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European Journal of Pharmacology

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Analgesia

Comparacin del efecto de analgsicos opioides y no opioides mediante el modelo de ---------- en ratones albinos. Mrquez Gonzlez Jimena, Mora Snchez Martha Viviana, Romero Vzquez Lucero, Solis Camacho Carlos, Soto Ramos Jorge Armando.Farmacologa especial, Departamento de bioqumica y farmacologa, Departamento de ciencias biolgicas, Facultad de Estudios Superiores Cuautitln, UNAM

Article i nfo

Article history:Received 10 November 2014Received in revised form 10 November 2014Accepted November 2014Available online November 2014

Keywords:

Abstract

2014 Elsevier B.V. All rights reserved.

1. Introduction

Opioids, in particular mu-opioid receptor agonists (e.g. morphine, hydrocodone, oxycodone), are considered to be the primary drugs for the treatment of moderate to severe acute, chronic and cancer pain. Despite their effectiveness they have several clinically signicant side- effects including, sedation (cognitive dysfunction), nausea, vomiting, respiratory depression, cardiovascular depression, constipation, hyperalgesia. In addition, mu-opioids have a limited spectrum of action, being effective for nociceptive pain (e.g. postoperative pain, low back pain) but having less efcacy for neuropathic pain states (e.g. radicular pain, tumor invasion in neural plexus) (Ballantine and Mao, 2003, review). Doses of mu-opioids used for neuropathic pain are typically signicantly higher than for nociceptive pain, thereby being associated with greater side-effects. The search for a long-acting opioid, in particular after neuroaxial (intrathecal, epidural) adminis- tration, with effectiveness in both nociceptive as well as neuropathic pain states and with a more favorable side-effect prole than a

Corresponding author. Department of Anesthesiology, Chandler Medical Center, College of Medicine, 800 Rose Street, N-202, Lexington, KY 40536-0293, USA. Tel.: +1859 257 4487; fax: +1 859 323 1924.E-mail address: epwala@uky.edu (E.P. Wala).

prototypical mu-opioid, morphine, continues to be an important priority in analgesic drug development.Morphine analogs substituted at the O-6 position, such as morphine-6--glucuronide and morphine-6-O-sulfate, have generat- ed interest as potentially more promising analgesics. Several studies had examined morphine-6--glucuronide, which is more potent and apparently less toxic than morphine (Smith and South, 2001; van Dorp et al., 2006, reviews). Less attention has been directed towards the study of morphine-6-O-sulfate, which also binds to mu-opioid receptors (Zuckerman et al., 1999); however, it is not a biotransfor- mation product of morphine (Donnerer et al., 1987; Nagano et al.,2000). Morphine-6-O-sulfate had a signicantly higher potency (approximately 30-fold) then morphine after central (intracerebro- ventricular, i.c.v.) administration in a rodent model of nociception (the tail-ick test) (Brown et al., 1985; Houdi et al., 1996; Mori et al.,1972; Zuckerman et al., 1999). In addition, it had reduced (2-fold) afnity at mu- but enhanced afnity (30-fold) at delta-opioid receptors compared to morphine in rat brain membranes (Oguri et al., 1987). This appears of importance as opioids active at delta- receptors have been shown to enhance analgesic activity while lacking typical morphine-like side-effects, including respiratory depression and inhibition of gastrointestinal transit. Furthermore, delta-receptor activity has been associated with analgesia in

0014-2999/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.08.034J.R. Holtman Jr. et al. / European Journal of Pharmacology 648 (2010) 879492

92J.R. Holtman Jr. et al. / European Journal of Pharmacology 648 (2010) 8794

Facultad de Estudios Superiores Cuautitln. UNAM

neuropathic and inammatory pain (Petrillo et al., 2003). The above promising ndings lead us to further characterize morphine-6-O- sulfate with regard to its analgesic effect, in particular in neuropathic pain, and its toxicity.The purpose of the present study in the rat was three-fold: 1) to determine if morphine-6-O-sulfate had antinociceptive activity after acute neuroaxial, parenteral and oral routes of administration, as well as to determine if tolerance develops with repeated doses in the tail- ick test; 2) to determine if morphine-6-O-sulfate had effectiveness in chronic pain; and specically, if the drug was able to attenuate enhanced pain sensitivity (hyperalgesia, allodynia) in a chronic constriction nerve injury model of neuropathy and to inhibit inching behavior in a formalin model of inammatory pain; 3) to determine the morphine-6-O-sulfate side-effect prole by observing gastroin- testinal motility (charcoal transit), coordination (rotarod) and locomotor activity. Findings on morphine-6-O-sulfate were compared to results using morphine as a positive control in some experiments.

2. Materiales y mtodos

2.1.Animales

Ratones albinos machos (10), aproximadamente un mes de edad, con un peso promedio de 50 gr (Ciudad Universitaria), fueron puestos en una caja grande transparente con viruta, los animales estuvieron a una temperatura ambiente con acceso libre a comida (Nutricubos) y agua. Los ratones estuvieron bajo stas condiciones una semana. Next, they were familiarized with the experimental environment/apparatus (three times) before the experiments were begun. Al terminar el experimento se les aplic la eutanasia al introducirlos en una cmara de anestesia saturada de cloroformo.

2.2. Frmacos y material

cido acetilsaliclico (500mg) y Tramadol (300mg) fueron adquiridos en Farmacias del Ahorro. Los frmacos fueron disueltos y aforados en agua destilada y fueron administrados va oral DOSIS.cido actico fue adquirido en la facultad y fue disuelto en agua destilada para tener una solucin al 1%.cido pcrico neutralizado

2.3. Comparacin de efectos analgsicos

El efecto analgsico fue comparado al administrar a ratones albinos un frmaco opioide (Tramadol) y un frmaco no opioide (c. Acetilsaliclico). Para sto, primero se marcaron los animales con cido pcrico neutralizado, enseguida se pesaron y despus se repartieron los ratones en tres lotes; Lote control, Lote no opioide y Lote opioide; sta distribucin se realiz mediante el mtodo de curva culebra japonesa.Para evaluar ste efecto al lote control se administr cido actico va intraperitoneal.Para el lote no opioide se administr cido acetilsaliclico va oral y treinta minutos despus de administrar el analgsico se administr cido actico va intraperitoneal.Para el lote opioide se administr tramadol va oral y treinta minutos despus de administrar dicho analgsico se administr cido actico va intraperitoneal.En los tres lotes se evaluaron

Four groups of rats were used to characterize the antinociceptive properties of morphine-6-O-sulfate in the tail ick test. 1) Rats were treated with morphine-6-O-sulfate (0.25, 0.5, 1, 1.5 mg/kg) adminis- tered by the i.p. route (randomized doses, weekly intervals). 2) Rats were treated with morphine-6-O-sulfate (1, 5, 10, 15, 20 mg/kg) administered orally with help of gavage feeding needle after overnight fasting (randomized doses; weekly intervals). 3) Rats were subjected to chronic catheterization of the spinal subarachnoid space performed according to Yaksh and Rudy (1976) with modication. Briey, a21 cm long P-10 polyethylene tubing (10 l) which extended 8.5 cm beyond an incision in the atlanto-occipital membrane was secured to the scull with acrylic cement. The catheter tip rested in the vicinity of T-12 at the rostral face of the lumbar cord enlargement. One week after surgery, rats were administered with morphine-6-O-sulfate (0.125,0.25, 0.5 g) by the i.t. route. 4) Rats were treated with repeated doses of morphine-6-O-sulfate (1.5 mg/kg) administered twice daily (ap- proximately at 0900 and 1700) by the i.p. route for 25 days. Responsiveness was determined 30, 45 and 60 min after injection. Each rat was considered tolerant when the maximum possible effect, % MPE, was equal or lower than 5%, as assessed by TFL (Holtman et al.,2004). In addition, morphine was administered acutely by i.t. (3, 10,30 g), i.p. (2, 3, 5, 7 mg/kg) and p.o. (10, 15, 20 mg/kg) routes, as well as chronically (7 mg/kg i.p., twice a day for 12 days) for comparison (the tail ick test).

2.4. Neuropathic pain (chronic constriction nerve injury)

2.4.1. SurgeryA well-established chronic constriction nerve injury (CCI) rodent model of neuropathic pain (Bennett and Xie, 1988) was utilized to determine the antihyperalgesic and antiallodynic effects of morphine-6-O-sulfate. Briey, rats were subjected to a sciatic nerve ligation (left paw) and sham surgery (right paw) as follows. Proximal to the sciatic trifurcation, the nerve (approximately 7 mm) was freed from adhering tissue and four loose ligatures were tied around the nerve (1 mm apart) using 4.0 chromic catgut, barely constricting the diameter of the nerve. In sham surgery, the nerve was exposed but was not ligated. The incision was closed in layers with silk thread 3.0. This procedure results in enhanced sensitivity to both noxious stimuli (hyperalgesia) and normally non-noxious stimuli (allodynia) that develops typically within 7 days and last approximately 28 days after surgery in a CCI paw. There are no signicant changes in the sham- operated paw.

2.4.2. Mechanical hyperalgesiaThe presence of mechanical hyperalgesia was determined by the paw pressure test (32 g/s; cut-off 300 g) (Randall and Selitto, 1957) utilizing the Basile Analgesimeter (UGO Basile, Italy). Vocalization was used as the end point (vocalization threshold, VT, g). Experiments were carried out prior to (pre-surgery baseline) and on post-surgery days 7, 9, 11 and 14 when abnormal pain behavior was at a stable maximum. Typically, VT values were equal to 200 g and 100 g prior to and after CCI, respectively, in control rats. Each rat received four doses of morphine-6-O-sulfate (0.1, 0.5, 0.75, 1 mg/kg i.p.; randomized doses). Responsiveness to mechanical noxious stimuli (VT) was assessed prior to (baseline) and at several time points (15180 min) after injection in both CCI and sham paws. In addition, morphine (3, 5,7 mg/kg i.p.) was tested for comparison in CCI rats.

2.4.3. Tactile allodyniaResponsiveness to normally non-noxious stimuli (allodynia) was assessed using von Frey laments (Stoelting, Wood Dale, IL) with incremental stiffness (4, 8, 15 g) (Flatters and Bennett, 2004). Briey, each von Frey hair was applied (and held for 5 s) to the plantar surface of each hind paw (10 times/paw) prior to (day 0) and on post-surgery days 9, 11, 14 and 16 when enhanced pain sensitivity was at a stableFacultad de Estudios Superiores Cuautitln. UNAM

maximum. A positive response was regarded as the sharp withdrawal of the paw (paw withdrawal frequency, PWF, expressed as an overall percentage response). Typically, PWF values were equal to 20% versus90% (4 g), 50% versus 90% (8 g) and 80% versus 100% (15 g) prior to and after CCI, respectively, in control (saline) rats. Thus, enhanced responsiveness to 4 g lament (a normally innocuous stimulus) can be described as tactile allodynia, responses to 15 g lament are probably best described as hyperalgesia (heightened pain response from normally noxious punctuate stimulus) and the responses to 8 g lament are intermediate. Rats were treated with morphine-6-O- sulfate (0.1, 0.5, 0.75, 1 mg/kg i.p.; randomized doses) and tested with von Frey laments prior to (baseline) and 60 min after injection. Effect of morphine (1, 2, 4 mg/kg i.p.) also was assessed in this paradigm.

2.5. Inammatory pain (the formalin test)

The ability of morphine-6-O-sulfate to block formalin-induced inching behavior was assessed using the well-established formalin test (Wheeler-Aceto and Cowan, 1991). Briey, 50 l of formalin (5%) was injected subcutaneously (s.c.) into the dorsal surface of the left hind paw. This procedure typically produces a biphasic behavioral response consisting of inching, lifting and licking in control rats. The rst phase (010 min) is thought to result from direct stimulation of nociceptors (nociceptive pain) whereas the second phase (2060 min) involves central sensitization. Herein, rats were injectedwith morphine-6-O-sulfate (0.01, 0.1, 0.5, 1 mg/kg i.p.) 15 min prior to formalin injection (s.c.). Incidences of formalin-induced inching were counted continuously in 5 min intervals for 60 min. Each rat received only one treatment. Morphine (1, 2.5, 5 mg/kg i.p.) also was characterized in the formalin test.

2.6. Gastrointestinal motility (the charcoal test)

Inhibition of intestinal propulsion was tested using the charcoal meal test in the rat (Megens et al., 1998). After the overnight fasting, rats were given 2 ml of oral charcoal solution (5% charcoal and 10% gum Arabic) 30 min after i.p. administration of morphine-6-O-sulfate (0.5, 1.5, 3, 6, 15 mg/kg) or morphine (2.5, 5, 10 mg/kg). Thirty minutes later rats were euthanized (CO2 inhalation), stomach and intestine were removed, and the distance traveled by the charcoal was recorded as a percentage of the total length of the small intestine. Control rats were treated with saline.

2.7. Motor coordination (the rotarod test)

The motor effect of morphine-6-O-sulfate was determined using the rotarod performance test (Watzman et al., 1964). Intact rats were trained to run on the Rat Rota Rod (Ugo Basile, Comeno, Italy) at a constant speed (10 rpm) for 180 s at two consequent days. Thereafter, they were treated with morphine-6-O-sulfate (1.5, 3, 6, 15 mg/kg i.p.; randomized doses; weekly intervals) and latency to fall was assessed prior to and at several time points (15, 30, 45, 60, 90 and 120 min) after injection (cut-off = 180 s).

2.8. Locomotor activity

Locomotor activity was determined using the Opto-Varimex infrared photocell-based activity monitor (Columbus Instrument, Columbus, OH). Horizontal (ambulation) and vertical (rearing) activities were recorded during 5 min sessions prior to and after (30, 60, 90 and 120 min) administration of morphine-6-O-sulfate (1.5,3, 6, 15 mg/kg i.p.; randomized doses, weekly intervals) in intact rats.

2.9. Data and statistics

Data were normalized for baseline values for each rat at each time point. The areas under the curves (AUC0 t) were calculated for baseline-normalized data (the trapezoidal rule). Percent maximum possible effect (%MPE) was calculated as follows: tail-ick test: [(TFL baseline)/(10 baseline)] * 100; CCI (paw pressure test): [(PWT baseline postCCI)/(baseline preCCI baseline postCCI)] * 100; CCI (von Frey test): (baseline PWF/baseline) * 100; formalin test: (nchessaline inchesdrug/inchessaline) * 100; rotarod test: (latency to fall/180) * 100; locomotor activity (AUCsaline AUCdrug/AUCsaline) * 100. The percent of inhibition of gastrointestinal transit (%IGT) was calculated as (GTsaline GTdrug/GTsaline) * 100, where GTsaline and GTdrug is the percent of gastrointestinal transit in the saline- and drug-treated rats, respectively. Doseresponse curves were generated for %MPE (at peak time) or %IGT as a function of a log dose. The doseresponse curves were assessed by regression analysis. A dose of drug causing 50% MPE (ED50) and 95% condence limits (95% CL) were calculated using the method of Tallarida and Murray (1987). The data were analyzed using one-way analysis of variance (ANOVA) and post-hoc Student Newman Keuls (SNK) test (locomotor, charcoal), KruskalWallis ANOVA followed by Dunn's test (rotarod) and two-way RM ANOVA (tolerance). Statistical analysis was performed using SigmaPlot for Windows version 11.0 (Systat Software, Inc.). The level of signicance was P 0.05. All data are mean S.E.M. (n rats).

3. Results

3.1. Facultad de Estudios Superiores Cuautitln. UNAM

Fig. 1.

Fig. 2. Facultad de Estudios Superiores Cuautitln. UNAM

MPE = 99.7 0.2%).

4.Discusin de resultados

In an effort to identify an opioid with a better therapeutic index compared to the classical mu-opioid agonist, morphine, a relatively little-known morphine derivative, morphine-6-O-sulfate, was char- acterized with regard to analgesia and side-effect prole using a range of well-established rodent models. The present data demonstrated the potent analgesic activities of morphine-6-O-sulfate against acute nociception, neuropathic pain (hyperalgesia, allodynia) and inam- matory pain; a good separation based on dose (N 10-fold) between analgesia and side-effects tested (motor, gastrointestinal); and apparently a more favorable gastrointestinal effect/analgesic effect ratio for morphine-6-O-sulfate compared to morphine.The initial objective was to assess the analgesic effectiveness of morphine-6-O-sulfate with regard to dose, route and mode of administration utilizing several rodent models of pain. Our data in rats after neuroaxial (i.t.), systemic (i.p.) and oral routes of administration conrmed earlier reports in mice after central injection (i.c.v.) (Brown et al., 1985; Houdi et al., 1996; Mori et al., 1972; Zuckerman et al., 1999) that morphine-O-sulfate produced a dose- related antinociception in the tail-ick test. A novel nding with the present study was that morphine-6-O-sulfate alleviated pain sensiti- zation occurring as the result of an injury to nerve or tissue. Specically, it attenuated, in a dose-related manner, an enhanced sensitivity to a noxious (hyperalgesia) and previously non-noxious (allodynia) stimulus in a CCI model of peripheral neuropathy, as well as inhibited both the early and the late nociceptive responses in a formalin model of inammatory pain. Of note, the high analgesic potency of morphine-6-O-sulfate was observed irrespective of the rat model of pain used, i.e. nociception (the tail ick test) and neuropathy (CCI). However, the later appeared to be related to the modality of mechanical hyperalgesia tested (non-punctuate N punctuate). Opioids (e.g. morphine) seem differently depress responses triggered by different bers (Le Bars et al., 2001).The next objective was to characterize the side-effect prole of morphine-6-O-sulfate. This was done by assessing motor function (rotarod), gastrointestinal transit (charcoal) and locomotor activity in intact rats. A consistent nding with this study was that morphine-6- O-sulfate produced motor incoordination, hypolocomotion and inhibition of gastrointestinal motility at doses signicantly higher (10- to 100-fold) than those responsible for analgesia in rodent models of thermal nociception, nerve injury-evoked peripheral neuropathy and inammatory pain following formalin injection. Whether this is also the case with respiratory depression (severe side-effect of opioids) warrants further study. Previous data with other clinically used mu-opioids (morphine, codeine, oxycodone,

hydrocodone) showed that the side-effects of such drugs (motor, gastrointestinal, respiratory) occurred at doses similar to the analgesic effects in rodents (Meert and Vermeirsh, 2005).The nal objective was to compare the effects of morphine-6-O- sulfate with those of morphine (a prototypical mu-opioid), specically the analgesic potency and a common adverse effect, opioid-induced bowel dysfunction. 1) Analgesic potency. The previous nding of a greater antinociceptive potency of morphine-6-O-sulfate compared to morphine at the supraspinal level (30-fold, i.c.v.) in mice (Zuckerman et al., 1999) was conrmed herein both at the spinal level (10-fold, i.t.) and in the periphery (5-fold, i.p.) in rats (tail ick test). As can be seen, the ratio of potencies (morphine-6-O-sulfate/morphine) was mark- edly greater after central delivery compared to delivery via the spinal and systemic routes, which may suggest that these compounds differ in their distribution within the CNS. The previous studies (micro- dialysis) showed that morphine and its 6--glucuronide conjugate accumulated respectively in the intra- and extra-cellular uids, which in turn, likely dened their availability at, and interactions with, neuronal opioid receptors (Smith and South, 2001, review). This may be the case with morphine-6-O-sulfate. Importantly, the present data also showed that morphine-6-O-sulfate was more potent than morphine in alleviation of pain in rodent models of neuropathy (6- to 8-fold, CCI) and inammatory pain (3-fold, formalin test) (Table 1). This is a potentially meaningful nding, as chronic pain conditions, in particular neuropathic pain, are typically regarded as less responsive to mu-opioids (Ballantine and Shin, 2008; Martin and Eisenach, 2001; Przewlocki and Przewlocka, 2001, reviews). The reason for these differences is not clear. For example, it has been proposed that morphine-6-O-sulfate (like morphine-6--glucuronide) mediates its pain-relieving effects through a different mu receptor subtype (sensitive to 3-methoxynaltrexone) than morphine does (Pasternak,2001, review). Indeed, the obviously greater analgesic potency of morphine-6-O-sulfate than morphine (in particular after central administration; 30-fold) was not supported by the traditional in vitro binding data showing that this morphine derivative had only slightly improved (~ 3-fold) afnity (Ki, nM) compared to a parent drug for mu- (0.9 0.01 versus 2.5 0.3), delta- (18 0.4 versus 58 3.8) and kappa-3- (6.3 0.3 versus 13.9 4.1) opioid receptors in guinea pig brain homogenates (Crooks et al., 2006). On another note, the earlier work demonstrated similar afnities (Ki, nM) for mu- (1.8 0.4 versus 0.8 0.1) but signicantly different afnities for delta- (4.8 0.4 versus 161 10.2) receptors for morphine-6-O- sulfate compared to morphine in rat brain homogenates (morphine-6--glucuronide had the Ki values equal to 10.4 2.4 and 88.5 10.7 nM for these receptors) (Oguri et al., 1987). Use of different species may explain discrepancy in these ndings. The binding prole (delta/mu) was more favorable for morphine-6-O-sulfate than mor- phine (Oguri et al., 1987); thus, it is plausible to speculate that delta- opioid receptors may also play a role in morphine-6-O-sulfate pharmacology; i.e. enhanced potency in neuropathic and inamma- tory pain in rats. This possible mechanism of action should be further tested with selective delta-receptor antagonists. 2) Duration of analgesia. A prolonged duration of action, in particular after neuroaxial (intrathecal, epidural) administration, is an appreciated property of an analgesic drug in the clinical setting. Morphine-6-O-sulfate had a longer time-course of activity compared to morphine after direct administration to the CNS, either i.c.v. (Brown et al., 1985) or i.t. (present study). Morphine-6-O-sulfate (a polar molecule) was expected to have a delayed distribution into the brain and a consequently slower onset and duration of analgesia than morphine. Neither was observed after the parenteral or oral administration (similar onsets and durations of activity for both drugs). Overall oral efcacy was more favorable for morphine compared to the more charged molecules, morphine-6-O-sulfate (ceiling effect; present study) and morphine-6--glucuronide (van Dorp et al., 2006). Thus, the above ndings can only be explained in part by the differences inFacultad de Estudios Superiores Cuautitln. UNAM

the physicochemical properties of these compounds. 3) Tolerance. Diminished analgesic responsiveness (tolerance) frequently occurs with a long-term exposure to mu-opioids, and this undesired phenomenon leads typically to dose escalation and accompanying adverse effects (cognitive, motor, gastrointestinal). The development of tolerance to antinociception was notably slower for morphine-6-O- sulphate than morphine (25 versus 10 days) when these drugs were administered repeatedly at equipotent doses (%MPE ~ 100%, Day 1) in the tail ick test. This nding appears to support a general belief that the degree of tolerance is inversely correlated to potency of mu- opioids (Frey and Latasch, 2003; Pawar et al., 2007). 4) Therapeutic index (toxicity/analgesia). Opioid-induced bowel dysfunction is a signicant clinical problem, as it occurs typically at analgesic doses, and contrary to the other side-effects of opioids (e.g. respiratory depression), does not decline with repeated dosing (a lack of tolerance). Herein, morphine and its sulfate conjugate inhibited, with similar potencies, the propulsive activity (charcoal test) in the rat. However, morphine-6-O-sulfate had a signicant separation based on dose (at least 10-fold) between the undesirable gastrointestinal effect and the desirable analgesic activities in all pain models tested. This separation was signicantly less for morphine (only 2-fold) either in rats (present study) or mice (Paul et al., 1989). This is a promising nding for morphine-6-O-sulfate especially in view of the fact that morphine-6--glucuronide, which is recognized for its potential superiority over morphine (Smith and South, 2001; van Dorp et al.,2006, reviews), delayed gastrointestinal motility at doses far less than those required to produce antinociception (ED50 = 0.065 and2.7 mg/kg s.c., respectively) in mice (Paul et al., 1989). In addition,morphine-6--glucuronide inhibited gastrointestinal transit with great- er potency (10-fold) than morphine (Paul et al., 1989). The complexity of mu-opioid pharmacology is well recognized. The analgesic (suprasp- inal) and gastrointestinal effects of mu-opioids (i.e. morphine) are thought to be mediated by different subsets of receptors, likely mu-1 and mu-2, respectively (Pasternak, 2001; review). Previous data showed that morphine-6-O-sulfate had a greater afnity (~ 10-fold) for mu-1 than mu-2 receptor subtype (Ki, nM = 0.8 0.01 versus 10.2 0.9) in mouse brain homogenates (Zuckerman et al., 1999). On the other hand, both morphine and its major active metabolite, morphine-6-- glucuronide, competed with similar IC50 (nM) values for mu-1 and mu-2 receptor subtypes (morphine: 3.4 0.8 versus 4.2 1.1; mor- phine-6--glucuronide: 13.3 3.2 versus 14.5 2.7) in the same binding assay (Paul et al., 1989).In conclusion, the present study in rats demonstrated potential clinical advantages of morphine-6-O-sulfate compared to morphine, based on the following ndings: a signicantly greater (5- to 10-fold) analgesic potency when assessed using varies routes of administra- tion (i.t., i.p.) across a range of nociceptive assays (nociceptive, neuropathic, inammatory); a slower onset of tolerance development to antinociception (~ 3-fold) and reduced side-effect liability mani- fested by a considerable greater separation between inhibition of gastrointestinal transit (charcoal test) and analgesia. Together, these preclinical data suggest that morphine-6-O-sulfate may be an interesting potential drug for further study and use for management of nociceptive, neuropathic and mixed pain states.5. Conclusiones

Acknowledgements

Insys Therapeutic, Inc. provided the nancial support to this research.

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