1521-0103/359/1/171–181$25.00 http://dx.doi.org/10.1124/jpet.116.233551THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 359:171–181, October 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

New Morphine Analogs Produce Peripheral Antinociceptionwithin a Certain Dose Range of Their Systemic Administration

Erzsébet Lackó, Pál Riba, Zoltán Giricz, András Váradi, Laura Cornic, Mihály Balogh,Kornél Király, Kata Csek}o, Shaaban A. Mousa, Sándor Hosztafi, Michael Schäfer,Zoltán Sándor Zádori, Zsuzsanna Helyes, Péter Ferdinandy, Susanna Fürst,and Mahmoud Al-KhrasaniDepartments of Pharmacology and Pharmacotherapy (E.L., P.R., Z.G., L.C., M.B., K.K., Z.S.Z., P.F., S.F., M.A.) andPharmaceutical Chemistry (A.V., S.H.), Faculty of Medicine, Semmelweis University, Budapest, Hungary; Department ofAnaesthesiology and Intensive Care Medicine, Charité University Berlin, Campus Virchow Klinikum and Campus Charité Mitte,Berlin, Germany (S.A.M., M.S.); Department of Pharmacology and Pharmacotherapy, Medical School, Szentágothai ResearchCentre (K.C., Z.H.), and MTA-PTE NAP B Chronic Pain Research Group, Faculty of Medicine (Z.H.), University of Pécs, Pécs,Hungary

Received March 16, 2016; accepted July 18, 2016

ABSTRACTGrowing data support peripheral opioid antinociceptive effects,particularly in inflammatory pain models. Here, we examinedthe antinociceptive effects of subcutaneously administered,recently synthesized 14-O-methylmorphine-6-O-sulfate (14-O-MeM6SU) compared with morphine-6-O-sulfate (M6SU) in a ratmodel of inflammatory pain induced by an injection of completeFreund’s adjuvant and in a mouse model of visceral painevoked by acetic acid. Subcutaneous doses of 14-O-MeM6SUand M6SU up to 126 and 547 nmol/kg, respectively, producedsignificant and subcutaneous or intraplantar naloxone methio-dide (NAL-M)–reversible antinociception in inflamed paws com-pared with noninflamed paws. Neither of these doses significantlyaffected thiobutabarbital-induced sleeping time or rat pulmo-nary parameters. However, the antinociceptive effects of higherdoses were only partially reversed by NAL-M, indicating contri-bution of the central nervous system. In the mouse writhing test,

14-O-MeM6SU was more potent than M6SU after subcutane-ous or intracerebroventricular injections. Both displayed highsubcutaneous/intracerebroventricular ED50 ratios. The antinoci-ceptive effects of subcutaneous 14-O-MeM6SU and M6SU upto 136 and 3043 nmol/kg, respectively, were fully antagonizedby subcutaneous NAL-M. In addition, the test compoundsinhibited mouse gastrointestinal transit in antinociceptivedoses. Taken together, these findings suggest that systemicadministration of the novel compound 14-O-MeM6SU similarto M6SU in specific dose ranges shows peripheral antinoci-ception in rat and mouse inflammatory pain models withoutcentral adverse effects. These findings apply to male animalsand must be confirmed in female animals. Therefore, titrationof systemic doses of opioid compounds with limited accessto the brain might offer peripheral antinociception of clinicalimportance.

IntroductionOpioid analgesics are the cornerstone in treatment of

moderate-to-severe pain. They exert their antinociceptiveaction by activating opioid receptors. Themajority of clinicallyused opioid analgesics have central adverse effects such asrespiratory depression, development of opioid tolerance anddependence, and addiction liabilities. These effects stronglylimit clinical use of these drugs. In addition to central opioidreceptors, several studies support the existence of functionalopioid receptors in the periphery as well (Stein et al., 1993;

Nagasaka et al., 1996; Coggeshall et al., 1997; Tegeder et al.,2003; Bergström et al., 2006). Pharmacological evidence indi-cates that activation of these receptors also results in painmitigation (Stein et al., 1995; Kalso et al., 2002; Fürst et al.,2005; Al-Khrasani et al., 2007; Khalefa et al., 2012; Stein, 2013).In addition, peripheral opioid receptors are reported to beupregulated in inflamed tissues (Stein et al., 1989; Schäferet al., 1995). To note, the peripheral antinociception of opioidsin humans has been investigated in several randomizedcontrolled clinical trials (Kalso et al., 1997; Gupta et al.,2001). Pooled analyses of data from 19 studies suitable formetaanalysis showed only a moderate analgesic effect afteradministration of intra-articular morphine compared withplacebo in patients who underwent arthroscopic knee surgery(Kalso et al., 1997; Gupta et al., 2001). Morphine-6-glucuronide(M6G) was reported to have peripheral antihyperalgesic effects

This research was supported by Semmelweis University [GrantAOK/DH/148-7/2015 and Doctoral Scholarship (to E.L. and M.A.K.)] and theRichter Gedeon Talentum Foundation [(to K.C.)].

E.L. and P.R. contributed equally to this work.dx.doi.org/10.1124/jpet.116.233551.

ABBREVIATIONS: 14-O-MeM6SU, 14-O-methylmorphine-6-O-sulfate; ANOVA, analysis of variance; CFA, complete Freund’s adjuvant; CNS,central nervous system; M6G, morphine-6-glucuronide; M6SU, morphine-6-O-sulfate; MOR, m-opioid receptor; NAL-M, naloxone methiodide; PPT,paw pressure threshold; WBP, whole-body plethysmography.

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after its systemic administration in human volunteers (Tegederet al., 2003). Because of its high hydrophilicity, M6G has aconsiderable delay between peak plasma concentrations andpeak central opioid effects such that peripheral antinociceptiveeffects can be detected within this time window (Skarke et al.,2003; Tegeder et al., 2003). M6G was recently described as apartial agonist with respect to its antinociceptive effects but asa full agonist regarding its central effect of respiratorydepression (Kuo et al., 2015), which may prevent its use as aperipheral analgesic. In addition, the number of m-opioidreceptor (MOR) agonists with high efficacy but restricted accessto the central nervous system (CNS) is very low. Therefore, thedesign of opioids with high hydrophilicity and high efficacymay provide analgesic agents of high clinical value.Animal models used to study the peripheral antinociceptive

action of opioids include complete Freund’s adjuvant (CFA)–induced or acetic acid–induced inflammatory pain. Inducinginflammation by injecting CFA into the hind paw of animals(e.g., rats) has long been used as a strategy to demonstrate theperipheral antinociceptive actions of opioid agonists (Stein et al.,1988; Zhou et al., 1998; Al-Khrasani et al., 2007). We recentlydesigned and synthesized a new MOR agonist, 14-O-methyl-morphine-6-O-sulfate (14-O-MeM6SU), which displays highaffinity for MOR and strong antinociceptive effects in acutethermal nociceptive tests (Lacko et al., 2012). 14-O-MeM6SUhashigher affinity than morphine-6-O-sulfate (M6SU) or morphinefor MORs or d-opioid receptors and similar affinity for k-opioidreceptors. However, the d-opioid receptor/MOR affinity ratio of14-O-MeM6SU was smaller than that of M6SU, whereas thek-opioid receptor/MOR ratio was higher for 14-O-MeM6SU.14-O-MeM6SU acted as a full agonist in a functional 59-O-(3-[35S]thio)triphosphate binding assay, whereas morphine andM6SU were partial agonists (Lacko et al., 2012).We recently showed that topical administration of 14-O-

MeM6SU strongly attenuates CFA-induced inflammatorypain in rats (Khalefa et al., 2013). However, to our knowledge,its peripheral antinociceptive action after systemic (subcuta-neous) administration has not yet been tested.M6SUhas beensuggested as a potential candidate for the development ofnovel opioid agents to treat nociceptive, neuropathic, andmixed pain states (Holtman et al., 2010). In addition, M6SUwas recently reported to have a good safety profile, since thereis a clear separation between the doses of produced antinoci-ception versus adverse effects (Holtman et al., 2010). Thesereports also supported our idea to develop M6SU analogs,among them 14-O-MeM6SU.This work aimed to demonstrate the antinociceptive effects

of 14-O-MeM6SU compared with M6SU by subcutaneousadministration in rats with CFA-induced inflammatory painand in mice with acetic acid–induced pain. Additional objec-tives were 1) to determine the site of analgesic action by use ofnaloxone methiodide (NAL-M), the peripherally restrictedopioid antagonist); 2) to study the barbiturate anesthesia-potentiating effect of 14-O-MeM6SU and M6SU to detect thecentral or peripheral target of the drugs; and 3) to test theaction of 14-O-MeM6SUandM6SU on gastrointestinal transitand respiratory function.

Materials and MethodsAnimals. MaleWistar rats (200–300g, AnimalHouse of Semmelweis

University) and male NMRI mice (20–30 g, Toxicoop, Hungary) were

used for all tests. Animals were housed in a roomwith cages linedwithground corncob bedding, with a temperature of 20 6 2°C under a12-hour/12-hour light/dark cycle, in the local animal house of theSemmelweis University Departments of Pharmacology and Pharma-cotherapy (Budapest, Hungary) and the University of Pécs Faculty ofMedicine (Pécs, Hungary). Food and water were freely available. Tominimize stress, rats were handled once daily for 3 subsequent daysprior to the experimentation day. Experiments were performed inaccordance with guidelines of the local animal care committee(PEI/001/276-4/2013) and the Ethical Board of Semmelweis Univer-sity based on the Declaration of the European Communities CouncilDirectives (86/609/ECC).

Drugs. The following drugs were used. 14-O-MeM6SU and M6SU(Fig. 1) were synthesized by S. Hosztafi at the Semmelweis UniversityDepartment of Pharmaceutical Chemistry. NAL-M (Sigma-Aldrich,Budapest, Hungary) and all other chemicals were of analytical gradeand were purchased from standard commercial sources. Drugs weredissolved in 0.9% NaCl solution. Drugs or saline were delivered asfollows: via subcutaneous administration (under skin over the neck),5 ml/kg for rats and 10ml/kg for mice; intravenous injection, 2.5 ml/kgfor rats; intraplantar administration, 100 ml/rat; and intracerebroven-tricular injection, 5 ml/mouse. A separate group of animals was usedfor each dose. Researchers performing the experiments were blindedto the drugs and doses applied.

Induction of Inflammation in Rats. Rats received an intra-plantar injection of 0.15mlCFA (Calbiochem, SanDiego, CA), awater-in-oil emulsion of inactivated mycobacterium administered into theright hind paw, while under brief isoflurane (Willy Rüsch GmbH,Böblingen, Germany) anesthesia. This treatment consistently pro-duces localized inflammation of the inoculated paw, characterized byan increase in paw volume, paw temperature, and infiltration withvarious types of immune cells (Rittner et al., 2001).

Induction of Pain in Mice (Mouse Writhing Test). The aceticacid–induced writhing test was performed as previously described(Koster et al., 1959). Mice were injected intraperitoneally with 0.2 ml0.6% acetic acid aqueous solution to produce the writhing reaction,characterized by contractions of the abdominal musculature followedby extension of the hind limbs.

Nociceptive Testing in Rats with CFA-Evoked Hyperalge-sia. On days 4 and 7 after the intraplantar CFA injection, baseline(pretest compound) paw pressure thresholds (PPTs) of inflamed andnoninflamed paws were assessed by paw pressure algesiometry(modified Randall–Selitto test; Ugo Basile, Comerio, Italy) as de-scribed in detail previously (Mousa et al., 2007; Khalefa et al., 2012).

Fig. 1. Chemical structures of morphine, M6SU, 14-O-MeM6SU, andNAL-M.

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PPTs were then reevaluated at 30, 60, and 120 minutes after subcu-taneous drug administration, using an arbitrary cut-off weight oftwice the control and expressed as percentages.

Dose-Response Relationships in Rats. The antinociceptiveeffects of subcutaneous 14-O-MeM6SU and M6SU were examined inthese experiments. After baseline measurements were obtained,separate groups of animals for each subcutaneous dose were used.Antinociception assessed with respect to the change in PPTs of bothpaws after subcutaneous drug administration was compared with thebaseline value obtained before drug treatment in ipsilateral orcontralateral paws. Doses of each drug that produced a 60%–80%antinociceptive effect in inflamed paws, without a significant effect onthe contralateral noninflamed paws, were selected for experimentsdesigned to analyze the antagonism of NAL-M. In these experimentsNAL-M (21.3mmol) was coadministered with the test compounds or at30 minutes, when the peak effect was achieved at 60 minutes. Inanother series of experiments, NAL-M (0.43 mmol/rat, intraplantarly)was injected 5 minutes prior to measurement (at 25 minutes or55 minutes, when the peak effect was achieved at 30 and 60 minutes,respectively).

Dose-Response Relationships in Mice. Groups of mice wereinjected subcutaneously or intracerebroventricularly with differentdoses of 14-O-MeM6SU or M6SU followed 15 minutes later by anintraperitoneal injection of 0.6% acetic acid solution. Each mouse wasthen placed in individual transparent Plexiglas chambers. Fiveminutes after the acetic acid injection, the number of writhes wascounted during a 10-minute observation period. To determine thenumber of writhes in control groups, animals were subcutaneously orintracerebroventricularly injected with 0.9% saline solution beforethey were intraperitoneally injected with 0.6% acetic acid using asimilar protocol as for the test drugs. Subcutaneous NAL-M wascoadministered with the respective agonist in experiments in whichantagonist action was assessed. Assessments were performed 20 min-utes after subcutaneous opioid agonists as described above by re-cording the number of writhes for each mouse for a 10-minute timeperiod beginning 5 minutes after the intraperitoneal injection ofacetic acid.

Determination of Thiobutabarbital-Induced Sleeping Time.Animals received intravenous saline or thiobutabarbital (153mmol/kg)and then were placed on their left side. Sleeping time in minutes wasdocumented and was considered to end when animals spontaneouslyturned to the opposite right position (“righting reflex”). Anesthesia-potentiating effects of test drugs were studied by subcutaneousadministration. Thiobutabarbital was injected intravenously at thetime of peak antinociceptive action of test compounds (60 minutesafter subcutaneous 14-O-MeM6SU and 30 minutes after subcutane-ous M6SU).

Determination the Effect of 14-O-MeM6SU and M6SU onGastrointestinal Transit. The effect of 14-O-MeM6SU and M6SUcompared with that of morphine on gastrointestinal transit wasmeasured in vivo by using the charcoal meal method (Scheibneret al., 2002). Briefly, male NMRI mice (20–25 g) were fasted 6 hoursprior to the experiments, with free access to water. At the time of theexperiment, a charcoal suspension (10% charcoal in 5% gum arabic)was given in a volume of 0.25ml permouse by an oral gavage, followedby subcutaneous administration of test compounds (0.1 ml/10 g). Micewere decapitated 30 minutes after drug or saline administration andtheir small intestines were removed. The distance traveled by thecharcoal suspension was expressed as a percentage of total smallintestine length. The doses that caused 50% inhibition of gastrointes-tinal transit (ID50) were calculated from the linear regression of dose-response curves.

Respiratory Function Tests. Respiratory function measure-ments were performed by unrestrained whole-body plethysmography(WBP) in conscious, spontaneously breathing animals 30 and 60 min-utes after a subcutaneous injection of saline, 14-O-MeM6SU, M6SU,or morphine. Rats were placed in the chamber of a whole-bodyplethysmograph (PLY 3213; Buxco Europe Ltd., Winchester, UK).

The flow transducers (TRD5700; Buxco Europe Ltd.) were connectedto the preamplifier module, which digitized the signals via an analog-to-digital converter (MAX2270; Buxco Europe Ltd.). Ventilation

Fig. 2. Time course of the antinociceptive effect of subcutaneouslyadministered 14-O-MeM6SU and M6SU in noninflamed and inflamedrat hind paws. Drugs were delivered in a volume of 5 ml/kg body weight.Each point represents the mean 6 S.E.M. (n = at least 4).

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parameters (frequency, tidal volume, minute ventilation, time ofinspiration, peak inspiratory flow, time of expiration, peak expiratoryflow, and relaxation time) were measured every 10 seconds during the15-minute acquisition times and were averaged by BioSystem XASoftware for Windows (Buxco Research Systems, Wilmington, NC).

Data Analysis. GraphPad Prism software (version 5.00 for Win-dows; GraphPad Software Inc., San Diego, CA) was used for dataanalysis. Two-way analysis of variance (ANOVA) with Bonferroni’spost hoc test was performed for comparison between inflamed andnoninflamed paws. Differences between animal groups that received

saline, drugs, or drugs plus NAL-M were determined by ANOVA,followed by the Newman–Keuls post hoc test. In the mouse writhingtest, the antinociceptive effect is presented as the percentage decreasein the number of writhes and is calculated according to the formula:Percent inhibition of writhing5 (C2 T)/ C� 100, where C is themeannumber of writhes in control animals and T is the number of writhes indrug-treated mice. Dose-response relationships of the percent in-hibition of writhing were constructed and the dose necessary toproduce a 50% effect (ED50) and 95% confidence limits were calcu-lated. Differences between groups were determined by ANOVA,followed by the Newman–Keuls post hoc test. Statistical analysis ofunrestrained WBP was performed by repeated-measures ANOVAfollowed by the Tukey multiple comparisons test (n 5 6 per group).Results were considered statistically significant when P , 0.05.

ResultsAntinociceptive Effects of 14-O-MeM6SU and M6SU

after Systemic Administration in Rats with CFA-Induced Inflammatory Pain. CFA treatment reduced thePPT to approximately 65% 6 2% (n 5 70) and 70% 6 2%(n5 55) of the control response after 4 and 7 days, respectively.The subcutaneous doses of 14-O-MeM6SU (32–1012 nmol/kg)and M6SU (137–8758 nmol/kg) were tested for their analgesicactions in CFA-induced inflammatory pain (Fig. 2). M6SUproduced peak analgesic effects at 30 minutes, whereas 14-O-MeM6SU did so at 60 minutes (Table 1). The antinociceptiveactions of the test compounds were significant in inflamedpaws comparedwith noninflamed paws at the doses presentedin Fig. 3. The analgesic effects of subcutaneous 14-O-MeM6SUand M6SU did not differ at doses over 506 nmol/kg and4379 nmol/kg, respectively. The antinociceptive actions ofsubcutaneous 14-O-MeM6SU (126, 253, and 506 nmol/kg)and M6SU (547, 1095, and 2189 nmol/kg) were furthertested for their peripheral analgesic actions in inflamedpaws inseparate experiments (see below). Treatment with saline orNAL-M alone had no effects on PPTs of inflamed paws ornoninflamed paws (Figs. 2, 4, and 5).Antagonist Effects of Subcutaneous and Intraplan-

tar NAL-M on the Antinociceptive Actions ofSubcutaneous 14-O-MeM6SU or M6SU in Rats withCFA-Induced Inflammatory Pain. The analgesic effectsproduced by subcutaneous 14-O-MeM6SU (126 nmol/kg) andM6SU (547 nmol/kg) were antagonized by subcutaneousadministration of NAL-M (21.3 mmol/kg) (Fig. 4). In otherexperiments, the antinociceptive effects of the same doses oftest agonists were also significantly reduced by intraplantarNAL-M (0.43 mmol/rat) (Fig. 4). No differences were observedbetween PPTs of animals injected with saline or NAL-M(ANOVA, followed by the Newman–Keuls post hoc test, Fig. 4).

TABLE 1Antinociceptive potencies of 14-O-MeM6SU and M6SU in inflamed (right) and noninflamed (left) paws in the Randall–Selitto test in rats aftersubcutaneous administration

Compound

ED50 (95% Confidence Limits)

30 min 60 min 120 min

Left Paw Right Paw Left Paw Right Paw Left Paw Right Paw

nmol/kg14-O-MeM6SU 305 (155–601) 86 (33–220) 388 (201–751) 45 (22–92)a 792 (388–1620) 110 (47–258)M6SU 2313 (1226–4364) 292 (114–749)a 2931 (1434–5998) 520 (222–1216) 8920 (1100–72,318) 1651 (583–4673)

aPeak of effect.

Fig. 3. Antinociceptive effects of subcutaneously administered 14-O-MeM6SU and M6SU (in nanomoles per kilogram, n = 5–12 per group).Drugs were delivered in a volume of 5 ml/kg body weight. Each valuerepresents the mean 6 S.E.M. Data were obtained 30 minutes afterinjection of M6SU and 60 minutes after injection of 14-O-MeM6SU. Thepound symbol represents significant differences between the lowest andhighest doses. ***p , 0.001 (significant differences between inflamed andnoninflamed paws, two-way ANOVA).

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In another experiment, we also tested the antagonist effectof subcutaneous NAL-M (21.3 mmol/kg) on the antinocicep-tive effects produced by subcutaneous 14-O-MeM6SU(253 or 507 nmol/kg) and M6SU (1095 or 2189 nmol/kg)(Fig. 5). In these experiments, NAL-M partially reversedthe antinociceptive effect of 14-O-MeM6SU and totallyreversed the antinociceptive effect of the 1095-nmol/kgM6SU dose. However, NAL-M failed to reverse the anti-nociceptive effect of the 2189-nmol/kg M6SU dose (Fig. 5).These results indicate the contribution of the CNS to thetotal antinociception of systemically administered testcompounds at higher doses.

Potentiation of Thiobutabarbital-Induced Anesthe-sia. Thiobutabarbital (153 mmol/kg i.v.) produced a sleepingtime of 10 6 3, 10 6 5, and 8 6 4 minutes in the presence ofsubcutaneous saline, 14-O-MeM6SU (126 nmol/kg), andM6SU (547 nmol/kg), respectively (Fig. 6). At higher agonistdoses, sleeping time was longer compared with saline (Fig. 6).Antinociceptive Effects of 14-O-MeM6SU Compared

with M6SU in the Mouse Writhing Test. Injection of a0.6% acetic acid solution into the peritoneal cavity of miceadministered subcutaneous or intracerebroventricular salineresulted in an average of 43.96 1.5 (n5 43) writhes during the10-minute period. As shown in Fig. 7, subcutaneous or

Fig. 4. The antagonist effect of NAL-M (21.3 mmol/kgs.c. or 0.43 mmol/rat i.pl.) against subcutaneous anti-nociceptive effects of 14-O-MeM6SU (126 nmol/kg) andM6SU (547 nmol/kg) in inflamed rat paws. Drugs weredelivered in a volume of 5 ml/kg body weight and100 ml/rat for subcutaneous and intraplantar adminis-tration, respectively. Each value represents the mean6S.E.M. Each data point was obtained 30 minutes afterinjection of M6SU (n = 17–18), saline (n = 18–35), orNAL-M (n = 10–12) and 60 minutes after injection of14-O-MeM6SU (n = 7–13), saline (n = 6–35), or NAL-M(n = 6–12). *P , 0.05; ***P , 0.001 (significantdifferences versus the effect of agonist in inflamedpaw; one-way ANOVA, Newman–Keuls post hoc test).

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intracerebroventricular administration (20 minutes beforetesting) of all of the agonists produced a dose-dependentantinociceptive action. Table 2 provides calculated ED50 valueswith 95% confidence intervals at 20 minutes. 14-O-MeM6SU

produced a more potent inhibitory effect than M6SU on aceticacid–induced writhing in mice after administration via sub-cutaneous and intracerebroventricular routes (Table 2).14-O-MeM6SU was about 23-fold more potent than M6SU

after subcutaneous administration, whereas 14-O-MeM6SUproved to be only 5-foldmore active thanM6SU in inhibition ofwrithing after intracerebroventricular administration. How-ever, large subcutaneous/intracerebroventricular potency ra-tios were calculated for M6SU or 14-O-MeM6SU (Table 2).NAL-M Antagonism on Systemic 14-O-MeM6SU or

M6SU Antinociception. To evaluate opioid specificity andthe site of action of 14-O-MeM6SU and M6SU in aceticacid–induced writhing in mice, the effects of the test agonistswere assessed after systemic coadministration with thequaternary opioid antagonist NAL-M (21.3 mmol/kg s.c). Ourresults show that a subcutaneous equipotent analgesic dose of14-O-MeM6SU (136 nmol/kg) and M6SU (3043 nmol/kg)significantly decreased the number of writhes at 20 minutesafter administration. Coadministration of NAL-M signifi-cantly antagonized the antinociceptive effect of test opioids,as indicated in Fig. 8. NAL-M treatment failed to affect thenumber of writhes and was found to be similar to the valuesobtained after saline treatment (Fig. 8). At higher doses, 14-O-MeM6SU (272 nmol/kg) also showed NAL-M–reversible anti-nociception (Fig. 8).Inhibitory Effect of Systemic 14-O-MeM6SU and

M6SU on Gastrointestinal Transit in Mice. Subcutane-ously administered 14-O-MeM6SU, M6SU, and morphineinhibited gastrointestinal transit of charcoal in dose-dependent manner. The calculated ID50 values and confidenceintervals were 250 nmol/kg (205–305), 325 nmol/kg (70–1517),and 2228 nmol/kg (666–7455) for 14-O-MeM6SU, M6SU, andmorphine, respectively. These results indicate that the testcompounds inhibit gastrointestinal transit in antinociceptivedoses.

Fig. 5. Antagonist effect of NAL-M (21.3 mmol/kg s.c.) against the subcu-taneous antinociceptive effects of 14-O-MeM6SU (253 and 506 nmol/kg) andM6SU (1095 and 2189 nmol/kg) in inflamed rat paws (n = 4–9 per group).Drugs were delivered in a volume of 5 ml/kg body weight. Each valuerepresents the mean 6 S.E.M. Each data point was obtained 30 minutesafter injection of M6SU or saline and 60 minutes after injection of 14-O-MeM6SU or saline. **P , 0.01 (versus saline in the inflamed paw, one-wayANOVA, Newman–Keuls post hoc test); ***P , 0.001 (versus saline in theinflamed paw, one-wayANOVA, Newman–Keuls post hoc test); ###P, 0.001(versus drug plus NAL-M); ++P, 0.01 (versus 1095 nmol drug plus NAL-M).

Fig. 6. Effect of subcutaneous 14-O-MeM6SU and M6SUon thiobutabarbital (153 mmol/kg i.v.)–induced sleepingtime (n = 5–10). *P , 0.05 versus saline (one-way ANOVA,Newman–Keuls post hoc test).

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Respiratory Effects of 14-O-MeM6SU and M6SUCompared with Morphine in Awake, UnrestrainedRats. Figure 9 depicts the effects of 14-O-MeM6SU(253 nmol/kg), M6SU (1095 nmol/kg), and morphine(7776 nmol/kg) on rat pulmonary parameters. None of therespiratory parameters determined by unrestrained WBP(frequency, tidal volume, minute ventilation, time of inspira-tion, peak inspiratory flow, time of expiration, peak expiratoryflow, and relaxation time) showed significant differencesbetween the saline-treated control or drug-treated groups30 and 60 minutes after subcutaneous injection; however, thismore clinically relevant test remains subject to more elaborate

future investigations. None of the drugs caused any sedativeeffects; the animals were at rest by the end of the measure-ments, but they woke when the WBP chambers were opened.

DiscussionThis study aimed to further investigate the recently syn-

thesized morphine analog, 14-O-MeM6SU (Lacko et al., 2012)compared with M6SU (Brown et al., 1985; Zuckerman et al.,1999; Crooks et al., 2006; Holtman et al., 2010) in a rat modelof inflammatory pain and amousemodel of visceral pain basedon our previous work (Al-Khrasani et al., 2007; Khalefa et al.,

Fig. 7. Antinociceptive dose-response curves of subcutane-ously (A) or intracerebroventricularly (B) injected 14-O-MeM6SU and M6SU in the mouse writhing test. Pointsrepresent the mean 6 S.E.M for groups of four to five mice.

TABLE 2Antinociceptive potencies of 14-O-MeM6SU, M6SU, and morphine against acetic acid–induced nociception in the mousewrithing test after 20 minutes of subcutaneous and intracerebroventricular administrationAt least four animals per dose group and three to four doses were used for each ED50 determination.

Compound

ED50 (95% Confidence Limits)Subcutaneous/

Intracerebroventricular ED50 RatioaSubcutaneousAdministration Intracerebroventricular Administration

nmol/kg pmol/mouse14-O-MeM6SU 87 (47–163) 1.7 (0.3–9.5) 51,177M6SU 1993 (1282–3101) 9 (15–54) 221,444

aCalculated as the subcutaneous administration (nmol/kg)/intracerebroventricular administration (pmol/mouse) ED50 ratio.

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2013). Our data clearly show that 14-O-MeM6SU and M6SUproduced peripheral antinociceptive effects in a CFA-inducedinflammatory pain model or acetic acid–evoked visceral painover a specific dose/concentration range of their systemic (sub-cutaneous) administration. The antinociceptive action on theformer test in certain doses was localized to the inflamed paw.Thus, beyond the antinociceptive effect elicited by the local

administration of 14-O-MeM6SU (Khalefa et al., 2013), pe-ripherally mediated antinociception of this compound and itsparent molecule, M6SU, could also be established by itssystemic application. It is important to emphasize thatantinociceptive effects of 14-O-MeM6SU and M6SU have notyet been tested in CFA-induced inflammatory pain and aceticacid–induced pain in rats and mice, respectively. Our dataconfirm previous reports on the peripheral antinociceptiveactions of systemic opioids in the inflammatory pain model(Stein et al., 1988; Al-Khrasani et al., 2012). These resultsseem to be in contrast with findings that intravenous mor-phine elicits antinociceptive effects exclusively by the activa-tion of a CNS MOR but not on peripheral sensory nerveterminals, since NAL-M antagonized these effects after intra-cerebroventricular and intrathecal administration but notafter intraplantar delivery (Khalefa et al., 2012). In our work,test compounds could also elicit central antinociception athigher doses. This was reflected by the phenomenon thatdifferences in the antinociceptive effects of 14-O-MeM6SUandM6SU between inflamed and noninflamed paws graduallydeclined but a clear peripheral action was demonstrated at alower dose range in the inflamed paw. These results show thatcareful dose titration of the MOR agonists 14-O-MeM6SU andM6SU during their systemic administration can reveal adistinct dose range inwhich antinociceptive effects are exertedexclusively by the activation of peripheral MOR at the in-flammation site. At these doses, PPTs on the contralateralside were not significantly elevated.

The possible explanation for why the test compounds pro-duced antinociception in inflamed paws compared with non-inflamed paws might be the increase in the number of opioidreceptors in the nerve endings (Schäfer et al., 1994) and thedisturbance of the perineural barrier during inflammation(Antonijevic et al., 1995). The latter condition might facilitatethe access of opioid agonists to their receptors, which seem tobe somewhat hidden (reserved) when inflammation is absent.The increase in the number of accessible opioid receptorsresults in enhanced peripheral opioid antinociceptive efficacyin inflammatory pain, as was previously reported (Antonijevicet al., 1995; Rittner et al., 2005, 2012).To demonstrate the peripheral antinociceptive action of

subcutaneous opioids, the peripherally acting opioid antago-nist NAL-M (Bianchi et al., 1982; Lewanowitsch and Irvine,2002; Riba et al., 2002) was used. Subcutaneous or intra-plantar NAL-M abolished the antinociceptive effects of 14-O-MeM6SU or M6SU in our experiments in rats (Fig. 4),indicating that these compounds (at the doses used) pro-duced antinociception of a peripheral origin. Our data are inagreement with previous studies using this experimentalmodel of pain and the same route of administration (Steinet al., 1988). However, test compounds at higher systemicdoses showed CNS-mediated antinociception, because sys-temic NAL-M failed to fully abolish it (Fig. 5).The obtained results in CFA-induced inflammatory pain

after systemic administration encouraged us to extend ourwork to examine the action of test compounds in other species(mice) to support or contradict the described effects of 14-O-MeM6SU or M6SU at the tested doses. The mouse writhingtest was used in these experiments. The acetic acid–evokedwrithing assay, one of the most well established and widelyused experimental models to assess the pain-relieving actionsof either nonsteroidal anti-inflammatory drugs or opioids, isalso suitable to demonstrate the central and peripheralcomponents of nociception. However, until now, the antinoci-ceptive effect of 14-O-MeM6SU compared with M6SU in thisanimal model of visceral pain has not been studied. In thismodel, the antinociceptive effects of subcutaneous or intra-cerebroventricular 14-O-MeM6SU were also studied andcompared with that of M6SU. 14-O-MeM6SU showed morepotent antinociceptive action than M6SU after both routes ofadministration, in accordance with data previously publishedby our group (Lacko et al., 2012). In addition, similarly to dataobtained in a rat model of inflammatory pain, systemic orcentral administration of 14-O-MeM6SU resulted in strongerantinociception than that of M6SU (Table 2). However, thesubcutaneous/intracerebroventricular ratio was higher forM6SU than for 14-O-MeM6SU. Regarding the antinociceptiveeffect, our results are in agreement with data we reportedpreviously in a thermal pain model (Lacko et al., 2012). 14-O-MeM6SUwas 23 timesmore potent thanM6SUafter systemicadministration but only 5 times more potent than M6SU aftercentral dosing. Previous reports (Frances et al., 1992; Al-Khrasani et al., 2007) indicate a lower subcutaneous/intracer-ebroventricular ratio formorphine (4215) and a larger ratio forM6G (5840) that is similar to 14-O-MeM6SU (Table 2). Datareported by Brown et al. (1985) attributed the weak anti-nociceptive action of subcutaneous M6SU to its limited accessto the CNS. It is worth noting that 14-O-MeM6SU alsodisplayed a high systemic/central dose ratio compared withother opioids such as morphine or fentanyl (Fürst et al., 2005;

Fig. 8. Antagonist action of coadministered NAL-M (21.3 mmol/kg) on theantinociceptive effect of 14-O-MeM6SU or M6SU after 20-minute sub-cutaneous administration in the writhing response induced by acetic acid(intraperitoneally) in mice. Data are expressed as the mean 6 S.E.M. forgroups of 4–12 mice. *P, 0.05; **P, 0.01; ***P, 0.001 (versus the othergroups, one-way ANOVA, Newman–Keuls post hoc test).

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Lacko et al., 2012). Therefore, 14-O-MeM6SU plausibly haslimited CNS penetration, similarly to M6SU. However, 14-O-MeM6SU has an advantage over M6SU because it displays

higher efficacy and affinity, reflecting its stronger antinoci-ceptive action as previously described (Lacko et al., 2012).M6SU and morphine are strong partial agonists in MOR-

Fig. 9. Respiratory function parameters determined by unrestrained WBP 30 and 60 minutes after subcutaneous injection of saline, M6SU (1095 nmol/kg),14-O-MeM6SU (253 nmol/kg), andmorphine (7776 nmol/kg). (A) Frequency (f). (B)Minute ventilation (MV). (C) Tidal volume (TV). (D) Time of inspiration (Ti).(E) Time of expiration (Te). (F) Peak inspiratory flow (PIF). (G) Peak expiratory flow (PEF). (H) Relaxation time (RT). n = 6 per group. *P , 0.05.

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mediated Gi protein activation and b-arrestin recruitment toopioid receptors (Frölich et al., 2011). Thus far, there are nodata on the effect of 14-O-MeM6SU on b-arrestin recruitmentto opioid receptors, but this is an area for future research.14-O-MeM6SU (136nmol/kg) orM6SU (3043nmol/kg) (Fig. 8)

showed peripheral antinociceptive effects in the mouse writh-ing assay after subcutaneous administration, since the coad-ministered quaternary opioid antagonist NAL-M significantlyreversed the effects of the test compounds. Systemicallyapplied quaternary opioid antagonists have been used tolocalize the site of opioid antinociceptive action because theydo not readily cross the blood–brain barrier.Another approach to study peripheral opioid activity might

be to assess whether test compounds evoke motor functionimpairment in antinociceptive doses by subcutaneous admin-istration. Vanderah et al. (2004) applied the latter method todemonstrate peripheral antinociception of D-amino acid tetra-peptide (D-Phe-D-Phe-D-Nle-D-Arg-NH2) without motor im-pairment in the mouse writhing test (Vanderah et al., 2004).We also studied the central action of anesthetics (e.g., in-travenous thiobutabarbital) in the presence of opioids. CNS-depressing drugs are reported to have longer action bycoadministration of opioids (Mcguire et al., 1978; Craft andLeitl, 2006). Therefore, we further extended our work toexamine the actions of systemically injected test compoundson thiobutabarbital-induced sleeping time in rats. NAL-M–reversible antinociceptive doses of 14-O-MeM6SU orM6SU failed to potentiate thiobutabarbital-induced sleepingtime (Fig. 6). At higher doses, both compounds lengthenedsleeping time. Previous studies showed that intraperitoneallyadministeredM6SU elicited motor impairment and decreasedlocomotor activity at doses of 5 mg/kg or higher (Holtmanet al., 2010). In our study, the dose of M6SU studied for itsperipheral antinociception was 547 nmol/kg (0.2 mg/kg),which is markedly lower compared with doses tested pre-viously (Holtman et al., 2010). Therefore, these datastrengthen our results that at this dose, M6SU activatesperipheral opioid receptors at the site of inflammation in rats.In our studies, 14-O-MeM6SU and M6SU inhibited gastroin-testinal peristalsis, which is in accordance with previousstudies (Holtman et al., 2010). However, these drugs alsoinduced significant peripheral antinociception at the samedose range, clearly indicating that they are superior to anotherperipherally acting opioid, loperamide, which failed to produceantinociception in doses producing constipation in mice(Sánchez-Fernández et al., 2014).Furthermore, test compounds and morphine in doses pro-

longing thiobutabarbital-induced sleeping time showed nosignificant alterations in respiratory parameters comparedwith the control group, indicating that the drugs did not causerespiratory depression in the tested dose range.In summary, in specific systemic dose ranges, 14-O-

MeM6SU or M6SU elicited significant peripheral antinoci-ception in mouse visceral pain and in inflamed rat pawscompared with noninflamed paws. These actions were con-firmed by the antagonism of the peripherally acting opioidantagonist NAL-M. In addition, these doses did not potentiatethe sleeping time of general anesthetic agent thiobutabarbitalnor did they affect respiration indicating poor access to theCNS at these doses. However, systemically administeredNAL-M partially antagonized the antinociception of testcompounds of higher doses, which indicates the contribution

of the CNS. Titration of systemic doses of opioid compoundswith limited access to the brain might offer peripheralanalgesia of clinical importance. These data indicate thatthe development of opioid drugs such as M6SU and itsanalogs, which target the pain in the periphery with a widesafety profile, may represent a new generation of opioids forthe treatment of inflammatory pain.

Acknowledgments

The authors thank Mária Molnár Pénzes for skilled technicalassistance.

Authorship Contributions

Participated in research design: Hosztafi, Ferdinandy, Fürst,Al-Khrasani.

Conducted experiments: Lackó, Riba, Giricz, Cornic, Balogh,Király, Csek}o.

Contributed new reagents or analytic tools: Váradi, Hosztafi.Performed data analysis: Csek}o, Mousa, Zádori.Wrote or contributed to the writing of the manuscript: Mousa,

Schäfer, Helyes, Ferdinandy, Fürst, Al-Khrasani.

References

Al-Khrasani M, Lackó E, Riba P, Király K, Sobor M, Timár J, Mousa S, Schäfer M,and Fürst S (2012) The central versus peripheral antinociceptive effects of m-opioidreceptor agonists in the new model of rat visceral pain. Brain Res Bull 87:238–243.

Al-Khrasani M, Spetea M, Friedmann T, Riba P, Király K, Schmidhammer H,and Furst S (2007) DAMGO and 6beta-glycine substituted 14-O-methyloxymorphonebut not morphine show peripheral, preemptive antinociception after systemic ad-ministration in a mouse visceral pain model and high intrinsic efficacy in the isolatedrat vas deferens. Brain Res Bull 74:369–375.

Antonijevic I, Mousa SA, Schäfer M, and Stein C (1995) Perineurial defect and pe-ripheral opioid analgesia in inflammation. J Neurosci 15:165–172.

Bergström J, Ahmed M, Li J, Ahmad T, Kreicbergs A, and Spetea M (2006) Opioidpeptides and receptors in joint tissues: study in the rat. J Orthop Res 24:1193–1199.

Bianchi G, Fiocchi R, Tavani A, and Manara L (1982) Quaternary narcotic antago-nists’ relative ability to prevent antinociception and gastrointestinal transit in-hibition in morphine-treated rats as an index of peripheral selectivity. Life Sci 30:1875–1883.

Brown CE, Roerig SC, Burger VT, Cody RB, Jr, and Fujimoto JM (1985) Analgesicpotencies of morphine 3- and 6-sulfates after intracerebroventricular administra-tion in mice: relationship to structural characteristics defined by mass spectrom-etry and nuclear magnetic resonance. J Pharm Sci 74:821–824.

Coggeshall RE, Zhou S, and Carlton SM (1997) Opioid receptors on peripheral sen-sory axons. Brain Res 764:126–132.

Craft RM and Leitl MD (2006) Potentiation of morphine antinociception by pento-barbital in female vs. male rats. Pain 121:115–125.

Crooks PA, Kottayil SG, Al-Ghananeem AM, Byrn SR, and Butterfield DA (2006)Opiate receptor binding properties of morphine-, dihydromorphine-, and codeine6-O-sulfate ester congeners. Bioorg Med Chem Lett 16:4291–4295.

Frances B, Gout R, Monsarrat B, Cros J, and Zajac JM (1992) Further evidence thatmorphine-6 beta-glucuronide is a more potent opioid agonist than morphine. JPharmacol Exp Ther 262:25–31.

Frölich N, Dees C, Paetz C, Ren X, Lohse MJ, Nikolaev VO, and Zenk MH (2011)Distinct pharmacological properties of morphine metabolites at G(i)-protein andb-arrestin signaling pathways activated by the human m-opioid receptor. BiochemPharmacol 81:1248–1254.

Fürst S, Riba P, Friedmann T, Tímar J, Al-Khrasani M, Obara I, Makuch W, SpeteaM, Schütz J, Przewlocki R, et al. (2005) Peripheral versus central antinociceptiveactions of 6-amino acid-substituted derivatives of 14-O-methyloxymorphone inacute and inflammatory pain in the rat. J Pharmacol Exp Ther 312:609–618.

Gupta A, Bodin L, Holmström B, and Berggren L (2001) A systematic review of theperipheral analgesic effects of intraarticular morphine. Anesth Analg 93:761–770.

Holtman JR, Jr, Crooks PA, Johnson-Hardy J, and Wala EP (2010) Antinociceptiveeffects and toxicity of morphine-6-O-sulfate sodium salt in rat models of pain. Eur JPharmacol 648:87–94.

Kalso E, Smith L, McQuay HJ, and Andrew Moore R (2002) No pain, no gain: clinicalexcellence and scientific rigour–lessons learned from IA morphine. Pain 98:269–275.

Kalso E, Tramèr MR, Carroll D, McQuay HJ, and Moore RA (1997) Pain relief fromintra-articular morphine after knee surgery: a qualitative systematic review. Pain71:127–134.

Khalefa BI, Mousa SA, Shaqura M, Lackó E, Hosztafi S, Riba P, Schäfer M,Ferdinandy P, Fürst S, and Al-Khrasani M (2013) Peripheral antinociceptive ef-ficacy and potency of a novel opioid compound 14-O-MeM6SU in comparison toknown peptide and non-peptide opioid agonists in a rat model of inflammatorypain. Eur J Pharmacol 713:54–57.

Khalefa BI, Shaqura M, Al-Khrasani M, Fürst S, Mousa SA, and Schäfer M (2012)Relative contributions of peripheral versus supraspinal or spinal opioid receptorsto the antinociception of systemic opioids. Eur J Pain 16:690–705.

180 Lackó et al.

at ASPE

T Journals on A

pril 13, 2020jpet.aspetjournals.org

Dow

nloaded from

Koster R, Anderson M, and De Beer E (1959) Acetic acid for analgesic screening. JFed Proc 18:412–415.

Kuo A, Wyse BD, Meutermans W, and Smith MT (2015) In vivo profiling of sevencommon opioids for antinociception, constipation and respiratory depression: notwo opioids have the same profile. Br J Pharmacol 172:532–548.

Lacko E, Varadi A, Rapavi R, Zador F, Riba P, Benyhe S, Borsodi A, Hosztafi S,Timar J, Noszal B, et al. (2012) A novel m-opioid receptor ligand with high in vitroand in vivo agonist efficacy. Curr Med Chem 19:4699–4707.

Lewanowitsch T and Irvine RJ (2002) Naloxone methiodide reverses opioid-inducedrespiratory depression and analgesia without withdrawal. Eur J Pharmacol 445:61–67.

Mcguire JL, Awouters F, and Niemegeers CJ (1978) Interaction of loperamide anddiphenoxylate with ethanol and methohexital. Arch Int Pharmacodyn Ther 236:51–59.

Mousa SA, Cheppudira BP, Shaqura M, Fischer O, Hofmann J, Hellweg R,and Schäfer M (2007) Nerve growth factor governs the enhanced ability of opioidsto suppress inflammatory pain. Brain 130:502–513.

Nagasaka H, Awad H, and Yaksh TL (1996) Peripheral and spinal actions of opioidsin the blockade of the autonomic response evoked by compression of the inflamedknee joint. Anesthesiology 85:808–816.

Riba P, Ben Y, Nguyen TM, Furst S, Schiller PW, and Lee NM (2002) [Dmt(1)]DALDA is highly selective and potent at mu opioid receptors, but is not cross-tolerant with systemic morphine. Curr Med Chem 9:31–39.

Rittner HL, Amasheh S, Moshourab R, Hackel D, Yamdeu RS, Mousa SA, FrommM, Stein C, and Brack A (2012) Modulation of tight junction proteins in theperineurium to facilitate peripheral opioid analgesia. Anesthesiology 116:1323–1334.

Rittner HL, Brack A, Machelska H, Mousa SA, Bauer M, Schäfer M, and Stein C(2001) Opioid peptide-expressing leukocytes: identification, recruitment, and si-multaneously increasing inhibition of inflammatory pain. Anesthesiology 95:500–508.

Rittner HL, Machelska H, and Stein C (2005) Leukocytes in the regulation of painand analgesia. J Leukoc Biol 78:1215–1222.

Sánchez-Fernández C, Montilla-García Á, González-Cano R, Nieto FR, Romero L,Artacho-Cordón A, Montes R, Fernández-Pastor B, Merlos M, Baeyens JM, et al.(2014) Modulation of peripheral m-opioid analgesia by s1 receptors. J PharmacolExp Ther 348:32–45.

Schäfer M, Carter L, and Stein C (1994) Interleukin 1 beta and corticotropin-releasing factor inhibit pain by releasing opioids from immune cells in inflamedtissue. Proc Natl Acad Sci USA 91:4219–4223.

Schäfer M, Imai Y, Uhl GR, and Stein C (1995) Inflammation enhances peripheralmu-opioid receptor-mediated analgesia, but not mu-opioid receptor transcription indorsal root ganglia. Eur J Pharmacol 279:165–169.

Scheibner J, Trendelenburg AU, Hein L, Starke K, and Blandizzi C (2002) Alpha2-adrenoceptors in the enteric nervous system: a study in alpha 2A-adrenoceptor-deficient mice. Br J Pharmacol 135:697–704.

Skarke C, Darimont J, Schmidt H, Geisslinger G, and Lötsch J (2003) Analgesiceffects of morphine and morphine-6-glucuronide in a transcutaneous electrical painmodel in healthy volunteers. Clin Pharmacol Ther 73:107–121.

Stein C (2013) Opioids, sensory systems and chronic pain. Eur J Pharmacol 716:179–187.

Stein C, Hassan AH, Lehrberger K, Giefing J, and Yassouridis A (1993) Local an-algesic effect of endogenous opioid peptides. Lancet 342:321–324.

Stein C, Millan MJ, Shippenberg TS, Peter K, and Herz A (1989) Peripheral opioidreceptors mediating antinociception in inflammation. Evidence for involvement ofmu, delta and kappa receptors. J Pharmacol Exp Ther 248:1269–1275.

Stein C, Millan MJ, Yassouridis A, and Herz A (1988) Antinociceptive effects of mu-and kappa-agonists in inflammation are enhanced by a peripheral opioid receptor-specific mechanism. Eur J Pharmacol 155:255–264.

Stein C, Schäfer M, and Hassan AH (1995) Peripheral opioid receptors. Ann Med 27:219–221.

Tegeder I, Meier S, Burian M, Schmidt H, Geisslinger G, and Lötsch J (2003) Pe-ripheral opioid analgesia in experimental human pain models. Brain 126:1092–1102.

Vanderah TW, Schteingart CD, Trojnar J, Junien JL, Lai J, and Riviere PJ (2004)FE200041 (D-Phe-D-Phe-D-Nle-D-Arg-NH2): a peripheral efficacious kappa opioidagonist with unprecedented selectivity. J Pharmacol Exp Ther 310:326–333.

Zhou L, Zhang Q, Stein C, and Schäfer M (1998) Contribution of opioid receptors onprimary afferent versus sympathetic neurons to peripheral opioid analgesia. JPharmacol Exp Ther 286:1000–1006.

Zuckerman A, Bolan E, de Paulis T, Schmidt D, Spector S, and Pasternak GW (1999)Pharmacological characterization of morphine-6-sulfate and codeine-6-sulfate.Brain Res 842:1–5.

Address correspondence to: Dr. Mahmoud Al-Khrasani, Department ofPharmacology and Pharmacotherapy, Faculty of Medicine, SemmelweisUniversity, Nagyvárad tér 4, H-1089 Budapest, Hungary. E-mail: al-khra-sani.mahmoud@med.semmelweis-univ.hu

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