Low dose Naltrexone for induction of remission in inflammatory bowel disease patients

Abstract

Background: Around 30% of patients with inflammatory bowel disease (IBD) are refractory to current IBD drugs or relapse over time. Novel treatments are called for, and low dose Naltrexone (LDN) may provide a safe, easily accessible alternative treatment option for these patients. We investigated the potential of LDN to induce clinical response in therapy refractory IBD patients, and investigated its direct effects on epithelial barrier function.

Methods: Patients not in remission and not responding to conventional therapy were offered to initiate LDN as a concomitant treatment. In total 47 IBD patients prescribed LDN were followed prospectively for 12 weeks. Where available, endoscopic remission data, serum and biopsies were collected. Further the effect of Naltrexone on wound healing (scratch assay), cytokine production and endoplasmic reticulum (ER) stress (GRP78 and CHOP western blot analysis, immunohistochemistry) were investigated in HCT116 and CACO2 intestinal epithelial cells, human IBD intestinal organoids and patient samples.

Results: Low dose Naltrexone induced clinical improvement in 74.5%, and remission in 25.5% of patients. Naltrexone improved wound healing and reduced ER stress induced by Tunicamycin, lipopolysaccharide or bacteria in epithelial barriers. Inflamed mucosa from IBD patients showed high ER stress levels, which was reduced in patients treated with LDN. Cytokine levels in neither epithelial cells nor serum from IBD patients were affected.

Conclusions: Naltrexone directly improves epithelial barrier function by improving wound healing and reducing mucosal ER stress levels. Low dose Naltrexone treatment is effective and safe, and could be considered for the treatment of therapy refractory IBD patients.

Keywords: Crohn’s disease; Endoplasmic reticulum stress; Naltrexone; Ulcerative colitis; Wound healing.

Fig. 1

Changes in individual endoscopic inflammation scores values. The change in endoscopic score value for patients in clinical remission after week 12 and patients not in clinical remission after week 12 are displayed. Each dot represents an individual. The horizontal line represents the group median. The difference in group medians is statistically significant (− 1.5 versus 1.0, for clinical remission and not in clinical remission respectively, p = 0.005)

Fig. 2

Naltrexone improves epithelial wound healing. a Expression of the μ-opioid receptor (MOR) was tested by rt-PCR, using -RT (i.e. RNA) controls. Ribosomal protein 2 (RP2) was used as cDNA quality control. b HCT116 (left panel) and CACO2 (right panel) cultures were scratched in the presence or absence of 1 μM Naltrexone (NTX), and wounds were photographed at t = 0, 24 and 48. Mean percentage wound size of three independent experiments is shown. c Migration of wound edges at t = 24 in pixels presented as mean of three experiments

Fig. 3

Naltrexone does not affect IL-8 levels in epithelial cell lines and patient sera. Stimulation of HCT116 (a) and CACO2 (b) cell layers for 24 h with bacteria results in significantly increased IL-8 levels in culture supernatants as determined by ELISA. Co-treatment with 1 μg/mL Naltrexone does not affect basal levels or bacteria-induced levels of IL-8 production in these cell lines. c–f Serum from patients was taken before low dose Naltrexone (NTX) treatment, and 3 or 6 months into treatment. IL-8 was detectable in 6 of 7 patients c by ELISA, whereas TNFα was detectable in 5 patients (d). There was no significant difference in the mean IL-8 (e) or TNFα (f) levels between responders and non-responders to low dose Naltrexone

Fig. 4

ER stress in epithelial cell lines is decreased by Naltrexone. a ER stress was induced in HCT116 cells by treatment with 2 μM Tunicamycin (Tuni), resulting in an upregulation of GRP78 expression levels as detected by Western Blot analysis. Co-treatment of cells with 1 μg/mL Naltrexone (NTX) reduces the amount of Tunicamycin-induced GRP78 expression. Upper graph: mean densitometry values of two independent experiments, GRP78 expression is corrected for Actin, to control for equal loading. Representative example is shown in the bottom panels. b Treatment of HCT116 cells with bacteria results in a significant upregulation of GRP78 expression as detected by Western blot analysis, which is reduced by co-treatment cells by treatment of cells with 1 μg/mL Naltrexone. Mean densitometry values of four independent experiments is shown. c Treatment of HCT116 cells with LPS results in a significant upregulation of CHOP expression as detected by Western blot analysis, which is reduced by co-treatment cells by treatment of cells with 1 μg/mL Naltrexone. Mean densitometry values of three independent experiments is shown. d Treatment of organoids with LPS results in a significant upregulation of GRP78 expression as detected by Western blot analysis, which is reduced by co-treatment cells by treatment of cells with 1 μg/mL Naltrexone. Mean densitometry values are shown of experiments performed on organoids derived from two individual donors, with two independent experiments each

Fig. 5

High ER stress in mucosa from IBD patients is reduced by low dose Naltrexone treatment. a Example of GRP78, showing high ER stress marker expression in crypts as well as lamina propria. b GRP78 intensity was scored in lamina propria and crypts from biopsies taken from 13 patients before start of low dose Naltrexone and 5 patients 3 months into treatment. Lower levels of GRP78 expression were observed in lamina propria, although this did not reach statistical significance. c Paired biopsies were available from three patients. All three showed reduction of GRP78 expression in the lamina propria upon treatment with low dose Naltrexone. d Two paired samples are shown. Patient A was a non-responder, Patient B did show clinical response to low dose Naltrexone