**Background:** Short bowel syndrome (SBS) results from surgical resection of the intestine, leading to malabsorption and often requiring parenteral support. Glucagon-like peptide-2 (GLP-2) is known to have proadaptive properties including stimulation of intestinal mucosal growth, blood flow, and angiogenesis. Glepaglutide is a long-acting GLP-2 analog. The authors previously reported that daily subcutaneous glepaglutide improved intestinal absorptive function in SBS patients. This paper reports secondary and exploratory endpoints from that trial, focusing on intestinal morphology and perfusion.
**Methods:** This was a randomized, double-blind, dose-finding, single-center, phase 2 crossover trial conducted at Rigshospitalet, Denmark between February 2016 and May 2017. Eighteen patients with SBS (fecal output ≥1500 g/day) were randomized to receive two of three doses (0.1, 1, or 10 mg) of glepaglutide as daily subcutaneous injections for three weeks in a crossover design with a 4-8 week washout. Assessments included: (1) fasting plasma citrulline measured by HPLC; (2) intestinal mucosa biopsies obtained via stoma enteroscopy for immunohistochemistry (villus height, crypt depth, epithelium height, chromogranin A-positive cells, GLP-2-positive cells) and q-PCR for mRNA expression of MKI67, ACTA2, CD68, SLC5A1, SLC5A2, SLC5A5, CLDN7, CLDN15, and OCEL1; (3) intestinal perfusion assessed by laser speckle contrast imaging (LSCI) at 0 and 15 minutes and quantitative fluorescence angiography with indocyanine green (q-ICG) measuring slope and normalized slope. Statistical analysis used ANCOVA with estimates, 95% CIs, and P values; q-PCR results were analyzed by Wilcoxon signed rank test.
**Key Results:** Mean baseline plasma citrulline was 45.3 ± 16.0 µmol/L. Glepaglutide significantly increased plasma citrulline in the 1 mg group (15.3 µmol/L, P = 0.001, 33% relative increase) and 10 mg group (15.6 µmol/L, P = 0.001, 36% relative increase), with a significant dose-dependent effect (P = 0.013). No significant change occurred in the 0.1 mg group. No carryover effect was observed (P = 0.712). Biopsies were collected in 9 patients. In the 10 mg group, trends toward morphological improvements included increased crypt depth of 99 µm (P = 0.019, 51% relative increase) and epithelium height of 14 µm (P = 0.005, 54% relative increase). Villus height increased numerically by 84 µm but did not reach significance (P = 0.427). The 10 mg dose was associated with a reduction in chromogranin A-positive cells (by 31 cells/mm², P = 0.008) and GLP-2-positive cells (by 3 cells/mm², P = 0.023). No significant changes in mRNA gene expressions were observed for any marker in any dose group. No significant changes in intestinal perfusion were detected by LSCI or q-ICG in any dose group. Plasma citrulline at baseline correlated with small-bowel length (r = 0.58, P = 0.0007) and macronutrient absorption (carbohydrate r = 0.38, P = 0.027; protein r = 0.44, P = 0.010; lipid r = 0.44, P = 0.009). Changes in plasma citrulline correlated with reductions in fecal wet weight output (r = -0.65, P < 0.0001) and increases in intestinal wet weight absorption (r = 0.69, P < 0.0001).
**Clinical Implications:** The increases in plasma citrulline and morphological improvements (crypt depth, epithelium height) suggest glepaglutide increases enterocyte mass and absorptive surface area, which may partly explain the previously reported improvements in intestinal absorptive function. The lack of significant perfusion changes at week 3 may indicate that acute hemodynamic effects of GLP-2 diminish with longer treatment, or that the detection threshold of q-ICG and LSCI was insufficient to capture residual increases. The reduction in chromogranin A- and GLP-2-positive cells may reflect dilution due to enterocyte proliferation rather than true downregulation. Limitations include the small sample size, absence of a placebo arm, no sample size calculation, and lack of data on acute changes after glepaglutide administration. The intestinotrophic effects raise theoretical concerns about accelerating growth of existing intestinal neoplasms, warranting colonoscopy prior to treatment in patients with colon in continuity. Future studies should address longer treatment durations and acute hemodynamic changes.