Indiscriminate activation of opioid receptors provides pain relief but also severe central and intestinal side effects. We hypothesized that exploiting pathological (rather than physiological) conformation dynamics of opioid receptor-ligand interactions might yield ligands without adverse actions. By computer simulations at low pH, a hallmark of injured tissue, we designed an agonist that, because of its low acid dissociation constant, selectively activates peripheral μ-opioid receptors at the source of pain generation. Unlike the conventional opioid fentanyl, this agonist showed pH-sensitive binding, heterotrimeric guanine nucleotide-binding protein (G protein) subunit dissociation by fluorescence resonance energy transfer, and adenosine 3',5'-monophosphate inhibition in vitro It produced injury-restricted analgesia in rats with different types of inflammatory pain without exhibiting respiratory depression, sedation, constipation, or addiction potential.

Endometriosis occurs in approximately 10% of women and is associated with persistent pelvic pain. It is defined by the presence of endometrial tissue (lesions) outside the uterus, most commonly on the peritoneum. Peripheral neuroinflammation, a process characterized by the infiltration of nerve fibers and macrophages into lesions, plays a pivotal role in endometriosis-associated pain. Our objective was to determine the role of estradiol (E2) in regulating the interaction between macrophages and nerves in peritoneal endometriosis. By using human tissues and a mouse model of endometriosis, we demonstrate that macrophages in lesions recovered from women and mice are immunopositive for estrogen receptor b, with up to 20% being estrogen receptor a positive. In mice, treatment with E2 increased the number of macrophages in lesions as well as concentrations of mRNAs encoded by Csf1, Nt3, and the tyrosine kinase neurotrophin receptor, TrkB. By using in vitro models, we determined that the treatment of rat dorsal root ganglia neurons with E2 increased mRNA concentrations of the chemokine C-C motif ligand 2 that stimulated migration of colony-stimulating factor 1edifferentiated macrophages. Conversely, incubation of colony-stimulating factor 1 macrophages with E2 increased concentrations of brainderived neurotrophic factor and neurotrophin 3, which stimulated neurite outgrowth from ganglia explants. In summary, we demonstrate a key role for E2 in stimulating macrophage-nerve interactions, providing novel evidence that endometriosis is an estrogen-dependent neuroinflammatory disorder. Endometriosis affects 10% of reproductive age women and is associated with persistent pelvic pain. 1 It is defined by the presence of endometrial-like tissue (lesions) found outside the uterus, most commonly on the peritoneum. The mechanisms underlying endometriosis-associated pain are poorly understood, but it has been postulated that estrogen-dependent neuroinflammation may be involved. 2 Notably, the presence of endometrial tissue fragments on the peritoneum elicits an immune response, including recruitment of macrophages, 3 blood vessels, and nerve fibers into the resultant lesions. 4,5 Within the lesions, CD68 þ macrophages have been detected in close association with nerve fibers. 6 Studies investigating macrophage activation and recruitment have revealed that endometriosis-associated macrophages exhibit a phenotype consistent with the alternative end of the macrophage activation spectrum. 7,8 In a mouse model of endometriosis that included cell transfer of polarized macrophages, Bacci et al 7 reported that mice injected with proinflammatory macrophages [macrophage (interferon g)] developed microscopic lesions, but those injected with alternatively activated macrophages [macrophage (IL-4)] developed larger lesions with a well-developed vasculature. Our studies in a mouse model of endometriosis have revealed that macrophages resident in peritoneal lesions can originate from both the peritoneum and the endometrium. 9

Novel pain killers without adverse effects are urgently needed. Opioids induce central and intestinal side effects such as respiratory depression, sedation, addiction, and constipation. We have recently shown that a newly designed agonist with a reduced acid dissociation constant (pKa) abolished pain by selectively activating peripheral μ-opioid receptors (MOR) in inflamed (acidic) tissues without eliciting side effects. Here, we extended this concept in that pKa reduction to 7.22 was achieved by placing a fluorine atom at the ethylidene bridge in the parental molecule fentanyl. The new compound (FF3) showed pH-sensitive MOR affinity, [35S]-GTPγS binding, and G protein dissociation by fluorescence resonance energy transfer. It produced injury-restricted analgesia in rat models of inflammatory, postoperative, abdominal, and neuropathic pain. At high dosages, FF3 induced sedation, motor disturbance, reward, constipation, and respiratory depression. These results support our hypothesis that a ligand’s pKa should be close to the pH of injured tissue to obtain analgesia without side effects.

No abstract

The non-selective activation of central and peripheral opioid receptors is a major shortcoming of currently available opioids. Targeting peripheral opioid receptors is a promising strategy to preclude side effects. Recently, we showed that fentanyl-derived μ-opioid receptor (MOR) agonists with reduced acid dissociation constants (pKa) due to introducing single fluorine atoms produced injury-restricted antinociception in rat models of inflammatory, postoperative and neuropathic pain. Here, we report that a new double-fluorinated compound (FF6) and fentanyl show similar pKa, MOR affinity and [35S]-GTPγS binding at low and physiological pH values. In vivo, FF6 produced antinociception in injured and non-injured tissue, and induced sedation and constipation. The comparison of several fentanyl derivatives revealed a correlation between pKa values and pH-dependent MOR activation, antinociception and side effects. An opioid ligand’s pKa value may be used as discriminating factor to design safer analgesics.

Currently, opioids targeting mu-opioid receptors are the most potent drugs for acute and cancer pain. However, opioids produce adverse side effects such as constipation, respiratory depression, or addiction potential. We recently developed (±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide (NFEPP), a compound that does not evoke central or intestinal side effects due to its selective activation of mu-opioid receptors at low pH in peripheral injured tissues. Although we demonstrated that NFEPP effectively abolishes injury-induced pain, hyperalgesia, and allodynia in rodents, the efficacy of NFEPP in nonevoked ongoing pain remains to be established. Here, we examined reward, locomotor activity, and defecation in rats with complete Freund's adjuvant-induced paw inflammation to compare fentanyl's and NFEPP's potentials to induce side effects and to inhibit spontaneous pain. We demonstrate that low, but not higher, doses of NFEPP produce conditioned place preference but not constipation or motor disturbance, in contrast to fentanyl. Using a peripherally restricted antagonist, we provide evidence that NFEPP-induced place preference is mediated by peripheral opioid receptors. Our results indicate that a low dose of NFEPP produces reward by abolishing spontaneous inflammatory pain.

This Article contains a typographical error in the figure legend for Figure 5."Correlation between pK a values of compounds with antinociception and side effects. (A,B) Antinociceptive effects as net AUC (4-12 μg/kg) of PPT (negative values result from subtraction of pre-CFA baseline PPT) at 15 min after i.v. injection of NFEPP, FF3, FF6 and Fen in inflamed (A) and contralateral, noninflamed (B) paws. AUC values were derived from curves generated by use of n = 9 (NFEPP and FF3 from 3,15 ), n = 8-10 (FF6, see also Fig. 3) and n = 19 (Fen) animals. (C) Constipation, as assessed by number of fecal boli 1 h after s.c. injection of agonists (30 μg/kg) in relation to the pK a of the substance. Maximum and minimum numbers (with 95% confidence intervals) of boli in controls (vehicle-treated) are shown by the dashed line. Fen (n = 30) and controls (n = 34) (always included in all experiments) are averaged across all experiments; NFEPP (n = 11); FF3 (n = 12); FF6 (n = 10). (D) Locomotor activity as assessed by the total distance travelled during 30 min after s.c. injection of agonists (30 μg/kg) in relation to the pK a of the substance. Maximum and minimum travelled distances (with 95% CI) in controls (vehicle-treated) are shown by the dashed line. Fen (n = 31) and controls (n = 34) (always included in all experiments) are averaged across all experiments; NFEPP (n = 10); FF3 (n = 12); FF6 (n = 10). Graphs show means ± SEM (where available). " should read:"Correlation between pK a values of compounds with antinociception and side effects. (A,B) Antinociceptive effects as net AUC (4-12 μg/kg) of PPT (negative values result from subtraction of pre-CFA baseline PPT) at 15 min after i.v. injection of NFEPP, FF3, FF6 and Fen in inflamed (A) and contralateral, noninflamed (B) paws. AUC values were derived from curves generated by use of n = 9 (NFEPP and FF3 from 3,15 ), n = 8-10 (FF6, see also Fig. 3) and n = 19 (Fen) animals. (C) Constipation, as assessed by number of fecal boli 1 h after s.c. injection of agonists (30 μg/kg) in relation to the pK a of the substance. The dashed line represents vehicle-treated controls (mean of 3 independent experiments). Fen (n = 30) and controls (n = 34) (always included in all experiments) are averaged across all experiments; NFEPP (n = 11); FF3 (n = 12); FF6 (n = 10). (D) Locomotor activity as assessed by the total distance travelled during 30 min after s.c. injection of agonists (30 μg/kg) in relation to the pK a of the substance. The dashed line represents vehicle-treated controls (mean of 3 independent experiments). Fen (n = 31) and controls (n = 34) (always included in all experiments) are averaged across all experiments; NFEPP (n = 10); FF3 (n = 12); FF6 (n = 10). Graphs show means ± SEM (where available). " Additionally, the Article contains errors in Reference 12 which was incorrectly given as: Sun J., Chen S. R., Chen H., Pan H. L. mu-Opioid receptors in primary sensory neurons are essential for opioid analgesic effect on acute and inflammatory pain and opioid-induced hyperalgesia. J ...