwin, S., Houde, R.W., Bennett, D.R., Hendershot, L.C. and Seevers, M.H., The effect of morphine, methadone and meperidine on some reflex responses of some spinal animals to nociceptive stimulation, J. Pharmacol. Exp. Ther., 101 (1951) 132-143. [20] Juma, I., D~npfung repetitiver AktivierungsvorgS.ngean der spinalen Motorik durch Morphin. In R. Janzen, W.D. Keidel, A. Herz and C. Steichele (Eds.), Schmerz, Grundlagen, Pharmakologie, Therapie, Georg Thieme Verlag, Stuttgart, 1972, pp. 267-269. [21] Juma, I. and Grossmann, W., The effect of morphine on the activity evoked in ventrolateral tract axons of the cat spinal cord, Exp. Brain Res., 24 (1976) 473-484.
138
L Jurna et a l . / Brain Research 722 (1996) 132-138
[22] Jurna, I. and Heinz, G., Differential effects of morphine and opioid analgesics on A and C fibre-evoked activity in ascending axons of the rat spinal cord, Brain Res., 171 (1979) 573-576. [23] Jurna, I., Spohrer, B. and Bock, R., lntrathecal injection of acetylsalicylic acid, salicylic acid and indometacin depresses C fibre-evoked activity in the rat thalamus and spinal cord, Pain, 49 (1992) 249-256. [24] Kitahata, L.M., Kosaka, Y., Taub, A., Bonikos, K and Hoffert, M., Lamina-specific suppression of dorsal-horn unit activity by morphine sulfate, Anesthesiology, 41 (1974) 39-48. [25] Laizure, S.C., Miller, J.H., Stevens, R.C., Donahue. D.J., Laster, R.E. and Brown, D., The disposition and cerebrospinal fluid penetration of morphine and its two major glucuronided metabolites in adults undergoing lumbar myelogram, Pharmacotherapy, 13 (1993) 471-475. [26] LeBars, D., Men6trey, D., Conseiller, C. and Besson, J.M., Depressive effects of morphine upon lamina V cells activities in the dorsal horn of the spinal cat, Brain Res., 98 (1975) 261-277. [27] LeBars, D., Guilbaud, G., Jurna, I. and Besson, J.M., Differential effects of morphine on responses of dorsal horn lamina V type cells elicited by A and C fibre stimulation in the spinal cat, Brain Res., 115 (1976) 518-524. [28] Lipkowski, A.W., Can', D.B., Langlade, A., Osgood, P.F. and Szyfelbein, S.J., Morphine-3-glucuronide: silent regulator of morphine actions, Life Sci., 55 (1994) 149-154. [29] Mc Quay, H.J., Moore, R.A., Hand, C.W. and Sear, J.W., Potency of oral morphine, Lancet, 1I (1987) 1458-1459. [30] Osborne, R.J., Joel, S.P. and Slevin, M.L., Morphine intoxication in renal failure: the role of morphine-6-glucuronide, Br. Med. J., 292 (1986) 1548-1549. [31] Osborne, R., Joel, S., Trew, D. and Slevin, M., Analgesic activity of morphine-6-glucuronide, Lancet, I (1988) 828. [32] Osborne, R., Joel, S., Trew, D. and Slevin, M., Morphine and metabolite behaviour after different routes of morphine administration: demonstration of the importance of the active metabolite morphine-6-glucuronide, Clin. Pharmacol. Ther., 47 (1990) 12-19. [33] Pasternak, G.W., Bodnar, R.J., Clark, J.A. and Inturrisi, C.E., Morphine-6-glucuronide, a potent mu agonist, Life Sci., 41 (1987) 2845-2849. [34] Paul, D., Standifer, K.M., Inturrisi, C.E. and Pasternak, G.W., Pharmacological characterization of morphine-6/3-glucuronide, a very potent morphine metabolite, J. Pharmacol. Exp. Ther., 251 (1989) 477-483. [35] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, San Diego, 1986. [36] Peterson, G.M., Randall, C.T.C. and Paterson, J., Plasma levels of morphine and morphine glucuronides in the treatment of cancer apin: relationship to renal function and route of administration, Eur. J. Clin. Pharmacol., 38 (1990) 121-124.
[37] Regnard, C.F.B. and Twycross, R.G., Metabolism of narcotics, Br. Med. J., 288 (1984) 860. [38] S~iwe, J. and Odar-CederlSf, I., Kinetics of morphine in patients with renal failure, Eur. J. Clin. Pharmacol., 32 (1987) 377-382. [39] S~iwe, J., Svensson, J.O. and Odar-Cederl~Sf, I., Kinetics of morphine in patients with renal failure, Lancet, II (1985) 211. [40] Samuelsson, H., Hedner, T., Venn, R. and Michaikiewicz, A., CSF and plasma concentrations of morphine and morphine glucuronides in cancer patients receiving epidural morphine, Pain, 52 (1993) 179-185. [41] Sear, J.W., Hand, C.W., Moore, R.A. and McQuay, H.J., Studies on morphine disposition: influence of renal failure on the kinetics of morphine and its metabolites, Br. J. Anaesth., 62 (1989) 28-32. [42] Shimomura, K., Kamata, O., Ueki, S., Ida, S., Oguri, K., Yoshimura, H. and Tsukamoto, H., Analgesic effect of morphine glucuronides, Tohoku J. Exp. Med., 105 (1971) 45-52. [43] Smith, M.T., Watt, J.A. and Cramond, T., Morphine-3-glucuronide - a potent antagonist of morphine analgesia, Life Sci., 47 (1990) 579-585. [44] Sullivan, A.F., McQuay, H.J., Bailey, D. and Dickenson, A.H., The spinal antinociceptive actions of morphine metabolites morphine-6glucuronide and normorphine in the rat, Brain Res., 482 (1989) 219-224. [45] Toyooka, H., Kitahata, L.M., Dohi, S., Ohtani, M., Hanaoka, K. and Taub, A., Effects of morphine on the rexed lamina. VII. Spinal neuronal responses to graded noxious radiant heat stimulation, Exp. Neurol., 62 (1978) 146-158. [46] Van Dongen, R.T.M., Crul, B.J.P., Koopman-Kimenai, P.M. and Vree, T.B., Morphine and morphine-glucuronide concentrations in plasma and CSF during long-term administration of oral morphine, Br. J. Clin. Pharmacol., 38 (1994) 271-273. [47] Wahlstr~Sm, A., Winblad, B., Bixo, M. and Rane, A, Human brain metabolism of morphine and naloxone, Pain, 35 (1988) 121-127. [48] Wolff, T., Samuelsson, H. and Hedner, T., Morphine and morphine metabolite concentrations in cerebrospinal fluid and plasma in cancer patients after slow-release oral morphine administration, Pain, 62 (1995) 147-154. [49] Yaksh, T.L. and Rudy, T.A., Narcotic anaigetics: CNS sites and mechanisms of action as revealed by intracerebral injection techniques, Pain, 4 (1978) 299-359. [50] Yeung, J.C. and Rudy, T.A., Multiplicative interaction between narcotic agonisms expressed at spinal and supraspinal sites of antinociceptive action as revealed by concurrent intrathecal and intracerebroventricular injections of morphine, J. Pharmacol. Exp. Ther., 215 (1980) 633-642. [51] Yoshimura, H., Ida, S., Oguri, K. and Tsukamoto, H., Biochemical basis for analgesic activity of morphine-6-glucuronide. I. Penetration of morphine-6-glucuronide in the brain of rats, Biochem. Pharmacol., 22 (1973) 1423-1430.