Glucosamine activates intestinal P-glycoprotein inhibiting drug absorption

Abstract

P-glycoprotein (P-gp) is a crucial drug efflux transporter in the gastrointestinal tract, reducing drug uptake and expelling harmful xenobiotics to prevent pathological changes. Current P-gp enhancers primarily increase P-gp expression, requiring 1-3 days, thus missing the critical rescue window for acute poisoning. This study identifies glucosamine (GlcN) as a potent P-gp activator that swiftly enhances drug efflux, significantly reducing drug absorption without altering P-gp expression levels. GlcN directly binds to P-gp, boosting its transport efficiency. Only GlcN with a polymerization degree below 5 can activate P-gp, whereas higher polymerized chitooligosaccharides enhance drug absorption. Additionally, GlcN activation of P-gp has significant implications for cellular metabolism by expelling xenobiotics and metabolic by-products, maintaining cellular homeostasis. Our findings suggest GlcN's potential as an effective antidote for paraquat poisoning and offer a detoxification strategy. This research provides a foundational understanding for developing improved detoxification agents and metabolic modulators.

Fig. 1. Effect of GlcN on intestinal absorption of drugs.

a–d The pharmacokinetics and AUC of capecitabine (a), acyclovir (b), cimetidine (c), and omeprazole (d) were examined in Sprague-Dawley (SD) rats treated with or without GlcN (500 mg/kg). e The percentage decrease in the bioavailability of the four drugs, normalized to their respective control groups. f Schematic of inhibition of drug intestinal absorption by oral GlcN. Data are means ± Standard Error of the Mean (SEM) from six (n  =  6) biological independent replicates in (a−d) p-values were calculated using two-tailed Student’s t-test. ^***p < 0.001, ^****p < 0.0001. Created in BioRender. Qinghua, W. (2025) https://BioRender.com/c20pkwq.

Fig. 2. GlcN inhibits drug intestinal absorption by enhancing P-gp efflux activity.

a Viscosity of rat intestinal mucus treated with or without GlcN (50 mg/mL for 30 min) was examined by rheometer. b The tight junctions between intestinal epithelial cells treated with GlcN (250 mg/kg for 20 min) were detected by TEM. Scare bar, 1 μm. The image only represents the results of one biological sample. c Transcellular transport rate of capecitabine in Caco-2 cells treated with the indicated drugs was examined by transwell model. n = 3 biologically independent samples. d Schematic of validating the interaction between GlcN and P-gp through cell uptake and gene knockout. Created in BioRender. Qinghua, W. (2025) https://BioRender.com/c20pkwqe, f Accumulation (e) and efflux (f) of Rh123 in Caco-2 cells treated with or without GlcN at the indicated concentrations were determined by a multimode reader (Ex = 485 nm, Em = 528 nm). n = 6 biologically independent samples. g Expression of P-gp in the indicated Caco-2 cells was determined by immunoblot. h Cellular uptake of Rh123 in control and P-gp silenced Caco-2 cells treated with or without GlcN at the indicated concentrations was determined (Ex = 485 nm, Em = 528 nm). n = 6 biologically independent samples (After-silencing, p-value for Verapamil = 0.3034, 1 mM = 0.9996, 10 mM = 0.7150, 15 mM = 0.9993). i, k Pharmacokinetic behavior of digoxin in wild-type (wt) FVB mice (i) and abcb1^(-/-) mice (k) in the presence or absence of GlcN (500 mg/kg). n = 6 biologically independent mice. j Schematic of the abcb1 gene knockout process in mice. Created in BioRender. Qinghua, W. (2025) https://BioRender.com/c20pkwq. Data are means ± SEM and p-values were calculated using one-way ANOVA in c−h. ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001, NS represent not significant.

Fig. 3. Activator role of GlcN in P-gp enhancement.

a Thermal stability of P-gp treated with or without GlcN (10 mM for 30 min) at indicated temperatures was determined by CETSA, each experiment was repeated three times independently with similar results. b The protein content of CETSA results was quantified, normalized to 45 °C. n = 3 biologically independent experiments. c Schematic of recombinant “inside-out” inverted vesicle model to determine the P-gp transport efficiency at a fixed protein level. Created in BioRender. Qinghua, W. (2025) https://BioRender.com/c20pkwq. d Uptake activity of P-gp treated with or without GlcN (10 mM for 5 min) was determined by recombinant “inside-out” inverted vesicle model. n = 3 independent experiments. e P-gp mediated ATPase activity in Caco-2 cells treated with the indicated conditions was determined in the presence or absence of sodium orthovanadate by ATPase test kit. n = 3 biologically independent samples. f CYP3A4 activity in Caco-2 cells treated with or without GlcN at indicated concentrations was determined by P450-Glo™ CYP3A4 Assay and Screening System. n = 6 biologically independent samples. Data are means ± SEM and p-values were calculated using one-way ANOVA in b−f. ^*p < 0.05, ^***p < 0.001, NS represent not significant.

Fig. 4. The role of GlcN’s polymerization degree in modulating drug absorption through P-gp binding.

a, b Three-dimensional overall view (a) and partial view (b) of the interaction between GlcN and P-gp protein (PDB code: 4Q9H). c Affinity between P-gp and COS with different degrees of polymerization (10 mM) was determined by SPR. d, e Pharmacokinetics (d) and AUC (e) of capecitabine in rats treated with COS (250 mg/kg) with indicated degrees of polymerization. n = 6 biologically independent mice. f Schematic of the varied effects of COS with different degrees of polymerization on drug intestinal absorption. Created in BioRender. Qinghua, W. (2025) https://BioRender.com/c20pkwq. Data are means ± SEM and p-values were calculated using one-way ANOVA in d and e. ^*p < 0.05, ^***p < 0.001, ^****p < 0.0001.

Fig. 5. GlcN can alleviate the toxicity of PQ and improve the survival rate of poisoned rats.

a Schematic of GlcN increasing the survival rate of paraquat-poisoned animals. Created in BioRender. Qinghua, W. (2025) https://BioRender.com/c20pkwq. b−d Pharmacokinetics of paraquat in SD rats (b), wt FVB mice (c) and abcb^1(-/-) mice (d) treated with or without GlcN (250 mg/kg in SD rat and 500 mg/kg in mice). n = 6 biologically independent experiments. e−g Survival rate of paraquat-poisoned rats (e), paraquat-poisoned FVB mice (f) and paraquat-poisoned abcb1^(-/-) mice (g) treated with GlcN (250 mg/kg in SD rat and 500 mg/kg in mice) or dexamethasone (100 mg/kg) 2 h post-poisoning. n = 10 biologically independent samples. Data are means ± SEM in b−d. p-values were calculated using Simple survival analysis (Kaplan-Meier) in e, f and g. ^*p < 0.05, ^**P < 0.01, NS represent not significant.