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Antinociceptive Interaction Between Alprazolam and Opioids
Brain Research Bulletin, Vol. 42, No. 3, pp. 239–243, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/97 $17.00 / .00
PII S0361-9230(96)00265-1
Antinociceptive Interaction Between Alprazolam and Opioids CHAIM G. PICK1 Department of Anatomy and Anthropology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, 69978 Israel [Received 2 April 1996; Revised 22 May 1996; Accepted 25 July 1996]
ABSTRACT: Alprazolam is a triazolobenzodiazepine, with a potent anxiolytic action and a short half-life. Alprazolam analgesia was measured, using the radiant heat tailflick assay in mice, which was administered alone or in combination with opioids. Intrathecally administered alprazolam produced a dose–response increase in the tailflick latency with an ED50 of 34 mg (19.4–72.5, 95% CL). There were almost no effects after intracerebroventricular injections. Naloxone almost completely abolished the analgesia response mediated by alprazolam. This sensitivity to naloxone indicates that at least some of the analgesic effects of alprazolam are mediated by an opioid mechanism of action. When administered together with various antagonists of opioid receptor subtypes, we found that the m antagonists, but not the d and k1 subtypes inhibited alprazolam analgesia significantly. No effect was found when alprazolam was coadministrated with k3 opioid agonists. In addition, we found a supra-additivity (synergistic) increase in analgesia when alprazolam was given with morphine. Competition binding assays show the highest affinity of alprazolam to the m1 subtype. In summary, we conclude that alprazolam mediates its analgesic effect, most probably via an m opiate mechanism of action. Copyright Q 1997 Elsevier Science Inc.
opioid peptides interact with multiple classes of receptors. Each subclass has its unique profile of ligand selectivity, regional distribution, and pharmacological actions, and each is capable of modulating the perception of pain. Classically, Mu ( m) receptors have been implicated in opiate analgesia, but recent studies indicate the ability of other opioid receptor systems to elicit analgesia. DPDPE clearly demonstrated d analgesic mechanisms. The highly k1-selective agonist U50,488 elicits selective k1 analgesia, and NalBzoH has been a useful tool in examining k3 analgesia [14]. The use of highly selective opioid drugs, which have minimal side effects in combination with BZs, can possibly enable more successful pain management. Interaction between BZs and opioids were demonstrated previously in the CNS. Biochemical studies have found that opioid agonists alter the pharmacodynamic and distribution properties of BZ receptors in the cortex, hippocampus, and some midbrain areas, such as the substantia nigra and central gray [19,22 ] . Acute BZ administration alters met5-enkephalin release [ 5 ] . Acute BZ administration increases pain threshold [ 26 ] . Naloxone, a selective opioid antagonist, blocks several behaviors that are induced by BZ, including hyperactivity, sedation, hyperphagia, hyperdipsia, anxiolysis, and learning acquisition. Several BZ derivatives, such as midazolam and diazepam, have been found to reduce responses to noxious stimulation [ 4,12,18,21,28 ] . Alprazolam is a triazolobenzodiazepine, with a potent anxiolytic effect and a short half-life. It is used in management of panic attacks, anxiety disorders, and premenstrual syndromes [9]. Alprazolam is one of the most frequently prescribed BZ and among the most widely used medications in the United States [9]. There are few studies dealing with the analgesic properties of alprazolam. It has been demonstrated that the use of alprazolam in cancer patients is associated with marked analgesic responses [6]. Another work showed that chronic administration of alprazolam resulted in a significant decrease in rats’ perception of pain in the hot-plate test [7]. The present study was designed to investigate the antinociceptive effects of alprazolam, administered alone or in combination with selective opioid drugs. An ultimate goal is to try and achieve a better way to reduce pain with fewer side effects.
KEY WORDS: Analgesia, Alprazolam, Opioid receptors, Receptor binding, Tailflick, Mice.
INTRODUCTION Pain is a common disabling symptom in cancer patients. Opioid analgesics are the mainstay of pain treatment in such patients. In spite of their advantages, side effects like tolerance, physical dependence, respiratory depression, and constipation greatly limit their usefulness [14 ] . In some patients, due to the development of tolerance or increasing intensity of the pain stimulus, the patient may reach a point where opioids can no longer provide adequate relief without unacceptable side effects. In addition, administration of high doses can lead to escalation of pain [16 ] . One way to overcome these problems is through coinjecting other drugs, such as benzodiazepines ( BZs ) with the opioids [18 ] . During recent years, multiple opioid receptor subtypes have been found that are capable of eliciting analgesia independently [14]. Like most putative neurotransmitters, the opiates and
1 Requests for reprints should be addressed to Chaim G. Pick, Department of Anatomy and Anthropology, Sackler Faculty of Medicine Tel-Aviv University, Ramat Aviv, Tel Aviv, 69978, Israel. Fax 972-36408287; e-mail [email protected].
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Animals and Surgery Male CD-1 mice (25–35 g; Charles River Breeding Laboratories, Wilmington, MA) were maintained on a 12:12 h (L:D) cycle with Purina rodent chow and water available ad lib. Mice were housed in groups of five until testing. Central injections were made under light halothane anesthesia, using a Hamilton 10-ml syringe fitted to a 30-gauge needle with V1 tubing. Intracerebroventricular (ICV) injections were administered approximately 2 mm caudal and 2 mm lateral to bregma at a depth of 3 mm [8] and intrathecal (IT) injections were given by lumbar puncture [10]. Agents Morphine sulfate was purchased from Mallinckroft (St. Louis, MO), halothane from Halocarbon Laboratories, Inc. (Hackensack, NJ), benzoylhydrazine from Aldrich Chemicals (Milwaukee, WI), ( D-Pen2,D-Pen5)enkephalin (DPDPE) and [ D-Ala 2,MePhe 4 ,Gly-(ol) 5 ]enkephalin (DAMGO) from Peninsula Laboratories (Belmont, CA). Naltrindole hydrochloride and nor-binaltorphamine (nor-BNI) were purchased from Research Biochemicals, Inc. (Natick, MA). b-funaltrexamine ( bFNA) was obtained from the Research Technology Branch of NIDA. Alprazolam and U50,488-H {trans-3,4-dchloro- Nmethyl- N- [ 2- ( 1-pyrrolindinyl ) -cyclohexyl ] -benzeneacetamide} were a generous gift from Upjohn Pharmaceutics (West Sussex, England). Naloxonazine and Naloxone Benzoylhydrazone (NalBzoH) were a generous gift from Dr. G. W. Pasternak. Radioligands and Formula 963 scintillation fluid were purchased from New England Nuclear (Boston, MA). Morphine, naltrindole, and nor-BNI doses were expressed as the salt and NalBzoH and U50,488 were expressed as the free base. High concentrations of NalBzoH were dissolved in 30% EtOH, which did not produce analgesia in control mice. Low concentrations of NalBzoH and all other drugs were dissolved in saline. All other compounds were purchased from commercial sources.
95% confidence limits which then can be compared to the line of additivity [23]. Binding Assays All binding assays were performed using the techniques reported previously [1,2]. Mu1 ( m1 ) binding was assessed in the calf thalamic membranes, using [ 3H][ D-Ala 2-D-Leu 5 ]enkephalin (DADL) in the presence of unlabeled DPDPE. Mu2 ( m2 ) binding was determined in calf thalamic membranes with DAMGO in the presence of DSLET, whereas d was measured with [ 3H]DPDPE in the calf frontal cortex. Kappa1 ( k1 ) binding was determined using guinea pig cerebellar membranes and [ 3H]U69,593 whereas [ 3H]NalBzoH was used to measure k3 with calf striatal membranes. Ki values were calculated from IC50 values. Brains were homogenized in 50 volumes of Tris (50 mM, pH 7.4, 257C) buffer containing PMSF (0.1 mM), Na2EDTA (1 mM), and NaCl (100 mM), incubated at 257C for 15 min to remove endogenous ligands, and centrifuged at 45,000 1 g at 47C for 30 min. The pellets were resuspended in 100 volumes of potassium phosphate buffer (50 mM, pH 7.4, 10 mg wet weight tissue/ml) and 1-ml aliquots was used in all binding assays. We selected the areas for the specific binding based on our previous experiments, as well as other studies using the same procedures [1,2], which indicated the species and areas in the brain in which we can receive the best binding results. All binding assays were performed at 257C. Kappa3 binding was determined, using [ 3H]NalBzoH for 60 min in the presence of K2EDTA (5 mM), [ 3H]DAMGO ( m) binding for 150 min in the presence of MgSO4 (1 mM) and [ 3H]U69,593 ( k1 ) binding for 45 min. When using [ 3H]U69,593, all filters were treated with polyethylenimine (1%) to decrease filter binding. Nonspecific binding was determined with levallorphan (1 mM). All points were determined in triplicate and only specific binding was reported. All values are presented as the mean / SEM of the stated number of independent determinations. RESULTS
Nocioceptive Tests
Opioid Receptor Binding by Alprazolam Competition Analysis
Analgesia was determined, using the radiant heat tailflick technique [3]. The latency to withdraw the tail from a focused light stimulus was measured electronically, using a photocell. Baseline latencies (2.0–3.0 s) were determined before experimental treatments for all animals and were expressed as the mean of the two trials. Post treatment latencies were determined as indicated for each experiment and a maximal latency of 10 s was used to minimize tissue damage. Analgesia was defined quantitatively as at least a doubling of baseline values for each mouse. For each dose (point), at least 10 different mice were checked and their scores were summarized to show the percentage of animals that achieved analgesia at each dose. Each mouse was checked once.
Competition studies were used in order to estimate the affinity of alprazolam to the various subtypes of opioid receptors. Overall, alprazolam showed low affinity to all the types and subtypes. The highest affinity of alprazolam was to the m1 subtype (labeled with the highly selective ligand [ 3H][ D-Ala 2-D-Leu 5 ]enkephalin), which was more than fourfold higher compared with the other subtypes (Table 1).
Data Analysis Dose–response curves were analyzed using a modification of the BLISS-20 computer program. This program maximizes the log-likelihood function to fit a parallel set of Gaussian normal sigmoid curves to the dose–response data. In addition, it was used to calculate the ED50 with 95% confidence limits [25]. Single-dose antagonist studies were analyzed, using the Fisher exact test. The presence of synergy was examined using isobolographic analysis. We administered a fixed dose of the drug in one location and determined the ED50 in the other. This provides an ED50 with
TABLE 1 INHIBITION OF OPIOID RECEPTOR BINDING BY ALPRAZOLAM Receptor Subtype
Ki (nM)
Mu1 Mu2 Delta Kappa1 Kappa3
316 ú1,667 ú1,250 ú1,250 ú1,364
(3) (4) (4) (4) (4)
A range of alprazolam concentrations were incubated in the specified binding assays (numbers in parentheses, numbers of assays). IC50 values were then determined and converted to Ki values.
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ANTINOCICEPTIVE INTERACTION BETWEEN ALPRAZOLAM AND OPIOIDS
FIG. 1. Dose-dependent effects of alprazolam in the tailflick assay. Groups of mice (n ¢ 10) received an IT or ICV injection of alprazolam at the indicated dose and were tested in the tailflick test 15 min later. The ED50 (95% CL) value for alprazolam IT was 34 mg (19.4, 72.5).
Alprazolam Analgesia Intrathecal administration of alprazolam induced analgesia in the tailflick assay with an ED50 of 34 mg (19.4–72.5). Intracerebroventricular administration did not elicit more than 13% analgesia (after 50 mg, Fig. 1). In a separate tailflick assay, alprazolam (60 mg IT) produced analgesia in 70% of the mice (n Å 10). Naloxone 1 mg/kg SC almost completely abolished (to 10%) the analgesic response (n Å 10; p õ 0.005), whereas a lower naloxone dose (0.1 mg/kg SC) had no significant effect. This sensitivity to naloxone indicates that at least some of the analgesic effects of alprazolam are mediated by an opioid mechanism of action. Alprazolam Analgesia and Opioid Receptor Subtypes In the next stage, the involvement of the selective antagonistic activity of m1 , m2 , d, and k1 receptors was assessed to evaluate their potential involvement in alprazolam analgesia. We examined several selective antagonists (Fig. 2). Administered 24 h prior to testing, b-FNA (40 mg/kg, SC) was found to be a selective m1 and m2 antagonist, whereas naloxonazine (35 mg/kg, SC) was shown to be a selective m1 antagonist [15]. Similarly, the d selective antagonist naltrindole (20 mg/kg, SC) blocked d analgesia [15]. Nor-BNI (10 mg/kg, SC) is a selective k1 antagonist [24]. All the antagonists do not mediate analgesia by themselves and do not change the latencies of the baselines of the pretreated animals. When we examined alprazolam analgesia, b-FNA and naloxonazine reversed alprazolam analgesia at the same dose that they reversed morphine analgesia, suggesting a role for m1 receptor in alprazolam analgesia (Fig. 2). The dose of naltrindole that reversed DPDPE analgesia [15] did not reverse alprazolam analgesia. Nor-BNI reversed k1 analgesia, mediated by U50,488H, but did not reverse alprazolam analgesia (Fig. 2). The activity of each of the antagonists was confirmed with its prototypic agonists (data not shown).
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FIG. 2. Effects of b-FNA, naloxonazine, naltrindole, and nor-BNI on alprazolam analgesia. Groups of mice (n ¢ 10) were treated with bFNA (40 mg/kg, SC) or with naloxonazine (35 mg/kg, SC) 24 h before alprazolam challenge. Naltrindole (20 mg/kg, SC), nor-BNI (10 mg/ kg, SC), or saline were treated immediately before alprazolam was injected and tested with the tailflick assay 30 min later. Both b-FNA and naloxonazine antagonized alprazolam analgesia significantly (p õ 0.05). Neither naltrindole nor nor-BNI antagonized alprazolam analgesia significantly.
without alprazolam was 44 mg/kg, SC (17–73, 95% CL), and with alprazolam was 42 mg/kg, SC (14–61). Alprazolam/Morphine Interactions To further examine the relationship between opioids and alprazolam, we continued working with morphine alone because of the clear relationship found between m antagonists and alprazolam (all the other subtypes did not effect alprazolam analgesia). Simultaneous injections of the two drugs, both that of ICV and IT, can activate both spinal and supraspinal systems. This dual mechanism of action may result in additive effects similar to those demonstrated previously with morphine and midazolam [13]. To test this possibility, we carried out an isobolographic analysis of the combination effect of IT alprazolam and ICV morphine (Fig. 4). If the action of morphine and alprazolam at both locations was simply additive, all the points would fall along the line, whereas points below the line would indicate synergism. The results reveal a marked synergy between the two sites of action. First we determined the ED50 of alprazolam (from Fig. 1) and that of morphine 0.56 mg, ICV (0.24–0.82). At the next stage, we used two fixed ICV morphine dose 0.23 and 0.11 mg
Alprazolam/NalBzoH Interactions The absence of a specific antagonist for k3 receptor subtype forced us to examine the relationship between alprazolam and the k3 subtype in a different manner. We gave NalBzoH a selective agonist of the k3 subtype. NalBzoH was given alone or with an inactive dose of alprazolam (0.5 mg, IT, Fig. 3). We did not find any shift in the dose–response curve; ED50 of NalBzoH
FIG. 3. NalBzoH–alprazolam interaction. Groups of mice (n ¢ 10) were treated with a stated dose of NalBzoH alone or with alprazolam (0.5 mg, IT) and tested in the tailflick test 30 min later. The ED50 of NalBzoH without alprazolam was 44 mg/kg, SC (17, 73) and with alprazolam was 42 mg/kg, SC (14, 61).
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FIG. 4. Isobolographic analysis of spinal alprazolam and supraspinal morphine. Groups of mice (n ¢ 10) were treated with a stated dose of morphine or with alprazolam or in combination. All mice were tested in the tailflick test 15 min later. An isobologram of ED50 values was plotted given either drug alone or in combination. ED50 of alprazolam (from Fig. 1) and ED50 of morphine 0.56 mg, ICV (0.24–0.82) were plotted on their respective axis and a line of additivity was drawn between them. At fixed ICV morphine doses (0.23 and 0.11 mg), the ED50 values for IT alprazolam were 2.5 mg (1.0, 4.1) and 6 mg (3.8, 9.8), respectively. Similarly, fixed IT alprazolan doses (17 and 8 mg) yield ED50 doses of 0.05 mg (0.045, 0.18) and 0.11 mg (0.04, 0.28) for morphine ICV, respectively. There was no overlap between the the expected value on the additive line and with the upper confidence limit of the experimental data. The results revealed a marked synergy between ICV morphine and IT alprazolam.
(1/2 and 1/4 of the ED50 of morphine). These fixed doses were given with three different doses of IT alprazolam. The same ratios were used with fixed IT alprazolam doses (17 and 8 mg; 1/2 and 1/4 of the ED50 of alprazolam) with three different doses of ICV morphine. At a fixed dose of ICV morphine of 0.23 mg, the ED50 of alprazolam was 2.5 mg (1.0; 4.1) and for 0.12 mg the ED50 for alprazolam was 6 mg (3.8; 9.8). The corresponding expected doses on the additive line (y Å 62.5 1 /35) were 21.7 and 27.5 mg, respectively. That is to say, the experimental data with fixed morphine is far apart from the expected values on the additivity line. Similarly, with a fixed dose of 17 mg alprazolam, the ED50 for morphine was 0.05 mg (0.045; 0.18) and 8 mg alprazolam yielded an ED50 of 0.11 mg (0.04; 0.28) for morphine ICV. The corresponding expected doses on the additive line (y Å 62.5 1 /35) were 0.288 and 0.432 mg respectively. Again, the expected value in the additive line does not overlap with the upper confidence limit of the experimental data. DISCUSSION Our results demonstrate that intrathecal injections of alprazolam can produce analgesia in the radiant heat tail-flick test. This analgesia was reversed by naloxone, indicating that the analgesic effect of alprazolam is mediated (at least partially) by an opioid mechanism of action. We found that only m1 and m2 , but
not d and k1 , selective antagonists block alprazolam analgesia, indicating that mainly the m receptor subtype interacts with alprazolam. Alprazolam showed low affinity to all types and subtypes of the opioid receptor. In competition binding analysis, the highest affinity of alprazolam was to the m1 subtype, the same subtype that is involved in alprazolam induced analgesia with the tailflick assay. In addition, we found that intrathecal alprazolam was able to synergystically enhance the increase in ICV morphine-induced antinociception in mice. No effect was found when a single dose of alprazolam was co-administered with the specific k3 opioid agonist NalBzoH. In the terminal stages of cancer, about half of the patients require two or more routes of opioid administration. One of the preferred ways of doing this is by introducing the drug intrathecally. Unfortunately, there is a limit to the amount of morphine that can be administrated intrathecally, and high dose can lead to escalation of pain [16]. Some non-opioid drugs have been studied clinically for their analgesic properties. Drugs such as clonidine, ketamine, midazolam, mianserin, and somatostatin produced analgesic activity, which was not as profound as morphine analgesia [16]. The synergism observed in the pervious work is similar to the one found by Yeung and Rudy [29]. They found that simultaneous injections of ICV and IT morphine induced significant synergistic effects. The analgesic effect of the combination of morphine and alprazolam suggested the possibility of using less morphine in comparison to the amount needed when morphine was injected alone. Other important consequences for the patients from this supra additive (synergism) effect is the sedation, muscle relaxation, and anxiolytic activity of alprazolam. These parameters may also have a possible clinical importance and there is a need to examine these effects in clinical trails. Despite the fact that BZs and opioids act at totally separate receptor sites and through different biochemical and pharmacological mechanisms, they obviously interact with each other at some level. However, this interaction is controversial and cannot be explained at the receptor complex level alone. We have demonstrated in the present study that the effect of alprazolam induced analgesia was antagonized by naloxone. The same phenomenon was observed after the injection of midazolam [18], as well as after the injection of the BZ antagonist Ro 15-1788 [4]. In spite of the above, another study showed that naloxone was able to antagonize only the synergistic effect of the two drugs and failed to reverse midazolam analgesia [28]. This sensitivity to naloxone indicates that at least some of the analgesic effects of BZs are mediated by an opioid mechanism of action. On the other hand, in contrast to what we found with alprazolam, some works showed that BZs can antagonize morphine analgesia. For instance, when midazolam [17] or diazepam [21] were given ICV, it was found to reduce analgesia created by morphine. Alprazolam is known to have a unique clinical spectrum among the BZs. Alprazolam is known to be a potent BZ and is used clinically in the treatment of panic disorders [11]. Clinical observations suggest that, in some types of pain syndromes, specifically those involving nerve injuries, alprazolam may be useful as an adjunct to narcotic analgesia for pain control [6]. In contrast to this observation, another study with chronic patients showed no beneficial effect of alprazolam [27]. In our competition studies, we have found that the highest affinity of alprazolam was for the m1 subtype, whereas the affinity to the other subtypes tested was relatively low. In the previous study, we used mainly calf membranes and guinea pig cortex. We did not use mice’s spinal cords due to technical difficulties as well as the large numbers of animals needed for these experiments. In addition, previous experiments have proven specific binding in these brain regions [1,2]. The use of different species
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ANTINOCICEPTIVE INTERACTION BETWEEN ALPRAZOLAM AND OPIOIDS of animals can explain the discrepancy between our results and that of others, which found high affinity for the k subtype [8,20] in midazolam and tifluadom. The midazolam binding assay was done using a spinal cord tissue of the rat [18]. In another study [21], which was performed with the forebrain of mice, midazolam and diazepam were almost inactive in the receptor binding assay. The differences in the binding results could be a consequence of the fact that the different BZs showed high affinity to different subclasses of the opioid receptor. Our study was done with alprazolam, whereas the others used mainly midazolam. In the present study, when BZ was given IT, the interactions between morphine and BZs were found to be supra additive at the spinal cord level. A number of works explain this interaction by an overlap in the anatomical distribution of opioid and BZ receptors in the spinal cord [18,28]. Perhaps this overlap is one of the reasons that leads to this supra additive enhancement. The same relationship between morphine and other BZs was found at the spinal cord level with midazolam [28]. We found involvement of the m1 subtype in the analgesic effect of alprazolam, a subtype that is located mainly in the brain. Therefore, alprazolam analgesia was detected mainly after IT injection. This in spite of the fact that the main m subtype that mediated morphine analgesia in the spinal cord was the m2 subtype [14]. Another mode of interaction between BZs and opioids can be seen on the GABA receptor complex, the site where the BZ receptor is located. It has been found that morphine downregulates the GABA-A receptor [11]. In conclusion, the findings of this study show that alprazolam given IT can induce potent analgesia. Marked enhancement in the analgesic effect of morphine was found when co-administered with alprazolam. However, further research is needed in order to establish the best route of administration of these drugs, the best sites for the injections, as well as the doses needed. All of these facts and data will enable us to use this synergistic effect between alprazolam and morphine in human patients. ACKNOWLEDGEMENTS
I express my gratitude to Dr. G.W. Pasternak from the Cotzias Laboratory of Neuro-Oncology, Memorial Sloan–Kettering Cancer Center, New York, for his support of these studies. I thank Jarrod P. Kaufman for his critical proofreading of this manuscript. This research was supported in part by the V. Schreiber Foundation of the Tel Aviv University.
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8. Haley, T. J.; McCormick, W. G. Pharmacological effects produced by intracerebral injections of drugs in the conscious mouse. Br. J. Pharmacol. Chemother. 12:12–15; 1957. 9. Huybrechts, I. The pharmacology of alprazolam: A review. Clinical Therapeutics 13:100–117; 1991. 10. Hylden, J. L.; Wilcox. G.L. Intrathecal morphine in mice: A new technique. Eur. J. Pharmacol. 67:313–316; 1980. 11. Jacquet, Y. F.; Saederup, E.; Squires, R. F. Non-stereospecific excitatory action of morphine may be due to GABA-A receptors blockade. Eur. J. Pharmacol. 138:285–288; 1987. 12. Kissin, I.; Brown, P. T.; Bradley, E. L. Sedative and hypnotic midazolam–morphine interactions in rats. Anesthesia Analgesia 71:137–143;1990. 13. Luger, T. J.; T. Hayashi, I. H.; Lorenz H.; Hill, F. Mechanism of the influence of midazolam on morphine antinociception at spinal and supraspinal levels in rats. Eur. J. Pharmacol. 271:421–431. 1994. 14. Pastenak, G. W. Pharmacological mechanisms of opioid analgesia. Clinical Neuropharmacol. 16:1–18;1993. 15. Paul, D.; Levison, J. A.; Howard, D. H.; Pick, C. G.; Hahn, E. F.; Pastenak, G. W. Naloxone Benzoylhydrazone (NalBzoH) analgesia. J. Pharmacol. Exp. Ther. 255:769–774; 1990. 16. Plummer, J. L.; Cmielewiski, P. L.; Gourlay, G. K.; Owen, H.; Cousins, M. J. Antinonciceptive and motor effects of intrathecal morphine combined with intrathecal clonidine, noradrenaline, carbachol or midazolam in rats. Pain 49:145–152; 1992. 17. Rady, J. J.; Fujimoto, J. M. Dynorphine A(1-17) mediates midazolam antagonism of morphine antinociception in mice. Pharmacol. Biochem. Behav. 46:331–339; 1993. 18. Rattan, A. K.; McDonald, J. S.; Tejwani, G. A. Differential effects of intrathecal midazolam on morphine-induced antinonciception in the rat: Role of spinal opioid receptors. Anesthesia Analgesia 73:124–131; 1991. 19. Rocha, L.; Tatsukawa, K.; Chugani, H. T.; Engel J. Jr. Benzodiazepine receptor binding following chronic treatment with naloxone, morphine and Met-enkephalin in normal rats. Brain Res. 612:247– 252;1993. 20. Romer, D.; Buscher, H. H.; Hill, R. C.; Maurer, R.; Petcher, T. J.; Zeugner, H.; Benson, W.; Finner, E.; Milkowski, W.; Thies, P. W. An opioid benzodiazepine. Nature 298:759–760; 1982. 21. Rosland, J. H.; Hole, K. Benzodiazepine-induced antagonism of opioid antinonciception may be abolished by spinalization or blockade of the benzodiazepine receptor. Pharmacol. Biochem. Behav. 37:505–509; 1990. 22. Smith, J. E.; Land, J. D. Limbic muscarinic cholinergic and benzodiazepine receptor changes with chronic intravenous morphine and self-administration. Pharmacol. Biochem. Behav. 20:443–450. 1984. 23. Tallarida, R. J.; Porreca, F.; Cowan, A. Statistical analysis of drugdrug and site-site interactions with isobolograms. Life Sciences 45:947–961.1989. 24. Takemori, A. E.; Ho, B. Y.; Naeseth, J. S.; Portoghase, P. S. NorBinaltrophimine, a highly selective Kappa-Opioid antagonist in analgesic and receptor binding assays. J. Phar. Exp. Ther. 246:255– 258; 1988. 25. Umans, J. G.; Inturrisi, C. E. Pharmacodynamics of subcutaneously administered diacetylmorphine, 6-acetylmorphine and morphine in mice. J. Pharmacol. Exp. Ther. 218:409–415; 1981. 26. Webster, J. L.; Polgar, W. E.; Brandt, S. R.; Berzetei-Gurske , I. P.; Toll, L. Comparisone of k2-opioid receptors in guinea pig brain and guinea pig ileum membranes. Eur. J. Pharmacol. 231:251–258; 1993. 27. Wilson, A. Comparison of flubiprofen and alprazolam in the management of chronic pain syndrome. Psychiat J. Univ. Ottawa 15:144–149; 1990. 28. Yanez, A.; Sabbe, M. B.; Stevens, C. W.; Yaksh, T. L. Interaction of midazolam and morphine in the spinal cord of the rat. Neuropharmacol. 29:359–364; 1990. 29. Yeung, J. C.; Rudy. T. A. Multiplicative interaction between narcotic agonism expressed at spinal and supraspinal sites antinociceptive action as revealed by concurrent intrathecal and intracerebroventricular injections of morphine. J. Pharmacol. Exp. Ther. 215:633–642; 1980.
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Antinociceptive Interaction Between Alprazolam and Opioids CHAIM G. PICK1 Department of Anatomy and Anthropology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, 69978 Israel [Received 2 April 1996; Revised 22 May 1996; Accepted 25 July 1996]
ABSTRACT: Alprazolam is a triazolobenzodiazepine, with a potent anxiolytic action and a short half-life. Alprazolam analgesia was measured, using the radiant heat tailflick assay in mice, which was administered alone or in combination with opioids. Intrathecally administered alprazolam produced a dose–response increase in the tailflick latency with an ED50 of 34 mg (19.4–72.5, 95% CL). There were almost no effects after intracerebroventricular injections. Naloxone almost completely abolished the analgesia response mediated by alprazolam. This sensitivity to naloxone indicates that at least some of the analgesic effects of alprazolam are mediated by an opioid mechanism of action. When administered together with various antagonists of opioid receptor subtypes, we found that the m antagonists, but not the d and k1 subtypes inhibited alprazolam analgesia significantly. No effect was found when alprazolam was coadministrated with k3 opioid agonists. In addition, we found a supra-additivity (synergistic) increase in analgesia when alprazolam was given with morphine. Competition binding assays show the highest affinity of alprazolam to the m1 subtype. In summary, we conclude that alprazolam mediates its analgesic effect, most probably via an m opiate mechanism of action. Copyright Q 1997 Elsevier Science Inc.
opioid peptides interact with multiple classes of receptors. Each subclass has its unique profile of ligand selectivity, regional distribution, and pharmacological actions, and each is capable of modulating the perception of pain. Classically, Mu ( m) receptors have been implicated in opiate analgesia, but recent studies indicate the ability of other opioid receptor systems to elicit analgesia. DPDPE clearly demonstrated d analgesic mechanisms. The highly k1-selective agonist U50,488 elicits selective k1 analgesia, and NalBzoH has been a useful tool in examining k3 analgesia [14]. The use of highly selective opioid drugs, which have minimal side effects in combination with BZs, can possibly enable more successful pain management. Interaction between BZs and opioids were demonstrated previously in the CNS. Biochemical studies have found that opioid agonists alter the pharmacodynamic and distribution properties of BZ receptors in the cortex, hippocampus, and some midbrain areas, such as the substantia nigra and central gray [19,22 ] . Acute BZ administration alters met5-enkephalin release [ 5 ] . Acute BZ administration increases pain threshold [ 26 ] . Naloxone, a selective opioid antagonist, blocks several behaviors that are induced by BZ, including hyperactivity, sedation, hyperphagia, hyperdipsia, anxiolysis, and learning acquisition. Several BZ derivatives, such as midazolam and diazepam, have been found to reduce responses to noxious stimulation [ 4,12,18,21,28 ] . Alprazolam is a triazolobenzodiazepine, with a potent anxiolytic effect and a short half-life. It is used in management of panic attacks, anxiety disorders, and premenstrual syndromes [9]. Alprazolam is one of the most frequently prescribed BZ and among the most widely used medications in the United States [9]. There are few studies dealing with the analgesic properties of alprazolam. It has been demonstrated that the use of alprazolam in cancer patients is associated with marked analgesic responses [6]. Another work showed that chronic administration of alprazolam resulted in a significant decrease in rats’ perception of pain in the hot-plate test [7]. The present study was designed to investigate the antinociceptive effects of alprazolam, administered alone or in combination with selective opioid drugs. An ultimate goal is to try and achieve a better way to reduce pain with fewer side effects.
KEY WORDS: Analgesia, Alprazolam, Opioid receptors, Receptor binding, Tailflick, Mice.
INTRODUCTION Pain is a common disabling symptom in cancer patients. Opioid analgesics are the mainstay of pain treatment in such patients. In spite of their advantages, side effects like tolerance, physical dependence, respiratory depression, and constipation greatly limit their usefulness [14 ] . In some patients, due to the development of tolerance or increasing intensity of the pain stimulus, the patient may reach a point where opioids can no longer provide adequate relief without unacceptable side effects. In addition, administration of high doses can lead to escalation of pain [16 ] . One way to overcome these problems is through coinjecting other drugs, such as benzodiazepines ( BZs ) with the opioids [18 ] . During recent years, multiple opioid receptor subtypes have been found that are capable of eliciting analgesia independently [14]. Like most putative neurotransmitters, the opiates and
1 Requests for reprints should be addressed to Chaim G. Pick, Department of Anatomy and Anthropology, Sackler Faculty of Medicine Tel-Aviv University, Ramat Aviv, Tel Aviv, 69978, Israel. Fax 972-36408287; e-mail [email protected].
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Animals and Surgery Male CD-1 mice (25–35 g; Charles River Breeding Laboratories, Wilmington, MA) were maintained on a 12:12 h (L:D) cycle with Purina rodent chow and water available ad lib. Mice were housed in groups of five until testing. Central injections were made under light halothane anesthesia, using a Hamilton 10-ml syringe fitted to a 30-gauge needle with V1 tubing. Intracerebroventricular (ICV) injections were administered approximately 2 mm caudal and 2 mm lateral to bregma at a depth of 3 mm [8] and intrathecal (IT) injections were given by lumbar puncture [10]. Agents Morphine sulfate was purchased from Mallinckroft (St. Louis, MO), halothane from Halocarbon Laboratories, Inc. (Hackensack, NJ), benzoylhydrazine from Aldrich Chemicals (Milwaukee, WI), ( D-Pen2,D-Pen5)enkephalin (DPDPE) and [ D-Ala 2,MePhe 4 ,Gly-(ol) 5 ]enkephalin (DAMGO) from Peninsula Laboratories (Belmont, CA). Naltrindole hydrochloride and nor-binaltorphamine (nor-BNI) were purchased from Research Biochemicals, Inc. (Natick, MA). b-funaltrexamine ( bFNA) was obtained from the Research Technology Branch of NIDA. Alprazolam and U50,488-H {trans-3,4-dchloro- Nmethyl- N- [ 2- ( 1-pyrrolindinyl ) -cyclohexyl ] -benzeneacetamide} were a generous gift from Upjohn Pharmaceutics (West Sussex, England). Naloxonazine and Naloxone Benzoylhydrazone (NalBzoH) were a generous gift from Dr. G. W. Pasternak. Radioligands and Formula 963 scintillation fluid were purchased from New England Nuclear (Boston, MA). Morphine, naltrindole, and nor-BNI doses were expressed as the salt and NalBzoH and U50,488 were expressed as the free base. High concentrations of NalBzoH were dissolved in 30% EtOH, which did not produce analgesia in control mice. Low concentrations of NalBzoH and all other drugs were dissolved in saline. All other compounds were purchased from commercial sources.
95% confidence limits which then can be compared to the line of additivity [23]. Binding Assays All binding assays were performed using the techniques reported previously [1,2]. Mu1 ( m1 ) binding was assessed in the calf thalamic membranes, using [ 3H][ D-Ala 2-D-Leu 5 ]enkephalin (DADL) in the presence of unlabeled DPDPE. Mu2 ( m2 ) binding was determined in calf thalamic membranes with DAMGO in the presence of DSLET, whereas d was measured with [ 3H]DPDPE in the calf frontal cortex. Kappa1 ( k1 ) binding was determined using guinea pig cerebellar membranes and [ 3H]U69,593 whereas [ 3H]NalBzoH was used to measure k3 with calf striatal membranes. Ki values were calculated from IC50 values. Brains were homogenized in 50 volumes of Tris (50 mM, pH 7.4, 257C) buffer containing PMSF (0.1 mM), Na2EDTA (1 mM), and NaCl (100 mM), incubated at 257C for 15 min to remove endogenous ligands, and centrifuged at 45,000 1 g at 47C for 30 min. The pellets were resuspended in 100 volumes of potassium phosphate buffer (50 mM, pH 7.4, 10 mg wet weight tissue/ml) and 1-ml aliquots was used in all binding assays. We selected the areas for the specific binding based on our previous experiments, as well as other studies using the same procedures [1,2], which indicated the species and areas in the brain in which we can receive the best binding results. All binding assays were performed at 257C. Kappa3 binding was determined, using [ 3H]NalBzoH for 60 min in the presence of K2EDTA (5 mM), [ 3H]DAMGO ( m) binding for 150 min in the presence of MgSO4 (1 mM) and [ 3H]U69,593 ( k1 ) binding for 45 min. When using [ 3H]U69,593, all filters were treated with polyethylenimine (1%) to decrease filter binding. Nonspecific binding was determined with levallorphan (1 mM). All points were determined in triplicate and only specific binding was reported. All values are presented as the mean / SEM of the stated number of independent determinations. RESULTS
Nocioceptive Tests
Opioid Receptor Binding by Alprazolam Competition Analysis
Analgesia was determined, using the radiant heat tailflick technique [3]. The latency to withdraw the tail from a focused light stimulus was measured electronically, using a photocell. Baseline latencies (2.0–3.0 s) were determined before experimental treatments for all animals and were expressed as the mean of the two trials. Post treatment latencies were determined as indicated for each experiment and a maximal latency of 10 s was used to minimize tissue damage. Analgesia was defined quantitatively as at least a doubling of baseline values for each mouse. For each dose (point), at least 10 different mice were checked and their scores were summarized to show the percentage of animals that achieved analgesia at each dose. Each mouse was checked once.
Competition studies were used in order to estimate the affinity of alprazolam to the various subtypes of opioid receptors. Overall, alprazolam showed low affinity to all the types and subtypes. The highest affinity of alprazolam was to the m1 subtype (labeled with the highly selective ligand [ 3H][ D-Ala 2-D-Leu 5 ]enkephalin), which was more than fourfold higher compared with the other subtypes (Table 1).
Data Analysis Dose–response curves were analyzed using a modification of the BLISS-20 computer program. This program maximizes the log-likelihood function to fit a parallel set of Gaussian normal sigmoid curves to the dose–response data. In addition, it was used to calculate the ED50 with 95% confidence limits [25]. Single-dose antagonist studies were analyzed, using the Fisher exact test. The presence of synergy was examined using isobolographic analysis. We administered a fixed dose of the drug in one location and determined the ED50 in the other. This provides an ED50 with
TABLE 1 INHIBITION OF OPIOID RECEPTOR BINDING BY ALPRAZOLAM Receptor Subtype
Ki (nM)
Mu1 Mu2 Delta Kappa1 Kappa3
316 ú1,667 ú1,250 ú1,250 ú1,364
(3) (4) (4) (4) (4)
A range of alprazolam concentrations were incubated in the specified binding assays (numbers in parentheses, numbers of assays). IC50 values were then determined and converted to Ki values.
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FIG. 1. Dose-dependent effects of alprazolam in the tailflick assay. Groups of mice (n ¢ 10) received an IT or ICV injection of alprazolam at the indicated dose and were tested in the tailflick test 15 min later. The ED50 (95% CL) value for alprazolam IT was 34 mg (19.4, 72.5).
Alprazolam Analgesia Intrathecal administration of alprazolam induced analgesia in the tailflick assay with an ED50 of 34 mg (19.4–72.5). Intracerebroventricular administration did not elicit more than 13% analgesia (after 50 mg, Fig. 1). In a separate tailflick assay, alprazolam (60 mg IT) produced analgesia in 70% of the mice (n Å 10). Naloxone 1 mg/kg SC almost completely abolished (to 10%) the analgesic response (n Å 10; p õ 0.005), whereas a lower naloxone dose (0.1 mg/kg SC) had no significant effect. This sensitivity to naloxone indicates that at least some of the analgesic effects of alprazolam are mediated by an opioid mechanism of action. Alprazolam Analgesia and Opioid Receptor Subtypes In the next stage, the involvement of the selective antagonistic activity of m1 , m2 , d, and k1 receptors was assessed to evaluate their potential involvement in alprazolam analgesia. We examined several selective antagonists (Fig. 2). Administered 24 h prior to testing, b-FNA (40 mg/kg, SC) was found to be a selective m1 and m2 antagonist, whereas naloxonazine (35 mg/kg, SC) was shown to be a selective m1 antagonist [15]. Similarly, the d selective antagonist naltrindole (20 mg/kg, SC) blocked d analgesia [15]. Nor-BNI (10 mg/kg, SC) is a selective k1 antagonist [24]. All the antagonists do not mediate analgesia by themselves and do not change the latencies of the baselines of the pretreated animals. When we examined alprazolam analgesia, b-FNA and naloxonazine reversed alprazolam analgesia at the same dose that they reversed morphine analgesia, suggesting a role for m1 receptor in alprazolam analgesia (Fig. 2). The dose of naltrindole that reversed DPDPE analgesia [15] did not reverse alprazolam analgesia. Nor-BNI reversed k1 analgesia, mediated by U50,488H, but did not reverse alprazolam analgesia (Fig. 2). The activity of each of the antagonists was confirmed with its prototypic agonists (data not shown).
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FIG. 2. Effects of b-FNA, naloxonazine, naltrindole, and nor-BNI on alprazolam analgesia. Groups of mice (n ¢ 10) were treated with bFNA (40 mg/kg, SC) or with naloxonazine (35 mg/kg, SC) 24 h before alprazolam challenge. Naltrindole (20 mg/kg, SC), nor-BNI (10 mg/ kg, SC), or saline were treated immediately before alprazolam was injected and tested with the tailflick assay 30 min later. Both b-FNA and naloxonazine antagonized alprazolam analgesia significantly (p õ 0.05). Neither naltrindole nor nor-BNI antagonized alprazolam analgesia significantly.
without alprazolam was 44 mg/kg, SC (17–73, 95% CL), and with alprazolam was 42 mg/kg, SC (14–61). Alprazolam/Morphine Interactions To further examine the relationship between opioids and alprazolam, we continued working with morphine alone because of the clear relationship found between m antagonists and alprazolam (all the other subtypes did not effect alprazolam analgesia). Simultaneous injections of the two drugs, both that of ICV and IT, can activate both spinal and supraspinal systems. This dual mechanism of action may result in additive effects similar to those demonstrated previously with morphine and midazolam [13]. To test this possibility, we carried out an isobolographic analysis of the combination effect of IT alprazolam and ICV morphine (Fig. 4). If the action of morphine and alprazolam at both locations was simply additive, all the points would fall along the line, whereas points below the line would indicate synergism. The results reveal a marked synergy between the two sites of action. First we determined the ED50 of alprazolam (from Fig. 1) and that of morphine 0.56 mg, ICV (0.24–0.82). At the next stage, we used two fixed ICV morphine dose 0.23 and 0.11 mg
Alprazolam/NalBzoH Interactions The absence of a specific antagonist for k3 receptor subtype forced us to examine the relationship between alprazolam and the k3 subtype in a different manner. We gave NalBzoH a selective agonist of the k3 subtype. NalBzoH was given alone or with an inactive dose of alprazolam (0.5 mg, IT, Fig. 3). We did not find any shift in the dose–response curve; ED50 of NalBzoH
FIG. 3. NalBzoH–alprazolam interaction. Groups of mice (n ¢ 10) were treated with a stated dose of NalBzoH alone or with alprazolam (0.5 mg, IT) and tested in the tailflick test 30 min later. The ED50 of NalBzoH without alprazolam was 44 mg/kg, SC (17, 73) and with alprazolam was 42 mg/kg, SC (14, 61).
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FIG. 4. Isobolographic analysis of spinal alprazolam and supraspinal morphine. Groups of mice (n ¢ 10) were treated with a stated dose of morphine or with alprazolam or in combination. All mice were tested in the tailflick test 15 min later. An isobologram of ED50 values was plotted given either drug alone or in combination. ED50 of alprazolam (from Fig. 1) and ED50 of morphine 0.56 mg, ICV (0.24–0.82) were plotted on their respective axis and a line of additivity was drawn between them. At fixed ICV morphine doses (0.23 and 0.11 mg), the ED50 values for IT alprazolam were 2.5 mg (1.0, 4.1) and 6 mg (3.8, 9.8), respectively. Similarly, fixed IT alprazolan doses (17 and 8 mg) yield ED50 doses of 0.05 mg (0.045, 0.18) and 0.11 mg (0.04, 0.28) for morphine ICV, respectively. There was no overlap between the the expected value on the additive line and with the upper confidence limit of the experimental data. The results revealed a marked synergy between ICV morphine and IT alprazolam.
(1/2 and 1/4 of the ED50 of morphine). These fixed doses were given with three different doses of IT alprazolam. The same ratios were used with fixed IT alprazolam doses (17 and 8 mg; 1/2 and 1/4 of the ED50 of alprazolam) with three different doses of ICV morphine. At a fixed dose of ICV morphine of 0.23 mg, the ED50 of alprazolam was 2.5 mg (1.0; 4.1) and for 0.12 mg the ED50 for alprazolam was 6 mg (3.8; 9.8). The corresponding expected doses on the additive line (y Å 62.5 1 /35) were 21.7 and 27.5 mg, respectively. That is to say, the experimental data with fixed morphine is far apart from the expected values on the additivity line. Similarly, with a fixed dose of 17 mg alprazolam, the ED50 for morphine was 0.05 mg (0.045; 0.18) and 8 mg alprazolam yielded an ED50 of 0.11 mg (0.04; 0.28) for morphine ICV. The corresponding expected doses on the additive line (y Å 62.5 1 /35) were 0.288 and 0.432 mg respectively. Again, the expected value in the additive line does not overlap with the upper confidence limit of the experimental data. DISCUSSION Our results demonstrate that intrathecal injections of alprazolam can produce analgesia in the radiant heat tail-flick test. This analgesia was reversed by naloxone, indicating that the analgesic effect of alprazolam is mediated (at least partially) by an opioid mechanism of action. We found that only m1 and m2 , but
not d and k1 , selective antagonists block alprazolam analgesia, indicating that mainly the m receptor subtype interacts with alprazolam. Alprazolam showed low affinity to all types and subtypes of the opioid receptor. In competition binding analysis, the highest affinity of alprazolam was to the m1 subtype, the same subtype that is involved in alprazolam induced analgesia with the tailflick assay. In addition, we found that intrathecal alprazolam was able to synergystically enhance the increase in ICV morphine-induced antinociception in mice. No effect was found when a single dose of alprazolam was co-administered with the specific k3 opioid agonist NalBzoH. In the terminal stages of cancer, about half of the patients require two or more routes of opioid administration. One of the preferred ways of doing this is by introducing the drug intrathecally. Unfortunately, there is a limit to the amount of morphine that can be administrated intrathecally, and high dose can lead to escalation of pain [16]. Some non-opioid drugs have been studied clinically for their analgesic properties. Drugs such as clonidine, ketamine, midazolam, mianserin, and somatostatin produced analgesic activity, which was not as profound as morphine analgesia [16]. The synergism observed in the pervious work is similar to the one found by Yeung and Rudy [29]. They found that simultaneous injections of ICV and IT morphine induced significant synergistic effects. The analgesic effect of the combination of morphine and alprazolam suggested the possibility of using less morphine in comparison to the amount needed when morphine was injected alone. Other important consequences for the patients from this supra additive (synergism) effect is the sedation, muscle relaxation, and anxiolytic activity of alprazolam. These parameters may also have a possible clinical importance and there is a need to examine these effects in clinical trails. Despite the fact that BZs and opioids act at totally separate receptor sites and through different biochemical and pharmacological mechanisms, they obviously interact with each other at some level. However, this interaction is controversial and cannot be explained at the receptor complex level alone. We have demonstrated in the present study that the effect of alprazolam induced analgesia was antagonized by naloxone. The same phenomenon was observed after the injection of midazolam [18], as well as after the injection of the BZ antagonist Ro 15-1788 [4]. In spite of the above, another study showed that naloxone was able to antagonize only the synergistic effect of the two drugs and failed to reverse midazolam analgesia [28]. This sensitivity to naloxone indicates that at least some of the analgesic effects of BZs are mediated by an opioid mechanism of action. On the other hand, in contrast to what we found with alprazolam, some works showed that BZs can antagonize morphine analgesia. For instance, when midazolam [17] or diazepam [21] were given ICV, it was found to reduce analgesia created by morphine. Alprazolam is known to have a unique clinical spectrum among the BZs. Alprazolam is known to be a potent BZ and is used clinically in the treatment of panic disorders [11]. Clinical observations suggest that, in some types of pain syndromes, specifically those involving nerve injuries, alprazolam may be useful as an adjunct to narcotic analgesia for pain control [6]. In contrast to this observation, another study with chronic patients showed no beneficial effect of alprazolam [27]. In our competition studies, we have found that the highest affinity of alprazolam was for the m1 subtype, whereas the affinity to the other subtypes tested was relatively low. In the previous study, we used mainly calf membranes and guinea pig cortex. We did not use mice’s spinal cords due to technical difficulties as well as the large numbers of animals needed for these experiments. In addition, previous experiments have proven specific binding in these brain regions [1,2]. The use of different species
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ANTINOCICEPTIVE INTERACTION BETWEEN ALPRAZOLAM AND OPIOIDS of animals can explain the discrepancy between our results and that of others, which found high affinity for the k subtype [8,20] in midazolam and tifluadom. The midazolam binding assay was done using a spinal cord tissue of the rat [18]. In another study [21], which was performed with the forebrain of mice, midazolam and diazepam were almost inactive in the receptor binding assay. The differences in the binding results could be a consequence of the fact that the different BZs showed high affinity to different subclasses of the opioid receptor. Our study was done with alprazolam, whereas the others used mainly midazolam. In the present study, when BZ was given IT, the interactions between morphine and BZs were found to be supra additive at the spinal cord level. A number of works explain this interaction by an overlap in the anatomical distribution of opioid and BZ receptors in the spinal cord [18,28]. Perhaps this overlap is one of the reasons that leads to this supra additive enhancement. The same relationship between morphine and other BZs was found at the spinal cord level with midazolam [28]. We found involvement of the m1 subtype in the analgesic effect of alprazolam, a subtype that is located mainly in the brain. Therefore, alprazolam analgesia was detected mainly after IT injection. This in spite of the fact that the main m subtype that mediated morphine analgesia in the spinal cord was the m2 subtype [14]. Another mode of interaction between BZs and opioids can be seen on the GABA receptor complex, the site where the BZ receptor is located. It has been found that morphine downregulates the GABA-A receptor [11]. In conclusion, the findings of this study show that alprazolam given IT can induce potent analgesia. Marked enhancement in the analgesic effect of morphine was found when co-administered with alprazolam. However, further research is needed in order to establish the best route of administration of these drugs, the best sites for the injections, as well as the doses needed. All of these facts and data will enable us to use this synergistic effect between alprazolam and morphine in human patients. ACKNOWLEDGEMENTS
I express my gratitude to Dr. G.W. Pasternak from the Cotzias Laboratory of Neuro-Oncology, Memorial Sloan–Kettering Cancer Center, New York, for his support of these studies. I thank Jarrod P. Kaufman for his critical proofreading of this manuscript. This research was supported in part by the V. Schreiber Foundation of the Tel Aviv University.
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