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Research ArticleBehavioral Pharmacology

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

Erzsébet Lackó, Pál Riba, Zoltán Giricz, András Váradi, Laura Cornic, Mihály Balogh, Kornél Király, Kata Csekő, Shaaban A. Mousa, Sándor Hosztafi, Michael Schäfer, Zoltán Sándor Zádori, Zsuzsanna Helyes, Péter Ferdinandy, Susanna Fürst and Mahmoud Al-Khrasani
Journal of Pharmacology and Experimental Therapeutics October 2016, 359 (1) 171-181; DOI: https://doi.org/10.1124/jpet.116.233551

Abstract

Growing data support peripheral opioid antinociceptive effects, particularly in inflammatory pain models. Here, we examined the 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 rat model of inflammatory pain induced by an injection of complete Freund’s adjuvant and in a mouse model of visceral pain evoked by acetic acid. Subcutaneous doses of 14-O-MeM6SU and M6SU up to 126 and 547 nmol/kg, respectively, produced significant and subcutaneous or intraplantar naloxone methiodide (NAL-M)–reversible antinociception in inflamed paws compared with noninflamed paws. Neither of these doses significantly affected thiobutabarbital-induced sleeping time or rat pulmonary parameters. However, the antinociceptive effects of higher doses were only partially reversed by NAL-M, indicating contribution of the central nervous system. In the mouse writhing test, 14-O-MeM6SU was more potent than M6SU after subcutaneous or intracerebroventricular injections. Both displayed high subcutaneous/intracerebroventricular ED50 ratios. The antinociceptive effects of subcutaneous 14-O-MeM6SU and M6SU up to 136 and 3043 nmol/kg, respectively, were fully antagonized by subcutaneous NAL-M. In addition, the test compounds inhibited mouse gastrointestinal transit in antinociceptive doses. Taken together, these findings suggest that systemic administration of the novel compound 14-O-MeM6SU similar to M6SU in specific dose ranges shows peripheral antinociception in rat and mouse inflammatory pain models without central adverse effects. These findings apply to male animals and must be confirmed in female animals. Therefore, titration of systemic doses of opioid compounds with limited access to the brain might offer peripheral antinociception of clinical importance.

Introduction

Opioid analgesics are the cornerstone in treatment of moderate-to-severe pain. They exert their antinociceptive action by activating opioid receptors. The majority of clinically used opioid analgesics have central adverse effects such as respiratory depression, development of opioid tolerance and dependence, and addiction liabilities. These effects strongly limit clinical use of these drugs. In addition to central opioid receptors, several studies support the existence of functional opioid 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 indicates that activation of these receptors also results in pain mitigation (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 be upregulated in inflamed tissues (Stein et al., 1989; Schäfer et al., 1995). To note, the peripheral antinociception of opioids in humans has been investigated in several randomized controlled clinical trials (Kalso et al., 1997; Gupta et al., 2001). Pooled analyses of data from 19 studies suitable for metaanalysis showed only a moderate analgesic effect after administration of intra-articular morphine compared with placebo 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 after its systemic administration in human volunteers (Tegeder et al., 2003). Because of its high hydrophilicity, M6G has a considerable delay between peak plasma concentrations and peak central opioid effects such that peripheral antinociceptive effects can be detected within this time window (Skarke et al., 2003; Tegeder et al., 2003). M6G was recently described as a partial agonist with respect to its antinociceptive effects but as a full agonist regarding its central effect of respiratory depression (Kuo et al., 2015), which may prevent its use as a peripheral analgesic. In addition, the number of µ-opioid receptor (MOR) agonists with high efficacy but restricted access to the central nervous system (CNS) is very low. Therefore, the design of opioids with high hydrophilicity and high efficacy may 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. Inducing inflammation by injecting CFA into the hind paw of animals (e.g., rats) has long been used as a strategy to demonstrate the peripheral antinociceptive actions of opioid agonists (Stein et al., 1988; Zhou et al., 1998; Al-Khrasani et al., 2007). We recently designed and synthesized a new MOR agonist, 14-O-methylmorphine-6-O-sulfate (14-O-MeM6SU), which displays high affinity for MOR and strong antinociceptive effects in acute thermal nociceptive tests (Lacko et al., 2012). 14-O-MeM6SU has higher affinity than morphine-6-O-sulfate (M6SU) or morphine for MORs or δ-opioid receptors and similar affinity for κ-opioid receptors. However, the δ-opioid receptor/MOR affinity ratio of 14-O-MeM6SU was smaller than that of M6SU, whereas the κ-opioid receptor/MOR ratio was higher for 14-O-MeM6SU. 14-O-MeM6SU acted as a full agonist in a functional 5′-O-(3-[35S]thio)triphosphate binding assay, whereas morphine and M6SU were partial agonists (Lacko et al., 2012).

We recently showed that topical administration of 14-O-MeM6SU strongly attenuates CFA-induced inflammatory pain in rats (Khalefa et al., 2013). However, to our knowledge, its peripheral antinociceptive action after systemic (subcutaneous) administration has not yet been tested. M6SU has been suggested as a potential candidate for the development of novel opioid agents to treat nociceptive, neuropathic, and mixed pain states (Holtman et al., 2010). In addition, M6SU was recently reported to have a good safety profile, since there is a clear separation between the doses of produced antinociception versus adverse effects (Holtman et al., 2010). These reports 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 subcutaneous administration in rats with CFA-induced inflammatory pain and in mice with acetic acid–induced pain. Additional objectives were 1) to determine the site of analgesic action by use of naloxone methiodide (NAL-M), the peripherally restricted opioid antagonist); 2) to study the barbiturate anesthesia-potentiating effect of 14-O-MeM6SU and M6SU to detect the central or peripheral target of the drugs; and 3) to test the action of 14-O-MeM6SU and M6SU on gastrointestinal transit and respiratory function.

Materials and Methods

Animals.

Male Wistar rats (200–300 g, Animal House of Semmelweis University) and male NMRI mice (20–30 g, Toxicoop, Hungary) were used for all tests. Animals were housed in a room with cages lined with ground corncob bedding, with a temperature of 20 ± 2°C under a 12-hour/12-hour light/dark cycle, in the local animal house of the Semmelweis University Departments of Pharmacology and Pharmacotherapy (Budapest, Hungary) and the University of Pécs Faculty of Medicine (Pécs, Hungary). Food and water were freely available. To minimize stress, rats were handled once daily for 3 subsequent days prior to the experimentation day. Experiments were performed in accordance with guidelines of the local animal care committee (PEI/001/276-4/2013) and the Ethical Board of Semmelweis University based on the Declaration of the European Communities Council Directives (86/609/ECC).

Drugs.

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

Fig. 1.
Fig. 1.

Chemical structures of morphine, M6SU, 14-O-MeM6SU, and NAL-M.

Induction of Inflammation in Rats.

Rats received an intraplantar injection of 0.15 ml CFA (Calbiochem, San Diego, CA), a water-in-oil emulsion of inactivated mycobacterium administered into the right hind paw, while under brief isoflurane (Willy Rüsch GmbH, Böblingen, Germany) anesthesia. This treatment consistently produces localized inflammation of the inoculated paw, characterized by an increase in paw volume, paw temperature, and infiltration with various types of immune cells (Rittner et al., 2001).

Induction of Pain in Mice (Mouse Writhing Test).

The acetic acid–induced writhing test was performed as previously described (Koster et al., 1959). Mice were injected intraperitoneally with 0.2 ml 0.6% acetic acid aqueous solution to produce the writhing reaction, characterized by contractions of the abdominal musculature followed by extension of the hind limbs.

Nociceptive Testing in Rats with CFA-Evoked Hyperalgesia.

On days 4 and 7 after the intraplantar CFA injection, baseline (pretest compound) paw pressure thresholds (PPTs) of inflamed and noninflamed paws were assessed by paw pressure algesiometry (modified Randall–Selitto test; Ugo Basile, Comerio, Italy) as described in detail previously (Mousa et al., 2007; Khalefa et al., 2012). PPTs were then reevaluated at 30, 60, and 120 minutes after subcutaneous drug administration, using an arbitrary cut-off weight of twice the control and expressed as percentages.

Dose-Response Relationships in Rats.

The antinociceptive effects of subcutaneous 14-O-MeM6SU and M6SU were examined in these 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 both paws after subcutaneous drug administration was compared with the baseline value obtained before drug treatment in ipsilateral or contralateral paws. Doses of each drug that produced a 60%–80% antinociceptive effect in inflamed paws, without a significant effect on the contralateral noninflamed paws, were selected for experiments designed to analyze the antagonism of NAL-M. In these experiments NAL-M (21.3 µmol) was coadministered with the test compounds or at 30 minutes, when the peak effect was achieved at 60 minutes. In another series of experiments, NAL-M (0.43 µmol/rat, intraplantarly) was injected 5 minutes prior to measurement (at 25 minutes or 55 minutes, when the peak effect was achieved at 30 and 60 minutes, respectively).

Dose-Response Relationships in Mice.

Groups of mice were injected subcutaneously or intracerebroventricularly with different doses of 14-O-MeM6SU or M6SU followed 15 minutes later by an intraperitoneal injection of 0.6% acetic acid solution. Each mouse was then placed in individual transparent Plexiglas chambers. Five minutes after the acetic acid injection, the number of writhes was counted during a 10-minute observation period. To determine the number of writhes in control groups, animals were subcutaneously or intracerebroventricularly injected with 0.9% saline solution before they were intraperitoneally injected with 0.6% acetic acid using a similar protocol as for the test drugs. Subcutaneous NAL-M was coadministered with the respective agonist in experiments in which antagonist action was assessed. Assessments were performed 20 minutes after subcutaneous opioid agonists as described above by recording the number of writhes for each mouse for a 10-minute time period beginning 5 minutes after the intraperitoneal injection of acetic acid.

Determination of Thiobutabarbital-Induced Sleeping Time.

Animals received intravenous saline or thiobutabarbital (153 µmol/kg) and then were placed on their left side. Sleeping time in minutes was documented and was considered to end when animals spontaneously turned to the opposite right position (“righting reflex”). Anesthesia-potentiating effects of test drugs were studied by subcutaneous administration. Thiobutabarbital was injected intravenously at the time of peak antinociceptive action of test compounds (60 minutes after subcutaneous 14-O-MeM6SU and 30 minutes after subcutaneous M6SU).

Determination the Effect of 14-O-MeM6SU and M6SU on Gastrointestinal Transit.

The effect of 14-O-MeM6SU and M6SU compared with that of morphine on gastrointestinal transit was measured in vivo by using the charcoal meal method (Scheibner et al., 2002). Briefly, male NMRI mice (20–25 g) were fasted 6 hours prior to the experiments, with free access to water. At the time of the experiment, a charcoal suspension (10% charcoal in 5% gum arabic) was given in a volume of 0.25 ml per mouse by an oral gavage, followed by subcutaneous administration of test compounds (0.1 ml/10 g). Mice were decapitated 30 minutes after drug or saline administration and their small intestines were removed. The distance traveled by the charcoal suspension was expressed as a percentage of total small intestine length. The doses that caused 50% inhibition of gastrointestinal transit (ID50) were calculated from the linear regression of dose-response curves.

Respiratory Function Tests.

Respiratory function measurements were performed by unrestrained whole-body plethysmography (WBP) in conscious, spontaneously breathing animals 30 and 60 minutes after a subcutaneous injection of saline, 14-O-MeM6SU, M6SU, or morphine. Rats were placed in the chamber of a whole-body plethysmograph (PLY 3213; Buxco Europe Ltd., Winchester, UK). The flow transducers (TRD5700; Buxco Europe Ltd.) were connected to the preamplifier module, which digitized the signals via an analog-to-digital converter (MAX2270; Buxco Europe Ltd.). Ventilation parameters (frequency, tidal volume, minute ventilation, time of inspiration, peak inspiratory flow, time of expiration, peak expiratory flow, and relaxation time) were measured every 10 seconds during the 15-minute acquisition times and were averaged by BioSystem XA Software for Windows (Buxco Research Systems, Wilmington, NC).

Data Analysis.

GraphPad Prism software (version 5.00 for Windows; GraphPad Software Inc., San Diego, CA) was used for data analysis. Two-way analysis of variance (ANOVA) with Bonferroni’s post hoc test was performed for comparison between inflamed and noninflamed 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 writhing test, the antinociceptive effect is presented as the percentage decrease in the number of writhes and is calculated according to the formula: Percent inhibition of writhing = (C − T)/ C × 100, where C is the mean number of writhes in control animals and T is the number of writhes in drug-treated mice. Dose-response relationships of the percent inhibition of writhing were constructed and the dose necessary to produce a 50% effect (ED50) and 95% confidence limits were calculated. Differences between groups were determined by ANOVA, followed by the Newman–Keuls post hoc test. Statistical analysis of unrestrained WBP was performed by repeated-measures ANOVA followed by the Tukey multiple comparisons test (n = 6 per group). Results were considered statistically significant when P < 0.05.

Results

Antinociceptive Effects of 14-O-MeM6SU and M6SU after Systemic Administration in Rats with CFA-Induced Inflammatory Pain.

CFA treatment reduced the PPT to approximately 65% ± 2% (n = 70) and 70% ± 2% (n = 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 analgesic actions in CFA-induced inflammatory pain (Fig. 2). M6SU produced peak analgesic effects at 30 minutes, whereas 14-O-MeM6SU did so at 60 minutes (Table 1). The antinociceptive actions of the test compounds were significant in inflamed paws compared with noninflamed paws at the doses presented in Fig. 3. The analgesic effects of subcutaneous 14-O-MeM6SU and M6SU did not differ at doses over 506 nmol/kg and 4379 nmol/kg, respectively. The antinociceptive actions of subcutaneous 14-O-MeM6SU (126, 253, and 506 nmol/kg) and M6SU (547, 1095, and 2189 nmol/kg) were further tested for their peripheral analgesic actions in inflamed paws in separate experiments (see below). Treatment with saline or NAL-M alone had no effects on PPTs of inflamed paws or noninflamed paws (Figs. 2, 4, and 5).

Fig. 2.
Fig. 2.

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

View this table:
TABLE 1

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

Fig. 3.
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 value represents the mean ± S.E.M. Data were obtained 30 minutes after injection of M6SU and 60 minutes after injection of 14-O-MeM6SU. The pound symbol represents significant differences between the lowest and highest doses. ***p < 0.001 (significant differences between inflamed and noninflamed paws, two-way ANOVA).

Fig. 4.
Fig. 4.

The antagonist effect of NAL-M (21.3 µmol/kg s.c. or 0.43 µmol/rat i.pl.) against subcutaneous antinociceptive effects of 14-O-MeM6SU (126 nmol/kg) and M6SU (547 nmol/kg) in inflamed rat paws. Drugs were delivered in a volume of 5 ml/kg body weight and 100 µl/rat for subcutaneous and intraplantar administration, respectively. Each value represents the mean ± S.E.M. Each data point was obtained 30 minutes after injection of M6SU (n = 17–18), saline (n = 18–35), or NAL-M (n = 10–12) and 60 minutes after injection of 14-O-MeM6SU (n = 7–13), saline (n = 6–35), or NAL-M (n = 6–12). *P < 0.05; ***P < 0.001 (significant differences versus the effect of agonist in inflamed paw; one-way ANOVA, Newman–Keuls post hoc test).

Fig. 5.
Fig. 5.

Antagonist effect of NAL-M (21.3 µmol/kg s.c.) against the subcutaneous antinociceptive effects of 14-O-MeM6SU (253 and 506 nmol/kg) and M6SU (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 value represents the mean ± S.E.M. Each data point was obtained 30 minutes after 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-way ANOVA, Newman–Keuls post hoc test); ***P < 0.001 (versus saline in the inflamed paw, one-way ANOVA, Newman–Keuls post hoc test); ###P < 0.001 (versus drug plus NAL-M); ++P < 0.01 (versus 1095 nmol drug plus NAL-M).

Antagonist Effects of Subcutaneous and Intraplantar NAL-M on the Antinociceptive Actions of Subcutaneous 14-O-MeM6SU or M6SU in Rats with CFA-Induced Inflammatory Pain.

The analgesic effects produced by subcutaneous 14-O-MeM6SU (126 nmol/kg) and M6SU (547 nmol/kg) were antagonized by subcutaneous administration of NAL-M (21.3 µmol/kg) (Fig. 4). In other experiments, the antinociceptive effects of the same doses of test agonists were also significantly reduced by intraplantar NAL-M (0.43 µmol/rat) (Fig. 4). No differences were observed between PPTs of animals injected with saline or NAL-M (ANOVA, followed by the Newman–Keuls post hoc test, Fig. 4). In another experiment, we also tested the antagonist effect of subcutaneous NAL-M (21.3 µmol/kg) on the antinociceptive 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 reversed the antinociceptive effect of 14-O-MeM6SU and totally reversed the antinociceptive effect of the 1095-nmol/kg M6SU dose. However, NAL-M failed to reverse the antinociceptive effect of the 2189-nmol/kg M6SU dose (Fig. 5). These results indicate the contribution of the CNS to the total antinociception of systemically administered test compounds at higher doses.

Potentiation of Thiobutabarbital-Induced Anesthesia.

Thiobutabarbital (153 µmol/kg i.v.) produced a sleeping time of 10 ± 3, 10 ± 5, and 8 ± 4 minutes in the presence of subcutaneous saline, 14-O-MeM6SU (126 nmol/kg), and M6SU (547 nmol/kg), respectively (Fig. 6). At higher agonist doses, sleeping time was longer compared with saline (Fig. 6).

Fig. 6.
Fig. 6.

Effect of subcutaneous 14-O-MeM6SU and M6SU on thiobutabarbital (153 μmol/kg i.v.)–induced sleeping time (n = 5–10). *P < 0.05 versus saline (one-way ANOVA, Newman–Keuls post hoc test).

Antinociceptive Effects of 14-O-MeM6SU Compared with M6SU in the Mouse Writhing Test.

Injection of a 0.6% acetic acid solution into the peritoneal cavity of mice administered subcutaneous or intracerebroventricular saline resulted in an average of 43.9 ± 1.5 (n = 43) writhes during the 10-minute period. As shown in Fig. 7, subcutaneous or intracerebroventricular administration (20 minutes before testing) of all of the agonists produced a dose-dependent antinociceptive action. Table 2 provides calculated ED50 values with 95% confidence intervals at 20 minutes. 14-O-MeM6SU produced a more potent inhibitory effect than M6SU on acetic acid–induced writhing in mice after administration via subcutaneous and intracerebroventricular routes (Table 2).

Fig. 7.
Fig. 7.

Antinociceptive dose-response curves of subcutaneously (A) or intracerebroventricularly (B) injected 14-O-MeM6SU and M6SU in the mouse writhing test. Points represent the mean ± S.E.M for groups of four to five mice.

View this table:
TABLE 2

Antinociceptive potencies of 14-O-MeM6SU, M6SU, and morphine against acetic acid–induced nociception in the mouse writhing test after 20 minutes of subcutaneous and intracerebroventricular administration

At least four animals per dose group and three to four doses were used for each ED50 determination.

14-O-MeM6SU was about 23-fold more potent than M6SU after subcutaneous administration, whereas 14-O-MeM6SU proved to be only 5-fold more active than M6SU in inhibition of writhing after intracerebroventricular administration. However, large subcutaneous/intracerebroventricular potency ratios 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 and the site of action of 14-O-MeM6SU and M6SU in acetic acid–induced writhing in mice, the effects of the test agonists were assessed after systemic coadministration with the quaternary opioid antagonist NAL-M (21.3 µmol/kg s.c). Our results show that a subcutaneous equipotent analgesic dose of 14-O-MeM6SU (136 nmol/kg) and M6SU (3043 nmol/kg) significantly decreased the number of writhes at 20 minutes after administration. Coadministration of NAL-M significantly antagonized the antinociceptive effect of test opioids, as indicated in Fig. 8. NAL-M treatment failed to affect the number of writhes and was found to be similar to the values obtained after saline treatment (Fig. 8). At higher doses, 14-O-MeM6SU (272 nmol/kg) also showed NAL-M–reversible antinociception (Fig. 8).

Fig. 8.
Fig. 8.

Antagonist action of coadministered NAL-M (21.3 µmol/kg) on the antinociceptive effect of 14-O-MeM6SU or M6SU after 20-minute subcutaneous administration in the writhing response induced by acetic acid (intraperitoneally) in mice. Data are expressed as the mean ± S.E.M. for groups of 4–12 mice. *P < 0.05; **P < 0.01; ***P < 0.001 (versus the other groups, one-way ANOVA, Newman–Keuls post hoc test).

Inhibitory Effect of Systemic 14-O-MeM6SU and M6SU on Gastrointestinal Transit in Mice.

Subcutaneously administered 14-O-MeM6SU, M6SU, and morphine inhibited gastrointestinal transit of charcoal in dose-dependent manner. The calculated ID50 values and confidence intervals were 250 nmol/kg (205–305), 325 nmol/kg (70–1517), and 2228 nmol/kg (666–7455) for 14-O-MeM6SU, M6SU, and morphine, respectively. These results indicate that the test compounds inhibit gastrointestinal transit in antinociceptive doses.

Respiratory Effects of 14-O-MeM6SU and M6SU Compared with Morphine in Awake, Unrestrained Rats.

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 the respiratory parameters determined by unrestrained WBP (frequency, tidal volume, minute ventilation, time of inspiration, peak inspiratory flow, time of expiration, peak expiratory flow, and relaxation time) showed significant differences between the saline-treated control or drug-treated groups 30 and 60 minutes after subcutaneous injection; however, this more clinically relevant test remains subject to more elaborate future investigations. None of the drugs caused any sedative effects; the animals were at rest by the end of the measurements, but they woke when the WBP chambers were opened.

Fig. 9.
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), and morphine (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.

Discussion

This study aimed to further investigate the recently synthesized 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 model of inflammatory pain and a mouse model of visceral pain based on our previous work (Al-Khrasani et al., 2007; Khalefa et al., 2013). Our data clearly show that 14-O-MeM6SU and M6SU produced peripheral antinociceptive effects in a CFA-induced inflammatory pain model or acetic acid–evoked visceral pain over a specific dose/concentration range of their systemic (subcutaneous) administration. The antinociceptive action on the former 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), peripherally mediated antinociception of this compound and its parent molecule, M6SU, could also be established by its systemic application. It is important to emphasize that antinociceptive effects of 14-O-MeM6SU and M6SU have not yet been tested in CFA-induced inflammatory pain and acetic acid–induced pain in rats and mice, respectively. Our data confirm previous reports on the peripheral antinociceptive actions of systemic opioids in the inflammatory pain model (Stein et al., 1988; Al-Khrasani et al., 2012). These results seem to be in contrast with findings that intravenous morphine elicits antinociceptive effects exclusively by the activation of a CNS MOR but not on peripheral sensory nerve terminals, since NAL-M antagonized these effects after intracerebroventricular and intrathecal administration but not after intraplantar delivery (Khalefa et al., 2012). In our work, test compounds could also elicit central antinociception at higher doses. This was reflected by the phenomenon that differences in the antinociceptive effects of 14-O-MeM6SU and M6SU between inflamed and noninflamed paws gradually declined but a clear peripheral action was demonstrated at a lower dose range in the inflamed paw. These results show that careful dose titration of the MOR agonists 14-O-MeM6SU and M6SU during their systemic administration can reveal a distinct dose range in which antinociceptive effects are exerted exclusively by the activation of peripheral MOR at the inflammation site. At these doses, PPTs on the contralateral side were not significantly elevated.

The possible explanation for why the test compounds produced antinociception in inflamed paws compared with noninflamed paws might be the increase in the number of opioid receptors in the nerve endings (Schäfer et al., 1994) and the disturbance of the perineural barrier during inflammation (Antonijevic et al., 1995). The latter condition might facilitate the access of opioid agonists to their receptors, which seem to be somewhat hidden (reserved) when inflammation is absent. The increase in the number of accessible opioid receptors results in enhanced peripheral opioid antinociceptive efficacy in inflammatory pain, as was previously reported (Antonijevic et al., 1995; Rittner et al., 2005, 2012).

To demonstrate the peripheral antinociceptive action of subcutaneous opioids, the peripherally acting opioid antagonist NAL-M (Bianchi et al., 1982; Lewanowitsch and Irvine, 2002; Riba et al., 2002) was used. Subcutaneous or intraplantar 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) produced antinociception of a peripheral origin. Our data are in agreement with previous studies using this experimental model of pain and the same route of administration (Stein et al., 1988). However, test compounds at higher systemic doses showed CNS-mediated antinociception, because systemic NAL-M failed to fully abolish it (Fig. 5).

The obtained results in CFA-induced inflammatory pain after systemic administration encouraged us to extend our work 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 writhing test was used in these experiments. The acetic acid–evoked writhing assay, one of the most well established and widely used experimental models to assess the pain-relieving actions of either nonsteroidal anti-inflammatory drugs or opioids, is also suitable to demonstrate the central and peripheral components of nociception. However, until now, the antinociceptive effect of 14-O-MeM6SU compared with M6SU in this animal model of visceral pain has not been studied. In this model, the antinociceptive effects of subcutaneous or intracerebroventricular 14-O-MeM6SU were also studied and compared with that of M6SU. 14-O-MeM6SU showed more potent antinociceptive action than M6SU after both routes of administration, in accordance with data previously published by our group (Lacko et al., 2012). In addition, similarly to data obtained in a rat model of inflammatory pain, systemic or central administration of 14-O-MeM6SU resulted in stronger antinociception than that of M6SU (Table 2). However, the subcutaneous/intracerebroventricular ratio was higher for M6SU than for 14-O-MeM6SU. Regarding the antinociceptive effect, our results are in agreement with data we reported previously in a thermal pain model (Lacko et al., 2012). 14-O-MeM6SU was 23 times more potent than M6SU after systemic administration but only 5 times more potent than M6SU after central dosing. Previous reports (Frances et al., 1992; Al-Khrasani et al., 2007) indicate a lower subcutaneous/intracerebroventricular ratio for morphine (4215) and a larger ratio for M6G (5840) that is similar to 14-O-MeM6SU (Table 2). Data reported by Brown et al. (1985) attributed the weak antinociceptive action of subcutaneous M6SU to its limited access to the CNS. It is worth noting that 14-O-MeM6SU also displayed a high systemic/central dose ratio compared with other opioids such as morphine or fentanyl (Fürst et al., 2005; Lacko et al., 2012). Therefore, 14-O-MeM6SU plausibly has limited CNS penetration, similarly to M6SU. However, 14-O-MeM6SU has an advantage over M6SU because it displays higher efficacy and affinity, reflecting its stronger antinociceptive action as previously described (Lacko et al., 2012). M6SU and morphine are strong partial agonists in MOR-mediated Gi protein activation and β-arrestin recruitment to opioid receptors (Frölich et al., 2011). Thus far, there are no data on the effect of 14-O-MeM6SU on β-arrestin recruitment to opioid receptors, but this is an area for future research.

14-O-MeM6SU (136 nmol/kg) or M6SU (3043 nmol/kg) (Fig. 8) showed peripheral antinociceptive effects in the mouse writhing assay after subcutaneous administration, since the coadministered quaternary opioid antagonist NAL-M significantly reversed the effects of the test compounds. Systemically applied quaternary opioid antagonists have been used to localize the site of opioid antinociceptive action because they do not readily cross the blood–brain barrier.

Another approach to study peripheral opioid activity might be to assess whether test compounds evoke motor function impairment in antinociceptive doses by subcutaneous administration. Vanderah et al. (2004) applied the latter method to demonstrate peripheral antinociception of d-amino acid tetrapeptide (d-Phe-d-Phe-d-Nle-d-Arg-NH2) without motor impairment in the mouse writhing test (Vanderah et al., 2004). We also studied the central action of anesthetics (e.g., intravenous thiobutabarbital) in the presence of opioids. CNS-depressing drugs are reported to have longer action by coadministration of opioids (Mcguire et al., 1978; Craft and Leitl, 2006). Therefore, we further extended our work to examine the actions of systemically injected test compounds on thiobutabarbital-induced sleeping time in rats. NAL-M–reversible antinociceptive doses of 14-O-MeM6SU or M6SU failed to potentiate thiobutabarbital-induced sleeping time (Fig. 6). At higher doses, both compounds lengthened sleeping time. Previous studies showed that intraperitoneally administered M6SU elicited motor impairment and decreased locomotor activity at doses of 5 mg/kg or higher (Holtman et al., 2010). In our study, the dose of M6SU studied for its peripheral antinociception was 547 nmol/kg (0.2 mg/kg), which is markedly lower compared with doses tested previously (Holtman et al., 2010). Therefore, these data strengthen our results that at this dose, M6SU activates peripheral opioid receptors at the site of inflammation in rats. In our studies, 14-O-MeM6SU and M6SU inhibited gastrointestinal peristalsis, which is in accordance with previous studies (Holtman et al., 2010). However, these drugs also induced significant peripheral antinociception at the same dose range, clearly indicating that they are superior to another peripherally acting opioid, loperamide, which failed to produce antinociception in doses producing constipation in mice (Sánchez-Fernández et al., 2014).

Furthermore, test compounds and morphine in doses prolonging thiobutabarbital-induced sleeping time showed no significant alterations in respiratory parameters compared with the control group, indicating that the drugs did not cause respiratory depression in the tested dose range.

In summary, in specific systemic dose ranges, 14-O-MeM6SU or M6SU elicited significant peripheral antinociception in mouse visceral pain and in inflamed rat paws compared with noninflamed paws. These actions were confirmed by the antagonism of the peripherally acting opioid antagonist NAL-M. In addition, these doses did not potentiate the sleeping time of general anesthetic agent thiobutabarbital nor did they affect respiration indicating poor access to the CNS at these doses. However, systemically administered NAL-M partially antagonized the antinociception of test compounds of higher doses, which indicates the contribution of the CNS. Titration of systemic doses of opioid compounds with limited access to the brain might offer peripheral analgesia of clinical importance. These data indicate that the development of opioid drugs such as M6SU and its analogs, which target the pain in the periphery with a wide safety profile, may represent a new generation of opioids for the treatment of inflammatory pain.

Acknowledgments

The authors thank Mária Molnár Pénzes for skilled technical assistance.

Authorship Contributions

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

Conducted experiments: Lackó, Riba, Giricz, Cornic, Balogh, Király, Csekő.

Contributed new reagents or analytic tools: Váradi, Hosztafi.

Performed data analysis: Csekő, Mousa, Zádori.

Wrote or contributed to the writing of the manuscript: Mousa, Schäfer, Helyes, Ferdinandy, Fürst, Al-Khrasani.

Footnotes

    • Received March 16, 2016.
    • Accepted July 18, 2016.

  • E.L. and P.R. contributed equally to this work.

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

  • 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
µ-opioid receptor
NAL-M
naloxone methiodide
PPT
paw pressure threshold
WBP
whole-body plethysmography

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Research ArticleBehavioral Pharmacology

Novel Morphine Analog with Peripheral Antinociception

Erzsébet Lackó, Pál Riba, Zoltán Giricz, András Váradi, Laura Cornic, Mihály Balogh, Kornél Király, Kata Csekő, Shaaban A. Mousa, Sándor Hosztafi, Michael Schäfer, Zoltán Sándor Zádori, Zsuzsanna Helyes, Péter Ferdinandy, Susanna Fürst and Mahmoud Al-Khrasani
Journal of Pharmacology and Experimental Therapeutics October 1, 2016, 359 (1) 171-181; DOI: https://doi.org/10.1124/jpet.116.233551
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