British Journal of Pharmacology (1997) 121, 834 ± 840 1997 Stockton Press All rights reserved 0007 ± 1188/97 $12.00 Antinociceptive and immunosuppressive eects of opiate drugs: a structure-related activity study 1Paola Sacerdote, Barbara Manfredi, Paolo Mantegazza & Alberto E. Panerai Department of Pharmacology, University of Milano, via Vanvitelli 32, 20129, Milano, Italy 1 Although it is well known that morphine induces signi®cant immunosuppression, the potential immunosuppressive activity of morphine derived drugs commonly used in the treatment of pain (codeine, hydromorphone, oxycodone) has never been evaluated. 2 We evaluated in the mouse the eect of the natural opiates (morphine and codeine) and synthetic derivatives (hydromorphone, oxycodone, nalorphine, naloxone and naltrexone) on antinociceptive thresholds and immune parameters (splenocyte proliferation, Natural Killer (NK) cell activity and interleukin-2 (IL-2) production). 3 Morphine displayed a potent immunosuppressive eect that was not dose-related to the antinociceptive eect, codeine possessed a weak antinociceptive eect and limited immunosuppressive activity; nalorphine, a m-antagonist and k-agonist, exerted a potent immunosuppressive eect, but had very weak antinociceptive activity. The pure k-antagonist nor-BNI antagonized the antinociceptive, but not the immunosuppresive eect of nalorphine. 4 Hydromorphone and oxycodone, potent antinociceptive drugs, were devoid of immunosuppressive eects. 5 The pure antagonists naloxone and naltrexone potentiated immune responses. 6 Our data indicate that the C6 carbonyl substitution, together with the presence of a C7-8 single bond potentiates the antinociceptive eect, but abolishes immunosuppression (hydromorphone and oxycodone). 7 The single substitution of an allyl on the piperidinic ring resulted in a molecule that antagonized the antinociceptive eect but maintained the immunosuppressive eect. 8 Molecules that carry modi®cations of C6, the C7-8 bond and C14, together with an allyl or caboxymethyl group on the piperidinic ring antagonized both the antinociceptive and the immunosuppressive eect of opiates and were themselves immunostimulants. Keywords: Morphine; codeine; oxycodone; hydromorphone; naloxone; naltrexone; nalorphine; immunosuppression; analgesia Introduction For many years chemists and pharmacologists have tried to drug, e.g. in the postoperative period, during treatment of obtain a molecule that had the same or better antinociceptive chronic pain, or in the intake for abuse (Starec et al., 1993). activity of morphine accompanied by less side eects. The The consequences of the immunosuppressive eect of the eort led to several new drugs, many of which were derived opiate could be an increased susceptibility to infections in from slight modi®cations of the natural opiates morphine and the postoperative period, a possible lack of defences in codeine (Reisine & Pasternak, 1996). These drugs include the cancer patients, and an increased susceptibility to HIV in- agonists hydromorphone and oxycodone, the oxidation pro- fection in drug abusers (Peterson et al., 1990). While these ducts of morphine and codeine, respectively. In these drugs, aspects of morphine pharmacology have been extensively the C6 hydroxyl group of the native opiate has been substituted studied in the last few years, very little or nothing is known with a carbonyl group, and a single bond introduced at C7-8, of the eects of the other morphine congeners on immunity. yielding an antinociceptive potency that exceeds that of ori- Therefore, we thought it worthwhile to compare the eects ginal compounds. Naloxone and naltrexone, that carry an allyl of the acute administration of morphine, codeine, hydro- or carboxymethyl substituent at the piperidinic ring of hy- morphone, oxycodone, nalorphine, naloxone and naltrexone droxymorphone and an hydroxyl group at C14, display a pure on immune responses. To this end, after acute administra- antagonistic eect. Finally, nalorphine, a morphine derivative tion of the drugs to mice, we evaluated analgesia (hot-plate), with an allyl substituent at the piperidinic, but that retains the concanavalin-A (Con-A)-induced proliferation of spleno- C6 hydroxyl group, and the C7-8 bond unchanged, displays a cytes, Natural Killer (NK) cell activity and interleukin-2 (IL- mixture of agonist (on the k receptor) and antagonist (on the m 2) production by splenocytes. receptor) properties. The eects of these derivatives have been well docu- mented as far as analgesia and respiratory depression are Methods concerned (Rundlett Beyer & Elliot, 1976). However, great interest has recently been shown in the immunosuppressive Animals eects induced by morphine (Bryant et al., 1988; Peterson et al., 1993; Carr et al., 1993; Carpenter et al., 1995; Fecho et Male Swiss mice (Charles River, Calco, Italy), 20 ± 25 g body al., 1996), since this could be relevant for the modulation of weight were used. Animals were housed at 22+28C, with a immune responses after both acute or chronic use of the light:dark cycle of 14 ± 10 h, food and water ad libitum. Each experimental group consisted of 8 animals. Dierent animals were used for the measurement of nociceptive thresholds and of immune parameters, in order to avoid the stressful eect of 1 Author for correspondence. handling on immune parameters. P. Sacerdote et al Structure related immune effects of opiates 835 Evaluation of the antinociceptive eect standardized by Pharmingen (San Diego, California U.S.A.). Brie¯y, the anti IL-2 capture monoclonal antibody (mAb) Nociceptive thresholds were measured by the hot plate (548C) was absorbed on a polystyrene 96 well plate and the IL-2 method, as previously described (Bianchi et al., 1992) and the present in the sample was bound to the antibody coated end point used was the licking of the hind paws. The results are wells. The biotinylated anti IL-2 detecting mAb was added expressed as percentage of the maximal possible eect (% to bind the IL-2 captured by the ®rst antibody. After MPE). MPE expresses the equation [(TL7BL)/ washing, avidin-peroxidase (Sigma) was added to the wells ML7BL)]6100, where BL is the mean basal latency (8.0 ± to detect the biotinylated detecting antibody and ®nally 2,2'- 10.0 s) measured before the ®rst treatment was applied; TL is azino-bis (3-ethylbenthiazoline-6-sulphonic acid) (ABTS, the test latency measured after treatments; ML is the maximal Sigma) substrate was added and a coloured product was latency accepted (30 s), chosen in order to avoid tissue damage formed in proportion to the amount of IL-2 present in the to the footpads. sample, which was measured at OD 405 nm. Collection of cells Drugs Spleen was removed by sterile procedures after cervical dis- The following opiate agonists and antagonists were used: location of animals, and cells were teased from the spleens by morphine hydrochloride, codeine phosphate, nalorphine hy- using 20-gauge sterile needles through an incision made in the drochloride, oxycodone hydrochloride, naloxone hydro- spleen cuticle (Manfredi et al., 1993). Cells obtained from chloride, naltrexone hydrochloride (S.A.L.A.R.S., Como, single spleens were checked for immune responses. Italy), hydromorphone hydrochloride (Dilaudid HP, Knoll Pharmaceuticals, Whippany, New Jersey), and the speci®c k Splenocyte proliferation receptor antagonist non-binaltorphimine hydrochloride (nor- BNI; RBI, U.S.A.). Morphine.HCl (m. wt.321.8), oxycodone. Microcultures of splenocytes were set up (46106 cells) in HCl (m. wt. 351.8), nalorphine.HCl (m. wt. 347.8), hydro- RPMI 1640, 10% FCS+Con-A (2.5 mgml71,5mgml71, morphone.HCl (m. wt. 321.7), naloxone.HCl (m. wt. 363.8) 10 mgml71). After 48 h incubation at 378C, 1.0 mCi [3H]-thy- and naltrexone.HCl (m. wt. 377.7) were administered at doses 71 71 midine (speci®c activity 2 Ci mmol , Amersham, U.K.) was of 2.5, 5, 10, 20 mg kg , s.c. Codeine.H2PO4 (m. wt. 397.4) added to all cultures. Eighteen hours later, cells were harvested at doses of 2.5, 5, 10, 20 and 100 mg kg71 s.c., and nor-BNI by an automated cell harvester (Skatron) and radioactivity was (m. wt. 737.8) administered at a dose of 10 mg kg71, s.c., two measured in a liquid scintillation counter (Packard, Downers hours before the administration of 10 mg kg71 nalorphine for Grove, IL, U.S.A.). Background values, i.e. thymidine in- the evaluation of immunosuppression and 20 mg kg71 of the corporation of resting cells, were subtracted from mitogen- opiate for antinociception. In the IL-2 experiments all drugs induced proliferation (Manfredi et al., 1993). were used at the highest dose. All drugs were dissolved in The three concentrations of Con A were selected to provide sterile distilled water and administered subcutaneously, sub-maximal as well as maximal stimulation of proliferation. 60 min before the animals were killed, for the evaluation of The maximal proliferative response occurred at 5 mgml71 immune parameters, or before the hot-plate test. The che- Con-A, and was selected for data analysis because it provided mical structure of the drugs used in the study is presented in the most accurate measure of the overall proliferative re- Table 1. sponses of the lymphocytes. However, similar results were obtained at all mitogen concentrations, including sub-maximal Statistical analysis concentrations. Statistical analysis for NK activity, cell proliferation and IL-2 Natural Killer cell activity production was performed by ANOVA, followed by Bonfer- roni test for multiple comparisons, while antinociceptive re-