Olorinab (APD371), a peripherally acting, highly selective, full agonist of the cannabinoid receptor 2, reduces colitis-induced acute and chronic visceral hypersensitivity in rodents

Abstract

Abdominal pain is a key symptom of inflammatory bowel disease and irritable bowel syndrome, for which there are inadequate therapeutic options. We tested whether olorinab-a highly selective, full agonist of the cannabinoid receptor 2 (CB2)-reduced visceral hypersensitivity in models of colitis and chronic visceral hypersensitivity (CVH). In rodents, colitis was induced by intrarectal administration of nitrobenzene sulfonic acid derivatives. Control or colitis animals were administered vehicle or olorinab (3 or 30 mg/kg) twice daily by oral gavage for 5 days, starting 1 day before colitis induction. Chronic visceral hypersensitivity mice were administered olorinab (1, 3, 10, or 30 mg/kg) twice daily by oral gavage for 5 days, starting 24 days after colitis induction. Visceral mechanosensitivity was assessed in vivo by quantifying visceromotor responses (VMRs) to colorectal distension. Ex vivo afferent recordings determined colonic nociceptor firing evoked by mechanical stimuli. Colitis and CVH animals displayed significantly elevated VMRs to colorectal distension and colonic nociceptor hypersensitivity. Olorinab treatment significantly reduced VMRs to control levels in colitis and CVH animals. In addition, olorinab reduced nociceptor hypersensitivity in colitis and CVH states in a concentration- and CB2-dependent manner. By contrast, olorinab did not alter VMRs nor nociceptor responsiveness in control animals. Cannabinoid receptor 2 mRNA was detected in colonic tissue, particularly within epithelial cells, and dorsal root ganglia, with no significant differences between healthy, colitis, and CVH states. These results demonstrate that olorinab reduces visceral hypersensitivity through CB2 agonism in animal models, suggesting that olorinab may provide a novel therapy for inflammatory bowel disease- and irritable bowel syndrome-associated abdominal pain.

Trial registration: ClinicalTrials.gov NCT03155945.

Conflict of interest statement

S. Schmiel, B. Lindstrom, and J. Adams are employees of Arena Pharmaceuticals, Inc. S.M. Brierley received research funding from Arena Pharmaceuticals to conduct the study. The remaining authors have no conflicts of interest to declare. Data from this study were previously presented in part at Digestive Disease Week (DDW) on May 2, 2020, as a virtual ePoster; DDW 2019 on May 18 to 21, 2019, in San Diego, CA; American Neurogastroenterology and Motility Society (ANMS) Annual Meeting on August 16 to 18, 2019, in Chicago, IL; United European Gastroenterology (UEG) Week on October 11, 2020, as a virtual presentation; and UEGW on October 19 to 23, 2019, in Barcelona, Spain.

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the International Association for the Study of Pain.

Figures

Figure 1.

Study design schematic: Overview of the timing of colitis induction with intracolonic TNBS or DNBS administration, oral administration of olorinab or vehicle, and VMR recordings are outlined for (A) IBD (colitis) and (B) IBS (CVH) rodent models. BID, twice daily; CVH, chronic visceral hypersensitivity; DNBS, 2,4-dinitrobenzene sulfonic acid; IBD, inflammatory bowel disease; IBS, irritable bowel syndrome; TNBS, trinitrobenzene sulfonic acid; VMR, visceromotor response.

Figure 2.

Olorinab reversed colitis-induced hypersensitivity to CRD in colitis animals but had no effect in healthy animals. (A) Representative examples of EMG signals in response to each distension pressure for all animal cohorts. (B) In healthy rats, compared with vehicle treatment, olorinab administration at the highest dose of 30 mg/kg failed to alter the VMR to CRD. Comparison was not significant based on the generalized estimating equation method followed by the LSD post hoc test (P > 0.05). (C) Total AUC (sum of the AUC obtained at all distension pressures) of the VMR to CRD showed no difference in response between healthy rats treated with vehicle or the highest dose of olorinab (30 mg/kg). Comparison was not significant using a 2-tailed unpaired t test (P > 0.05). (D) Vehicle-treated colitis rats exhibited significantly enhanced VMR to CRD compared with vehicle-treated healthy control rats. Significant increases in colitis rats were observed across all distension pressures from 40 mm Hg. Olorinab treatment of colitis rats with doses of either 3 mg/kg or 30 mg/kg significantly reduced VMR to CRD relative to vehicle-treated colitis rats. Comparisons were performed using the generalized estimating equation method followed by the LSD post hoc test. **P < 0.01; †P < 0.001; ‡P < 0.0001. (E) Total AUC (sum of the AUC obtained at all distension pressures) of the VMR to CRD shows significantly elevated responses in colitis rats compared with control rats. Olorinab 3 mg/kg and 30 mg/kg significantly reduced the total AUC of the VMR to CRD relative to vehicle-treated colitis rats. Comparisons were performed using a one-way ANOVA followed by a Tukey multiple comparisons test. *P < 0.05; **P < 0.01; †P < 0.001. (F) No significant changes in colonic compliance were observed between healthy rats treated with vehicle and colitis rats treated with vehicle or olorinab at either dose. (G) No significant changes in colonic compliance were observed between healthy rats treated with vehicle or olorinab 30 mg/kg. All compliance comparisons were performed using the generalized estimating equation method followed by the LSD post hoc test, and results were not significant (P > 0.05). Data are presented as mean ± SEM. AUC was calculated as the difference of area values obtained predistension (20 seconds) minus those obtained during distension (20 seconds). aSum of the AUC obtained at all distension pressures. ANOVA, analysis of variance; AUC, area under the curve; CRD, colorectal distension; EMG, electromyography; LSD, least squares difference; VMR, visceromotor response.

Figure 3.

Olorinab reversed hypersensitivity to CRD in CVH mice but had no effect in healthy mice. (A) Representative examples of EMG signals in response to each distension pressure for all mouse cohorts. (B) Vehicle-treated CVH mice exhibited significantly enhanced VMR to CRD compared with vehicle-treated healthy control mice. Significant increases in CVH mice were observed for distension pressures higher than 40 mm Hg. Olorinab treatment of CVH mice with doses of 3 mg/kg, 10 mg/kg, or 30 mg/kg significantly reduced VMR to CRD relative to vehicle-treated colitis mice. Olorinab 1 mg/kg was not effective in reducing VMR to CRD in CVH mice. Comparisons were performed with generalized estimating equation using an LSD post hoc test. **P < 0.01; ‡P < 0.0001. (C) Total AUC of the VMR to CRD showed significantly elevated responses in CVH mice compared with control mice. Olorinab 3 mg/kg, 10 mg/kg, and 30 mg/kg significantly reduced the total AUC of the VMR to CRD relative to vehicle-treated CVH mice. Comparisons were performed using a one-way ANOVA followed by Tukey post hoc tests. *P < 0.05; †P < 0.001; ‡P < 0.0001. (D) No significant changes in colonic compliance were observed between healthy mice treated with vehicle and CVH mice treated with vehicle or olorinab at all doses. Data are presented as mean ± SEM. AUC was calculated as the difference of area values obtained predistension (20 seconds) minus those obtained during distension (20 seconds). aSum of all distension pressures. ANOVA, analysis of variance; AUC, area under the curve; CRD, colorectal distension; CVH, chronic visceral hypersensitivity; EMG, electromyography; LSD, least squares difference; VMR, visceromotor response.

Figure 4.

Olorinab had no effect on colonic nociceptors from healthy mice. (A) The mechanosensitive response of colonic nociceptors from healthy mice was unaffected by increasing concentrations of olorinab. Comparisons were performed using a one-way ANOVA followed by the Bonferroni post hoc test (P > 0.05). (B) The change in baseline response of healthy nociceptors in the presence of increasing concentrations of olorinab indicated no effect of olorinab on nociceptor firing even at high concentrations. (C) Single-unit colonic nociceptor recordings from healthy mice showed mechanical responsiveness was unchanged with olorinab application. Data are presented as mean ± SEM. ANOVA, analysis of variance; vfh, von Frey hair.

Figure 5.

Olorinab dose dependently inhibited colonic nociceptors from colitis mice through a CB2-dependent mechanism. (A) Application of increasing concentrations of olorinab to ex vivo colonic nociceptor endings isolated from colitis mice caused a dose-dependent decrease in action potential firing in response to mechanical stimulation (2 g vfh). Comparisons were performed using a one-way ANOVA followed by the Bonferroni post hoc test (*P < 0.05; **P < 0.01; †P < 0.001). (B) The change in colitis colonic nociceptor mechanosensitivity induced by olorinab compared with baseline responses indicated a dose-dependent decrease in nociceptor firing with increasing concentrations of olorinab. (C) Single-unit colonic nociceptor recordings from colitis mice showed mechanical responsiveness at baseline and dose-dependent inhibition with increasing concentrations of olorinab. (D) Application of the CB2 antagonist SR144528 had no effect on the baseline mechanosensitivity of colitis colonic nociceptors and prevented olorinab-induced inhibition of nociceptor hypersensitivity. Comparisons were performed using a one-way ANOVA followed by the Bonferroni post hoc test (P > 0.05). (E) The change in colitis colonic nociceptor mechanosensitivity compared with baseline demonstrated no inhibitory action of olorinab in the presence of the CB2 antagonist. (F) Single-unit colonic nociceptor recordings from colitis mice showed mechanical responsiveness was unchanged with olorinab application in the presence of the CB2 antagonist. Data are presented as mean ± SEM. ANOVA, analysis of variance; CB2, cannabinoid receptor 2; IBD, inflammatory bowel disease; vfh, von Frey hair.

Figure 6.

Olorinab dose dependently inhibited colonic nociceptors from CVH mice through a CB2-dependent mechanism. (A) Ex vivo application of increasing concentrations of olorinab to colonic nociceptor endings isolated from CVH mice caused a decrease in action potential firing in response to mechanical stimulation (2 g vfh; *P < 0.05, **P < 0.01, †P < 0.001). (B) The change in CVH colonic nociceptor mechanosensitivity induced by olorinab compared with baseline responses indicated a dose-dependent decrease in nociceptor response with increasing concentrations of olorinab. (C) Single-unit colonic nociceptor recordings from CVH mice showed mechanical responsiveness at baseline and dose-dependent inhibition with increasing concentrations of olorinab. (D) Application of the CB2 antagonist SR144528 had no effect on the baseline mechanosensitivity of CVH colonic nociceptors and prevented olorinab-induced inhibition of nociceptor action potential firing in response to mechanical stimulation (2 g vfh; P > 0.05). (E) The change in CVH colonic nociceptor mechanosensitivity induced by olorinab compared with baseline demonstrated no inhibitory action of olorinab in the presence of the CB2 antagonist. (F) Single-unit colonic nociceptor recordings from CVH mice showed the mechanical responsiveness was unchanged with olorinab application in the presence of the CB2 antagonist. Data are presented as mean ± SEM. Statistical analysis was performed using a one-way ANOVA with Bonferroni post hoc tests. ANOVA, analysis of variance; CB2, cannabinoid receptor 2; CVH, colonic visceral hypersensitivity; IBS, irritable bowel syndrome; vfh, von Frey hair.

Figure 7.

CB1 and CB2 mRNA expression in tissue from healthy, colitis, and CVH mice. (A) CB2 (CB2A+B isoforms) was predominantly expressed over CB1 in the colonic mucosa from healthy, colitis, and CVH mice, with CB2A as the most prevalent CB2 isoform. (B) In the colonic longitudinal and circular muscle also containing the myenteric plexus (ENS) from healthy, colitis, and CVH mice, CB1 had the highest relative abundance of all CB receptor transcripts compared with CB2A+B, CB2A, and CB2B. (C) In TL and LS DRG from healthy, colitis, and CVH mice, CB1 had the highest relative abundance of all CB receptor transcripts. CB2 mRNA was also detected in the TL and LS DRG, with the CB2A isoform as the predominantly expressed CB2 isoform. Expression profiles did not significantly differ between healthy, colitis, and CVH states in the (A) colonic mucosa, (B) colonic muscle + ENS, or (C) TL or LS DRG (P > 0.05). All comparisons shown were performed using a one-way ANOVA followed by the Tukey multiple comparison post hoc test (*P < 0.05; **P < 0.01; †P < 0.001; ‡P < 0.0001). CB1 and CB2 mRNA expression was measured relative to reference gene mRNA expression (Ppia, Gapdh, and β-actin) quantified by geometric mean. Data are presented as mean ± SEM. ANOVA, analysis of variance; CB1, cannabinoid receptor 1; CB2, cannabinoid receptor 2; DRG, dorsal root ganglia; ENS, enteric nervous system; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; IBD, inflammatory bowel disease; IBS, irritable bowel syndrome; LS, lumbosacral; mRNA, messenger ribonucleic acid; Ppia, peptidylprolyl isomerase A; TL, thoracolumbar.

Figure 8.

ISH analysis of CB2 mRNA expression in the mouse colon: (A) Positive control tissue for CB2 shows abundant expression of CB2 mRNA using ISH in cross-sections of the spleen (left panel), whereas the negative control probe (dapB) shows a lack of punctate staining (right panel). (B) CB2 labeling in the colon from a healthy mouse was prominent within epithelial cells lining the lumen edge and crypts (see arrows and inset) with sparser labeling observed in the lamina propria, muscularis mucosae, and myenteric plexus. Representative examples of in situ hybridizations for CB2 on cross-sections of the colon from (C) healthy, (D) colitis, and (E) CVH mice. (F) Quantitative analysis revealed that there was no significant difference in CB2 expression when expressed as CB2-positive area per section (left panel), average CB2-positive area per section (middle panel), or CB2-positive area per mouse (right panel). All comparisons shown were performed using a one-way ANOVA followed by the Tukey multiple comparison post hoc test (P > 0.05). Data are presented as mean ± SEM. CB2, cannabinoid receptor 2; cm, circular muscle; CVH, chronic visceral hypersensitivity; dapB, dihydrodipicolinate reductase; ISH, in situ hybridization; lm, longitudinal muscle; lp, lamina propria; LS, lumbosacral; mm, muscularis mucosae; mp, myenteric plexus.

Figure 9.

ISH of CB2 mRNA expression in DRG and proposed mechanism of action of olorinab: (A–C) Representative images of sections of thoracolumbar DRG from healthy mice that underwent hematoxylin staining (blue) and ISH labeling for (A, B) CB2 (brown dots) or (C) the negative probe dapB. Representative images of CB2 labeling in sections of (D) thoracolumbar and (E) lumbosacral DRG from healthy, acute colitis, and CVH mice. Scale bars = 20 μm. (F) Hypothesized mechanism of action of olorinab, which is a highly selective full agonist for CB2 that exhibits low brain penetration. To modulate abdominal pain, olorinab may activate CB2 located on one or multiple cell types including epithelial cells, immune cells, and afferent nerves within the gastrointestinal wall. Downstream or direct effects of CB2 activation may then reduce action potential firing of colonic nociceptors. This would reduce the nociceptive signal being sent from the gastrointestinal tract to the spinal cord, where this nociceptive information is processed and then sent to the brain where pain is perceived. CB2, cannabinoid receptor 2; CVH, chronic visceral hypersensitivity; DRG, dorsal root ganglia; ISH, in situ hybridization; LS, lumbosacral; TL, thoracolumbar.