Evolving acidic microenvironments during colitis provide selective analgesic targets for a pH-sensitive opioid

Abstract

Targeting the acidified inflammatory microenvironment with pH-sensitive opioids is a novel approach for managing visceral pain while mitigating side effects. The analgesic efficacy of pH-dependent opioids has not been studied during the evolution of inflammation, where fluctuating tissue pH and repeated therapeutic dosing could influence analgesia and side effects. Whether pH-dependent opioids can inhibit human nociceptors during extracellular acidification is unexplored. We studied the analgesic efficacy and side-effect profile of a pH-sensitive fentanyl analog, (±)- N -(3-fluoro-1-phenethylpiperidine-4-yl)- N -phenyl propionamide (NFEPP), during the evolution of colitis induced in mice with dextran sulphate sodium. Colitis was characterized by granulocyte infiltration, histological damage, and acidification of the mucosa and submucosa at sites of immune cell infiltration. Changes in nociception were determined by measuring visceromotor responses to noxious colorectal distension in conscious mice. Repeated doses of NFEPP inhibited nociception throughout the course of disease, with maximal efficacy at the peak of inflammation. Fentanyl was antinociceptive regardless of the stage of inflammation. Fentanyl inhibited gastrointestinal transit, blocked defaecation, and induced hypoxemia, whereas NFEPP had no such side effects. In proof-of-principle experiments, NFEPP inhibited mechanically provoked activation of human colonic nociceptors under acidic conditions mimicking the inflamed state. Thus, NFEPP provides analgesia throughout the evolution of colitis with maximal activity at peak inflammation. The actions of NFEPP are restricted to acidified layers of the colon, without common side effects in normal tissues. N -(3-fluoro-1-phenethylpiperidine-4-yl)- N -phenyl propionamide could provide safe and effective analgesia during acute colitis, such as flares of ulcerative colitis.

Fig. 1:. Effects of repeated NFEPP and fentanyl application on visceromotor responses (VMRs) to colorectal distension during acute DSS colitis.

A. Study design with injection regimen (color coded). VMRs were measured at day 3 and day 8. B-D. Effects of vehicle (B, 0.5% DMSO s.c., p=0.48, N=4), NFEPP (C, 0.4 mg/kg s.c., p<0.001, N=5) and fentanyl (D, 0.4 mg/kg s.c., p<0.001, N=4) on VMRs to colorectal distension at day 3 and day 8 with representative traces. Abbreviations: B, baseline. BID, bis in die (twice daily). DMSO, dimethyl sulfoxide. DSS, dextran sulphate sodium. *, # p<0.05. **, ##, †† p<0.01. ***, ### p<0.001. Two-way ANOVA, Tukeýs test.

Fig. 2:. Changes of colonic inflammation and NFEPP effects on visceromotor responses (VMRs) during acute DSS colitis.

A. DAI of DSS colitis mice over time (N=15–43, combined data of all cohorts, compared to day 1). B-E. Histology (B, H&E staining), histological damage score (C), MPO activity (D) and tissue pH (E) of the colon from healthy control (HC, N=8–11) and DSS colitis mice (N=5–10) at different time points. Arrows in (B) denote infiltrating inflammatory cells. F. Study design with injection regimen (color coded) and daily VMR measurements (80 μl colorectal distension, day 1 - day 5) after NFEPP administration (0.4 mg/kg s.c.) in DSS colitis mice (N=5). Abbreviations: BID, bis in die (twice daily). DAI, disease activity index. DSS, dextran sulphate sodium. H&E, haematoxylin and eosin. MPO, myeloperoxidase. * p<0.05. ** p<0.01. *** p<0.001. A, C, D: Kruskal-Wallis test, Dunńs test. E: Welch ANOVA, Dunnett T3 test. F: Friedman test, Dunńs test.

Fig. 3:. pHLIP sublocalization in the colon of healthy control and acute DSS colitis mice.

A. DAPI/ anti-Cy-FITC labelling in DSS colitis mice treated with pHLIP (representative images of N= 2 mice). Staining (arrows) is predominantly localized in the mucosal and submucosal layer. Inset shows the subcellular localization of pHLIP. See also Supplementary digital content Fig. 2 for expanded confocal images illustrating the insertion of pHLIP into cell membranes within the inflamed layers of the colon. B. pHLIP-FITC particle distribution within the colon wall of DSS colitis mice (N=8). C. DAPI/ anti-Cy-FITC labelling in a DSS colitis mouse that was not treated with pHLIP. D. DAPI/ anti-Cy-FITC labelling in a healthy control (HC) mouse treated with pHLIP. E. DAPI/ anti-Cy-FITC labelling and H&E staining in a DSS colitis mouse treated with pHLIP. Arrows denote the co-localization of pHLIP particles and infiltrating inflammatory cells. Abbreviations: Cy, Cyanin. DAPI, 4′,6-diamidino-2-phenylindole. DSS, dextran sulphate sodium. FITC, fluorescein isothiocyanate. H&E, haematoxylin and eosin. pHLIP, pH low insertion peptide. ** p<0.01. Paired t-test.

Fig. 4:. Effects of repeated NFEPP and fentanyl application on gastrointestinal (GI) transit and defaecation during acute DSS colitis.

A. Study design with injection regimen (color coded). B. Oral contrast- enhanced CT scans before (day 3, drug naive) and after (day 8) repeated dosing of vehicle (0.5% DMSO s.c., N=5), NFEPP (0.4 mg/kg s.c., N=5) or fentanyl (0.4 mg/kg s.c., N=6) BID. Arrows denote the cecum. C. Effects of NFEPP and fentanyl on CT-based GI transit. D. Effects of NFEPP and fentanyl administration (each opioid at 0.4 mg/kg s.c.) on defaecation. E-F. Effects of NFEPP and fentanyl on colon diameter (E) and cecum diameter (F). Abbreviations: BID, bis in die (twice daily). DMSO, dimethyl sulfoxide. DSS, dextran sulphate sodium. * p<0.05. *** p<0.001. Two-way ANOVA, Bonferroni test.

Fig. 5:. On-target side effects of repeated NFEPP and fentanyl application during acute DSS colitis at different time points.

A. Study design with injection regimen (color coded). Pulse oximeter measurements were performed at day 4, day 7 and day 8 in vehicle (0.5% DMSO s.c. BID, N=5), NFEPP (0.4 mg/kg s.c. BID, N=6) and fentanyl (0.4 mg/kg s.c. BID, N=6) treated DSS colitis mice. B-G. Effects of NFEPP and fentanyl on blood oxygen saturation (B-D) and heart rate (E-G) at different time points and concentrations. Abbreviations: BID, bis in die (twice daily). BPM, beats per minute. DMSO, dimethyl sulfoxide. DSS, dextran sulphate sodium. SpO2, blood oxygen saturation. *, # p<0.05. **, ## p<0.01. ***, ### p<0.001. Two-way ANOVA, Bonferroni test. D, G: fentanyl: N=3; NFEPP: N=5.

Fig. 6:. On-target side effects of increased NFEPP doses in healthy control and acute DSS colitis mice.

A. Study design. Tail immersion assay (N=8), locomotor activity (N=7) and pulse oximeter measurements (N=6) were performed in the same mice at different time points. B-H. Effects of increased doses of NFEPP (0.8 mg/kg s.c.) on withdrawal responses to noxious heat in a tail immersion assay (B), locomotor activity (C-F), blood oxygen saturation (G) and heart rate (H) in healthy control (HC) and DSS colitis mice. Abbreviations: BPM, beats per minute. DMSO, dimethyl sulfoxide. DSS, dextran sulphate sodium. OFT, open field test. SpO2, blood oxygen saturation. # p<0.05. **, ## p<0.01. *** p<0.001. B, G-H: Two-way ANOVA, Bonferroni test. C-F: One-way ANOVA

Fig. 7:. Effects of NFEPP on human colonic nociceptors.

A. Representative traces and histograms of colonic afferent nerve firing in response to von Frey filament probing (10 g) at pH 7.4 (control), pH 6.5 (control), in presence of NFEPP (300 nM) at pH 6.5 and after washout at pH 7.4. B-C. Summary bar charts of afferent nerve probing responses (N=3) presented as Hz (B) and as % of control at pH 7.4 (C). Abbreviations: Hz, Hertz. * p<0.05. Friedman test, Dunńs test.