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(S)-(+)-methadone is more immunosuppressive than the potent analgesic (R)-(−)-methadone
International Immunopharmacology 4 (2004) 1525 – 1530 www.elsevier.com/locate/intimp
(S)-(+)-methadone is more immunosuppressive than the potent analgesic (R)-()-methadone Mark R. Hutchinson*, Andrew A. Somogyi Department of Clinical and Experimental Pharmacology, University of Adelaide, Level 5, Medical School North, Frome Road, 5005, Adelaide, Australia Received 29 June 2004; received in revised form 8 July 2004; accepted 9 July 2004
Abstract Methadone is a widely used synthetic opioid which is administered as a racemic mixture of (R)-()- and (S)-(+)enantiomers, with only (R)-()-methadone possessing A opioid receptor agonist activity. Methadone inhibits numerous immune functions in vitro at concentrations above 10 AM in a nonstereoselective and naloxone-insensitive fashion, suggesting the presence of nonclassical opioid receptors on immune cells. No in vivo data on the effects of methadone’s enantiomers on immune function are available. Therefore, the stereoselectivity of methadone’s analgesia (hot plate latency) in vivo and immune suppression ex vivo (splenocyte proliferation) was investigated in groups of Balb/c mice. Significant analgesia was observed in animals that received racemic methadone ( P=0.0012, 52% MPE) and (R)-()-methadone ( P=0.0002, 70% MPE) when compared to saline-treated controls, while (S)-(+)-methadone was devoid of any such effect (4% MPE). In vivo (R)-()- and racemic methadone caused significant inhibition ( Pb0.001, greater than 70%) of basal proliferation compared to saline control. In stark contrast to analgesia, in vivo (S)-(+)-methadone caused significantly greater inhibition of basal proliferation ( Pb0.001, 130%) than (R)-()- and racemic methadone. The immune suppression caused by methadone is not purely a classical opioid response but involves nonclassical opioid receptors located at the central level, which have yet to be characterised. Moreover, the dose at which immune suppression occurred could be achieved clinically. D 2004 Elsevier B.V. All rights reserved. Keywords: Methadone; In vivo; Analgesia; Immune system; Stereoselectivity
1. Introduction
* Corresponding author. Tel.: +61- 8- 83035571; fax: +61- 882240685. E-mail address: [email protected] (M.R. Hutchinson). 1567-5769/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2004.07.011
Methadone is a widely used synthetic 3,3-diphenylpropylamine opioid which primarily acts at the A opioid receptor [1,2]. Its most common use is in substitution therapy for opioid dependence [3], but is also being increasingly used in the management of
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chronic pain [4]. Methadone is administered as a racemic mixture of equal amounts of (R)-()- and (S)(+)-enantiomers. Only (R)-()-methadone possesses significant A opioid receptor agonist activity with 10– 20-fold higher binding affinity than the (S)-(+)enantiomer [1,2]. It is this stereoselectivity which is characteristic of an opioid response [5]. Opioids can modulate the immune system at a cellular level in vitro [6,7] and indirectly in vivo [8,9]. Considerable debate surrounds the identity and bclassicalQ opioid nature (stereoselectivity, agonist sensitivity and rank order of potency of effect) of the receptor(s) that are responsible for these responses, and this has been recently highlighted in vitro [6,7]. Several factors must be consistent for a response to be deemed classically opioid, including ligand stereoselectivity, antagonist sensitivity, ion sensitivity and toxin sensitivity [5]. Previous studies have shown nonstereoselectivity and naloxone insensitivity for methadone’s in vitro immunomodulatory behaviour using various in vitro immunological assays and immune cell sources [6,10–12]. However, these responses have only occurred at very high ligand concentrations (10–100 AM) above the range of concentrations achieved clinically and so have been dismissed as physiologically insignificant [10]. In animal models, methadone has also been found to be less inhibitory than morphine in several immune functional assays [13–15]; however, the stereoselectivity of the effect of methadone on these responses has not been investigated. Therefore, the aim of this study was to determine if in vivo administration of methadone acts stereoselectively to inhibit concanavalin A-induced splenocyte proliferation ex vivo. In contrast to many previously applied models, a rapid nontoxic, sensitive proliferation detection method was used, combined with incubation conditions optimised for the detection of altered immune response by in vitro exposure to opioids.
2. Materials and methods 2.1. Animals Ethics approval to conduct the studies was obtained from the University of Adelaide Animal Ethics Committee. Male Balb/c mice 6–8 weeks old
(22F0.5 g) were purchased from Central Animal Supplies (Waite Campus, University of Adelaide, Adelaide, Australia) and used as donors of splenocytes for use in cell culture. Animals were provided with standard rodent feed and water ad libitum. Animals were housed in a standard 12-h light–dark cycle (starting at 7 AM) under constant room temperature of 22F2 8C. The observer was blinded to all treatments. 2.2. Chemicals RPMI 1640 with HEPES modification and lglutamine were purchased from Invitrogen (Mulgrave, Vic, Australia). Penicillin–streptomycin solution (10,000 U penicillin, 10 mg streptomycin/ml) and concanavalin A were purchased from Sigma (St. Louis, MO, USA). Racemic methadone hydrochloride was obtained from McFarlane Smith (Edinburgh, UK), while (R)-()- and (S)-(+)-methadone bases (less than 1% contamination by the opposite enantiomer) were obtained from the National Institute on Drug Abuse (Rockville, MD, USA). Foetal calf serum was obtained from Trace Scientific (Melbourne, Vic, Australia). All other reagents and chemicals were obtained from commercial sources and were of analytical grade quality. Compounds that were not supplied as a soluble salt were dissolved in an equimolar solution of hydrochloric acid (0.1 M), while all other compounds were dissolved in Milli Q water. The vehicle used for all in vivo drug delivery was normal saline (0.9% NaCl), and all injections were administered intraperitoneally (i.p.) in a volume of 10 ml kg1. All solutions were stored at 4 8C until used. 2.3. In vivo treatment Mice (n=3 for each treatment) received a single i.p. injection of (R)-()- (1.5 mg kg1), (S)-(+)- (1.5 mg kg1), racemic methadone (3 mg kg1) or saline. These doses were based on the results of an initial dose finding pilot study using racemic methadone (data not shown) as this dose (racemic methadone, 3 mg kg1) achieved a quantifiable analgesic response, but did not completely abolish the concanavalin A proliferative response by isolated splenocytes ex vivo. The analgesic responses to these treatments were then
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tested 20 min later by placing each animal once on a 50 8C hot plate and recording the time taken (seconds) until any jumping, paw shaking or paw licking was observed. Animals that did not respond in this fashion after 60 s were removed from the plate. 2.4. Lymphocyte preparation The animals that underwent the dosing regimen and antinociceptive testing described above were sacrificed by cervical dislocation 120 min after drug administration followed by prompt removal of the spleen. The method used to prepare splenocytes for culture is based on that used previously [6,7,16]. Briefly, the spleen was prepared as a single cell suspension by massaging and washing through a nylon mesh into a 15-ml tube with 10 ml of RPMI 1640 (HEPES modification, 0.3 mg ml1 l-glutamine, 5 ml penicillin–streptomycin solution l1). The cells were centrifuged at 4 8C for 5 min at 1000 rpm, the supernatant was discarded and the cells were resuspended in 1 ml of media followed by the addition of 10 ml of lysis buffer [ice-cold 1 ml of 20.56 g l1 tris base (pH 7.65), 9 ml 0.83% NH4Cl in H2O mix just prior to addition to cells; greater than 95% lymphocyte viability following lysis buffer treatment]. The suspension was placed on ice for 4 min, centrifuged (4 min at 1000 rpm) and supernatant was discarded. The cell suspensions were resuspended in 10 ml of media followed by centrifugation (4 min at 1000 rpm), removal of supernatant and resuspension in 5 ml of enriched RPMI 1640 (RPMI 1640 enriched with foetal calf serum). The number of viable lymphocytes in the suspension was counted using trypan blue and a haemocytometer. Cells were then diluted in enriched media to 1106 cells ml1 and 100 Al of this suspension was added to each well of the 96multiwell plates (Nunc, Roskilde, Denmark) and made up to 200 Al with 50 Al of concanavalin A (10 Ag ml1, final concentration of 2.5 Ag ml1 which produces submaximal proliferation) and/or 50 Al of (R)-()-, (S)-(+)- or racemic methadone (n=6; 0 AM control, 0.0004, 0.004, 0.04, 0.4, 4, 40 and 400 AM; the concentrations in the final incubation were 25% of those added). Unstimulated mitogen negative control wells were also prepared, using 50 Al of media instead of concanavalin A. The splenocyte proliferation assays of splenocytes from each animal were per-
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formed in separate plates (n=3 independent experiments). The plates were incubated at 37 8C, 5% CO2 in a humidified incubator (Thermoline, Sydney, Australia) for 24 h following which 25 Al of a diluted AlamarBlue solution (5 Al AlamarBlue, 20 Al cell medium) was added to each well. The plates were incubated for a further 4 h following which 175 Al of media was transferred from the clear 96-multiwell plates to white 96-multiwell plates (BMG Labtechnologies, Offenburg, Germany) for fluorescence quantification (Ex 545, Em 590) on a BMG Polarstar microplate reader (BMG Labtechnologies). 2.5. Data analysis The analgesic response was calculated as the percentage of maximum possible effect (%MPE) using the following equation [17]: %MPE ¼
test latency control latency 100 60 control latency
The control latency (20F3 s (n=3)) was obtained from untreated controls. The in vitro mitogenesis data comprised average baseline proliferation from saline-treated animals (n=3) (unstimulated proliferation) subtracted from all data from all treatment groups; the data were then expressed as the percent of the saline-treated animals’ mitogen control. All data are expressed as meanFS.E.M (n=3 independent experiments). Statistical significance was assessed using either a one- or two-way analysis of variance with Bonferroni post hoc test. Significance was set at Pb0.05.
3. Results Significant increases in hot plate latencies were observed in animals that received 3 mg kg1 racemic methadone ( P=0.0012) and 1.5 mg kg1 (R)-()methadone ( P=0.0002) when compared to salinetreated controls (Fig. 1A). However, (S)-(+)-methadone caused no increase in hot plate latency compared to saline control ( P=0.79) and therefore caused significantly less changes in hot plate latencies than racemic methadone ( P=0.0008) or (R)-()-methadone ( P=0.0001) (Fig. 1A).
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proliferative response to the seven other concentrations of the opioid agonists applied to the cultures. This ex vivo response was independent of in vivo treatment (Fig. 2). There were no significant differences between ex vivo responses to (R)-()-, (S)-(+)or racemic methadone in animals treated with racemic methadone ( P=0.22) or saline ( P=0.07) in vivo accounting for 0.38% and 0.73% of the variability, respectively, as assessed using a two-way ANOVA with Bonferroni post hoc. However, this statistical analysis did detect significant differences between ex vivo treatments in (R)-()-methadone and (S)-(+)methadone-treated animals, although post hoc analysis showed that these differences were only singular events and showed no trends and therefore were discounted.
4. Discussion
Fig. 1. (A) Hot plate latencies 20 min after single injection of saline, (S)-(+)- (1.5 mg kg1), (R)-()- (1.5 mg.kg1) or racemic methadone (3 mg kg1). Data represent meanFS.E.M. One-way ANOVA with Bonferroni post hoc test **Pb0.01, ***Pb0.001 vs. (S)-(+)-methadone; ##Pb0.01 vs. saline. No significant difference between (R)-()-methadone and racemic methadone was observed ( P=0.12). (B) Effect of (S)-(+)- (1.5 mg kg1), (R)-()- (1.5 mg kg1) and racemic methadone (3 mg kg1) on ex vivo concanavalin A-induced proliferation of splenocytes (n=3 independent experiments). ### represents Pb0.001 compared to saline control, while *** signifies Pb0.001 compared to (R)-()-methadone and racemic methadone.
The proliferative response of splenocytes isolated from each of the treatment groups of animals were quantified. Basal splenocyte proliferation was significantly suppressed in (R)-()-methadone (1.5 mg kg1) and racemic methadone (3 mg kg1) treated animals compared to saline controls ( Pb0.001, Fig. 1B). However, unlike the hot plate latency data, (S)(+)-methadone (1.5 mg kg1) caused significantly ( Pb0.001) greater basal inhibition than (R)-()methadone, racemic methadone and saline-treated animals (Fig. 1B). Ex vivo methadone treatment resulted in significant ( PN0.001) nonstereoselective inhibition of proliferation at 100 AM. The single data point below each cluster in Fig. 2 is the 100 AM response, with the other data points in the clusters representing the
Racemic methadone and (R)-()-methadone caused significant increases in hot plate latencies and hence caused antinociceptive responses in animals 20 min after drug administration. In contrast, (S)-(+)-methadone was devoid of any such antinociceptive response as has been reported previously due to its lack of significant A opioid receptor binding affinity [1,2]. This response of methadone was of a classical A opioid receptor nature. In stark contrast to the hot plate data, methadone in vivo and ex vivo acted nonstereoselectively to inhibit ex vivo concanavalin-A-induced splenocyte proliferation. Moreover, in vivo (S)-(+)-methadone administration caused significantly greater inhibition than racemic or (R)-()-methadone. Therefore, despite antinociception being mediated via the neuronal classical A opioid receptor, the immunomodulatory response appears not to be opioid related since (S)-(+)-methadone had no antinociceptive effect, but caused substantial inhibition of splenocyte proliferation. This result was unexpected since the magnitude of the response caused by the racemic dose (3 mg kg1) was less than the sum of the responses of each enantiomer, indicating a negative bsynergisticQ interaction between the two enantiomers. Therefore, it appears that (R)-()-methadone may dampen or eliminates the inhibitory potential of (S)-(+)-methadone.
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Fig. 2. Comparison of the ex vivo proliferative response following ex vivo exposure to eight concentrations of (R)-()-methadone (n), (S)-(+)methadone (E) and racemic methadone (.) (the lowest point in each cluster is the proliferative response 100 AM) in splenocytes from animals treated in vivo with saline (A), racemic methadone (B), (R)-()-methadone (C) and (S)-(+)-methadone (D) (n=3 independent experiments).
These responses are likely to be via bindirectQ mechanisms, such as activation of the HPA axis or autonomic nervous system, since the concentration of methadone achieved in vivo by the doses administered would be in the range where no in vitro immunomodulation occurred. Therefore, this response was mediated via nonclassical opioid receptors expressed at some higher centre, not on immune cells. Methadone is not only an opioid receptor agonist as it also possesses considerable antagonistic properties at NMDA receptors, equivalent to that of dextromethorphan [18]. Therefore, (R)-()-methadone or racemic methadone administration would result in antagonism of NMDA and agonism of opioid receptors, while (S)-(+)-methadone administration alone would cause little or no opioid stimulation but with exclusive NMDA antagonism. Therefore, the mechanism for the difference in the responses between each enantiomer and racemic methadone could be explained by the stereoselective agonism and nonstereoselective antagonism of multiple independent receptors. Since methadone is prescribed and administered in a racemic form, the influence of administering (S)-(+)-methadone alone will not be observed in a human population. However, due to the
fragility of the immunological status of methadone’s target population, this immunosuppressive nature of (S)-(+)-methadone must be considered. Further research is required to elucidate the mechanisms that mediate this response as it has potential significant clinical relevance for the use of methadone in cancer patients and HIV-positive or at-risk injecting drug users.
Acknowledgements A University of Adelaide Small Research Grant 2001 funded this study. Mark Hutchinson is the recipient of an Australian Postgraduate Award.
References [1] Kristensen K, Christensen CB, Christrup LL. The mu1, mu2, delta, kappa opioid receptor binding profiles of methadone stereoisomers and morphine. Life Sci 1995;56(2):PL45 – 50. [2] Codd EE, Shank RP, Schupsky JJ, Raffa RB. Serotonin and norepinephrine uptake inhibiting activity of centrally acting analgesics: structural determinants and role in antinociception. J Pharmacol Exp Ther 1995;274(3):1263 – 70.
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[3] Farrell M, Ward J, Mattick R, Hall W, Stimson GV, et al. Methadone maintenance treatment in opiate dependence: a review. BMJ 1994;309(6960):997 – 1001. [4] Ayonrinde OT, Bridge DT. The rediscovery of methadone for cancer pain management. Med J Aust 2000;173(10):536 – 40. [5] Sibinga NES, Goldstein A. Opioid peptides and opioid receptors in cells of the immune system. Annu Rev Immunol 1988;6:219 – 49. [6] Hutchinson MR, Somogyi AA. Opioid immunomodulation is not related to mu receptor binding affinity. Drug Alcohol Depend 2002;66(S1):S83. [7] Hutchinson MR, Somogyi AA. Relationship between 4,5epoxymorphinan structure and in vitro modulation of cell proliferation. Eur J Pharmacol 2004;494(2–3):251 – 62. [8] Fecho K, Maslonek KA, Dykstra LA, Lysle DT. Evidence for sympathetic and adrenal involvement in the immunomodulatory effects of acute morphine treatment in rats. J Pharmacol Exp Ther 1996;277(2):633 – 45. [9] Carrigan KA, Lysle DT. Morphine-6 beta-glucuronide induces potent immunomodulation. Int Immunopharmacol 2001;1(5): 821 – 31. [10] Ochshorn M, Novick DM, Kreek MJ. In vitro studies of the effect of methadone on natural killer cell activity. Isr J Med Sci 1990;26(8):421 – 5. [11] Singh VK. Suppressive effects of methadone on human blood lymphocytes. Immunol Lett 1980;2:177.
[12] Thomas PT, House RV, Bhargava HN. Direct cellular immunomodulation produced by diacetylmorphine (heroin) or methadone. Gen Pharmacol 1995;26(1):123 – 30. [13] LeVier DG, Brown RD, Musgrove DL, Butterworth LF, McCay JA, et al. The effect of methadone on the immune status of B6C3F1 mice. Fundam Appl Toxicol 1995;24(2): 275 – 84. [14] de Waal EJ, Van Der Laan JW, Van Loveren H. Effects of prolonged exposure to morphine and methadone on in vivo parameters of immune function in rats. Toxicology 1998; 129(2–3):201 – 10. [15] Pacifici R, Patrini G, Venier I, Parolaro D, Zuccaro P, Gori E. Effect of morphine and methadone acute treatment on immunological activity in mice: pharmacokinetic and pharmacodynamic correlates. J Pharmacol Exp Ther 1994;269(3): 1112 – 6. [16] Hutchinson MR, Somogyi AA. Diacetylmorphine degradation to 6-monoacetylmorphine and morphine in cell culture: implications for in vitro studies. Eur J Pharmacol 2002;453: 27 – 32. [17] Carmody J. Avoiding fallacies in nociceptive measurements. Pain 1995;63(1):136. [18] Ebert B, Andersen S, Krogsgaard-Larsen P. Ketobemidone, methadone and pethidine are non-competitive N-methyl-daspartate (NMDA) antagonists in the rat cortex and spinal cord. Neurosci Lett 1995;187(3):165 – 8.
(S)-(+)-methadone is more immunosuppressive than the potent analgesic (R)-()-methadone Mark R. Hutchinson*, Andrew A. Somogyi Department of Clinical and Experimental Pharmacology, University of Adelaide, Level 5, Medical School North, Frome Road, 5005, Adelaide, Australia Received 29 June 2004; received in revised form 8 July 2004; accepted 9 July 2004
Abstract Methadone is a widely used synthetic opioid which is administered as a racemic mixture of (R)-()- and (S)-(+)enantiomers, with only (R)-()-methadone possessing A opioid receptor agonist activity. Methadone inhibits numerous immune functions in vitro at concentrations above 10 AM in a nonstereoselective and naloxone-insensitive fashion, suggesting the presence of nonclassical opioid receptors on immune cells. No in vivo data on the effects of methadone’s enantiomers on immune function are available. Therefore, the stereoselectivity of methadone’s analgesia (hot plate latency) in vivo and immune suppression ex vivo (splenocyte proliferation) was investigated in groups of Balb/c mice. Significant analgesia was observed in animals that received racemic methadone ( P=0.0012, 52% MPE) and (R)-()-methadone ( P=0.0002, 70% MPE) when compared to saline-treated controls, while (S)-(+)-methadone was devoid of any such effect (4% MPE). In vivo (R)-()- and racemic methadone caused significant inhibition ( Pb0.001, greater than 70%) of basal proliferation compared to saline control. In stark contrast to analgesia, in vivo (S)-(+)-methadone caused significantly greater inhibition of basal proliferation ( Pb0.001, 130%) than (R)-()- and racemic methadone. The immune suppression caused by methadone is not purely a classical opioid response but involves nonclassical opioid receptors located at the central level, which have yet to be characterised. Moreover, the dose at which immune suppression occurred could be achieved clinically. D 2004 Elsevier B.V. All rights reserved. Keywords: Methadone; In vivo; Analgesia; Immune system; Stereoselectivity
1. Introduction
* Corresponding author. Tel.: +61- 8- 83035571; fax: +61- 882240685. E-mail address: [email protected] (M.R. Hutchinson). 1567-5769/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2004.07.011
Methadone is a widely used synthetic 3,3-diphenylpropylamine opioid which primarily acts at the A opioid receptor [1,2]. Its most common use is in substitution therapy for opioid dependence [3], but is also being increasingly used in the management of
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chronic pain [4]. Methadone is administered as a racemic mixture of equal amounts of (R)-()- and (S)(+)-enantiomers. Only (R)-()-methadone possesses significant A opioid receptor agonist activity with 10– 20-fold higher binding affinity than the (S)-(+)enantiomer [1,2]. It is this stereoselectivity which is characteristic of an opioid response [5]. Opioids can modulate the immune system at a cellular level in vitro [6,7] and indirectly in vivo [8,9]. Considerable debate surrounds the identity and bclassicalQ opioid nature (stereoselectivity, agonist sensitivity and rank order of potency of effect) of the receptor(s) that are responsible for these responses, and this has been recently highlighted in vitro [6,7]. Several factors must be consistent for a response to be deemed classically opioid, including ligand stereoselectivity, antagonist sensitivity, ion sensitivity and toxin sensitivity [5]. Previous studies have shown nonstereoselectivity and naloxone insensitivity for methadone’s in vitro immunomodulatory behaviour using various in vitro immunological assays and immune cell sources [6,10–12]. However, these responses have only occurred at very high ligand concentrations (10–100 AM) above the range of concentrations achieved clinically and so have been dismissed as physiologically insignificant [10]. In animal models, methadone has also been found to be less inhibitory than morphine in several immune functional assays [13–15]; however, the stereoselectivity of the effect of methadone on these responses has not been investigated. Therefore, the aim of this study was to determine if in vivo administration of methadone acts stereoselectively to inhibit concanavalin A-induced splenocyte proliferation ex vivo. In contrast to many previously applied models, a rapid nontoxic, sensitive proliferation detection method was used, combined with incubation conditions optimised for the detection of altered immune response by in vitro exposure to opioids.
2. Materials and methods 2.1. Animals Ethics approval to conduct the studies was obtained from the University of Adelaide Animal Ethics Committee. Male Balb/c mice 6–8 weeks old
(22F0.5 g) were purchased from Central Animal Supplies (Waite Campus, University of Adelaide, Adelaide, Australia) and used as donors of splenocytes for use in cell culture. Animals were provided with standard rodent feed and water ad libitum. Animals were housed in a standard 12-h light–dark cycle (starting at 7 AM) under constant room temperature of 22F2 8C. The observer was blinded to all treatments. 2.2. Chemicals RPMI 1640 with HEPES modification and lglutamine were purchased from Invitrogen (Mulgrave, Vic, Australia). Penicillin–streptomycin solution (10,000 U penicillin, 10 mg streptomycin/ml) and concanavalin A were purchased from Sigma (St. Louis, MO, USA). Racemic methadone hydrochloride was obtained from McFarlane Smith (Edinburgh, UK), while (R)-()- and (S)-(+)-methadone bases (less than 1% contamination by the opposite enantiomer) were obtained from the National Institute on Drug Abuse (Rockville, MD, USA). Foetal calf serum was obtained from Trace Scientific (Melbourne, Vic, Australia). All other reagents and chemicals were obtained from commercial sources and were of analytical grade quality. Compounds that were not supplied as a soluble salt were dissolved in an equimolar solution of hydrochloric acid (0.1 M), while all other compounds were dissolved in Milli Q water. The vehicle used for all in vivo drug delivery was normal saline (0.9% NaCl), and all injections were administered intraperitoneally (i.p.) in a volume of 10 ml kg1. All solutions were stored at 4 8C until used. 2.3. In vivo treatment Mice (n=3 for each treatment) received a single i.p. injection of (R)-()- (1.5 mg kg1), (S)-(+)- (1.5 mg kg1), racemic methadone (3 mg kg1) or saline. These doses were based on the results of an initial dose finding pilot study using racemic methadone (data not shown) as this dose (racemic methadone, 3 mg kg1) achieved a quantifiable analgesic response, but did not completely abolish the concanavalin A proliferative response by isolated splenocytes ex vivo. The analgesic responses to these treatments were then
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tested 20 min later by placing each animal once on a 50 8C hot plate and recording the time taken (seconds) until any jumping, paw shaking or paw licking was observed. Animals that did not respond in this fashion after 60 s were removed from the plate. 2.4. Lymphocyte preparation The animals that underwent the dosing regimen and antinociceptive testing described above were sacrificed by cervical dislocation 120 min after drug administration followed by prompt removal of the spleen. The method used to prepare splenocytes for culture is based on that used previously [6,7,16]. Briefly, the spleen was prepared as a single cell suspension by massaging and washing through a nylon mesh into a 15-ml tube with 10 ml of RPMI 1640 (HEPES modification, 0.3 mg ml1 l-glutamine, 5 ml penicillin–streptomycin solution l1). The cells were centrifuged at 4 8C for 5 min at 1000 rpm, the supernatant was discarded and the cells were resuspended in 1 ml of media followed by the addition of 10 ml of lysis buffer [ice-cold 1 ml of 20.56 g l1 tris base (pH 7.65), 9 ml 0.83% NH4Cl in H2O mix just prior to addition to cells; greater than 95% lymphocyte viability following lysis buffer treatment]. The suspension was placed on ice for 4 min, centrifuged (4 min at 1000 rpm) and supernatant was discarded. The cell suspensions were resuspended in 10 ml of media followed by centrifugation (4 min at 1000 rpm), removal of supernatant and resuspension in 5 ml of enriched RPMI 1640 (RPMI 1640 enriched with foetal calf serum). The number of viable lymphocytes in the suspension was counted using trypan blue and a haemocytometer. Cells were then diluted in enriched media to 1106 cells ml1 and 100 Al of this suspension was added to each well of the 96multiwell plates (Nunc, Roskilde, Denmark) and made up to 200 Al with 50 Al of concanavalin A (10 Ag ml1, final concentration of 2.5 Ag ml1 which produces submaximal proliferation) and/or 50 Al of (R)-()-, (S)-(+)- or racemic methadone (n=6; 0 AM control, 0.0004, 0.004, 0.04, 0.4, 4, 40 and 400 AM; the concentrations in the final incubation were 25% of those added). Unstimulated mitogen negative control wells were also prepared, using 50 Al of media instead of concanavalin A. The splenocyte proliferation assays of splenocytes from each animal were per-
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formed in separate plates (n=3 independent experiments). The plates were incubated at 37 8C, 5% CO2 in a humidified incubator (Thermoline, Sydney, Australia) for 24 h following which 25 Al of a diluted AlamarBlue solution (5 Al AlamarBlue, 20 Al cell medium) was added to each well. The plates were incubated for a further 4 h following which 175 Al of media was transferred from the clear 96-multiwell plates to white 96-multiwell plates (BMG Labtechnologies, Offenburg, Germany) for fluorescence quantification (Ex 545, Em 590) on a BMG Polarstar microplate reader (BMG Labtechnologies). 2.5. Data analysis The analgesic response was calculated as the percentage of maximum possible effect (%MPE) using the following equation [17]: %MPE ¼
test latency control latency 100 60 control latency
The control latency (20F3 s (n=3)) was obtained from untreated controls. The in vitro mitogenesis data comprised average baseline proliferation from saline-treated animals (n=3) (unstimulated proliferation) subtracted from all data from all treatment groups; the data were then expressed as the percent of the saline-treated animals’ mitogen control. All data are expressed as meanFS.E.M (n=3 independent experiments). Statistical significance was assessed using either a one- or two-way analysis of variance with Bonferroni post hoc test. Significance was set at Pb0.05.
3. Results Significant increases in hot plate latencies were observed in animals that received 3 mg kg1 racemic methadone ( P=0.0012) and 1.5 mg kg1 (R)-()methadone ( P=0.0002) when compared to salinetreated controls (Fig. 1A). However, (S)-(+)-methadone caused no increase in hot plate latency compared to saline control ( P=0.79) and therefore caused significantly less changes in hot plate latencies than racemic methadone ( P=0.0008) or (R)-()-methadone ( P=0.0001) (Fig. 1A).
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proliferative response to the seven other concentrations of the opioid agonists applied to the cultures. This ex vivo response was independent of in vivo treatment (Fig. 2). There were no significant differences between ex vivo responses to (R)-()-, (S)-(+)or racemic methadone in animals treated with racemic methadone ( P=0.22) or saline ( P=0.07) in vivo accounting for 0.38% and 0.73% of the variability, respectively, as assessed using a two-way ANOVA with Bonferroni post hoc. However, this statistical analysis did detect significant differences between ex vivo treatments in (R)-()-methadone and (S)-(+)methadone-treated animals, although post hoc analysis showed that these differences were only singular events and showed no trends and therefore were discounted.
4. Discussion
Fig. 1. (A) Hot plate latencies 20 min after single injection of saline, (S)-(+)- (1.5 mg kg1), (R)-()- (1.5 mg.kg1) or racemic methadone (3 mg kg1). Data represent meanFS.E.M. One-way ANOVA with Bonferroni post hoc test **Pb0.01, ***Pb0.001 vs. (S)-(+)-methadone; ##Pb0.01 vs. saline. No significant difference between (R)-()-methadone and racemic methadone was observed ( P=0.12). (B) Effect of (S)-(+)- (1.5 mg kg1), (R)-()- (1.5 mg kg1) and racemic methadone (3 mg kg1) on ex vivo concanavalin A-induced proliferation of splenocytes (n=3 independent experiments). ### represents Pb0.001 compared to saline control, while *** signifies Pb0.001 compared to (R)-()-methadone and racemic methadone.
The proliferative response of splenocytes isolated from each of the treatment groups of animals were quantified. Basal splenocyte proliferation was significantly suppressed in (R)-()-methadone (1.5 mg kg1) and racemic methadone (3 mg kg1) treated animals compared to saline controls ( Pb0.001, Fig. 1B). However, unlike the hot plate latency data, (S)(+)-methadone (1.5 mg kg1) caused significantly ( Pb0.001) greater basal inhibition than (R)-()methadone, racemic methadone and saline-treated animals (Fig. 1B). Ex vivo methadone treatment resulted in significant ( PN0.001) nonstereoselective inhibition of proliferation at 100 AM. The single data point below each cluster in Fig. 2 is the 100 AM response, with the other data points in the clusters representing the
Racemic methadone and (R)-()-methadone caused significant increases in hot plate latencies and hence caused antinociceptive responses in animals 20 min after drug administration. In contrast, (S)-(+)-methadone was devoid of any such antinociceptive response as has been reported previously due to its lack of significant A opioid receptor binding affinity [1,2]. This response of methadone was of a classical A opioid receptor nature. In stark contrast to the hot plate data, methadone in vivo and ex vivo acted nonstereoselectively to inhibit ex vivo concanavalin-A-induced splenocyte proliferation. Moreover, in vivo (S)-(+)-methadone administration caused significantly greater inhibition than racemic or (R)-()-methadone. Therefore, despite antinociception being mediated via the neuronal classical A opioid receptor, the immunomodulatory response appears not to be opioid related since (S)-(+)-methadone had no antinociceptive effect, but caused substantial inhibition of splenocyte proliferation. This result was unexpected since the magnitude of the response caused by the racemic dose (3 mg kg1) was less than the sum of the responses of each enantiomer, indicating a negative bsynergisticQ interaction between the two enantiomers. Therefore, it appears that (R)-()-methadone may dampen or eliminates the inhibitory potential of (S)-(+)-methadone.
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Fig. 2. Comparison of the ex vivo proliferative response following ex vivo exposure to eight concentrations of (R)-()-methadone (n), (S)-(+)methadone (E) and racemic methadone (.) (the lowest point in each cluster is the proliferative response 100 AM) in splenocytes from animals treated in vivo with saline (A), racemic methadone (B), (R)-()-methadone (C) and (S)-(+)-methadone (D) (n=3 independent experiments).
These responses are likely to be via bindirectQ mechanisms, such as activation of the HPA axis or autonomic nervous system, since the concentration of methadone achieved in vivo by the doses administered would be in the range where no in vitro immunomodulation occurred. Therefore, this response was mediated via nonclassical opioid receptors expressed at some higher centre, not on immune cells. Methadone is not only an opioid receptor agonist as it also possesses considerable antagonistic properties at NMDA receptors, equivalent to that of dextromethorphan [18]. Therefore, (R)-()-methadone or racemic methadone administration would result in antagonism of NMDA and agonism of opioid receptors, while (S)-(+)-methadone administration alone would cause little or no opioid stimulation but with exclusive NMDA antagonism. Therefore, the mechanism for the difference in the responses between each enantiomer and racemic methadone could be explained by the stereoselective agonism and nonstereoselective antagonism of multiple independent receptors. Since methadone is prescribed and administered in a racemic form, the influence of administering (S)-(+)-methadone alone will not be observed in a human population. However, due to the
fragility of the immunological status of methadone’s target population, this immunosuppressive nature of (S)-(+)-methadone must be considered. Further research is required to elucidate the mechanisms that mediate this response as it has potential significant clinical relevance for the use of methadone in cancer patients and HIV-positive or at-risk injecting drug users.
Acknowledgements A University of Adelaide Small Research Grant 2001 funded this study. Mark Hutchinson is the recipient of an Australian Postgraduate Award.
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