Study On Absorption Of Gentamicin Across Pathological Intestinal And Bladder As Well As Its Toxicity

Gentamicin (GTM) is one of the aminoglycoside antibiotics with broad antibacterial spectrum, including Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus, Methicillin sensitive S. aureus, MSSA). But it can also induce serious ADRs, such as ototoxicity and nephrotoxicity, which restrict its use in clinic. But due to its low bioavailability (BA) and high concentration in intestinal tract after oral administration, GTM is extensively used in Gastroenterology and Paediatrics to prevent chronic gatritis, acute diarrhea and intestinal cleanning of ulcerative colitis. A wide accept is established that GTM oral administration is safe enough to use, however, after analyzing the literatures retrieved from CNKI, we found out that GTM oral administration could also induce ototoxicity and nephrotoxicity. The literature search was performed to identify all clinical trails reporting GTM oral administration, review studies were excluded. GTM another extensively used route of administration is bladder irrigation in Nephrology department, the aim of it is to heal or prevent urinary tract infections. But the safety of it is not comprehensively studied. So in this study, we tried to evaluate the safety of GTM oral administration and bladder irrigation, and explore the mechanisms of absorption of oral administration.1. Absorption of GTM oral administration in vitro:a study on Caco-2 cells.We investigated the absorption of GTM across Caco-2 cells, a widely used in vitro model of intestinal barrier. To mimics in vivo inflammation bowel diseases (IBDs), lipopolysaccharide (LPS) was used to stimulate Caco-2 cells. Before transport study, cytotoxicity assays were performed to determine the optical concentration of GTM and LPS. Caco-2 cells were incubated with different concentrations of GTM (8-512μ.g/ml) and LPS (0.5-12.8μg/ml), and CCK-8 kit was performed to access the proliferation of Caco-2 cells. Finally 200μg/ml of GTM and 5μg/ml of LPS were chosen. In transport study, we found that the apical (AP) to baslateral (BL) absorption of GTM in Caco-2 cells and LPS induced inflammatory Caco-2 cells showed no significant difference, as the apparent permeability coefficients (Papp) were 0.823±0.06x 10-6cm/s,0.884±0.05x 10-6 cm/s, respectively, with P>0.05.2. Intestinal tract administration of GTM:rats and human studies.This section of study was completed in three methods. (i) Pharmacokinetics experiment carried on rats. The acute colitis model was induced by 1ml acetic acid directly put into the intestinal, and HE staining was used to justify the histopathological changes. And then,16.8mg/kg GTM was given by intragastric, 500μl blood was then withdrawn from jugular sinus at designed times and the concentration of GTM in serum was tested by FPIA method. Program DAS2.0 was applied to calculate the pharmacokinetic parameters. The result showed that the pharmacokinetic parameters of AUC, MRT, t1/2, and Cmax of model group and control group had no significant difference (P>0.05). (ii) GTM accumulation study in normal rats. The dosage of GTM was 8.4mg/kg and given twice a day in 9:00am and 5:00pm, respectively, for 10days. In day 5 and 10, after intragastric 30min,500μl blood was withdrawn and tested. The results showed that the mean serum concentration of D5 was 0.31±0.08μg/ml and 0.32±0.07μg/ml of D10, respectively, with P>0.05. (iii) Clinical trail.2ml blood was withdrawn from the patients who were prescribed GTM 160mg or 240mg coloclysis to heal the ulcerative colitis. The blood was withdrawn after GTM coloclysis 60min. The results showed that the concentrations of GTM serum were all<2μg/ml (concentration of GTM>2μg/ml is considered to be potentially toxic). In conclusion, GTM intestinal tract administration is safe to use.3. Safety of GTM bladder irrigation in rats and human. This section of study was completed in two methods. (i) Absorption of GTM in hemorrhagic cystitis rats. The hemorrhagic cystitis was induced by 140mg/kg CTX direct intraperitoneal injection, and HE staining was used to justify the histopathological changes. After 24h of CTX intraperitoneal injection, the model group was then given 8.4mg/kg GTM intravesical instillation,30min later 500μl blood was then withdrawn from jugular sinus and tested. The results showed that the mean concentration of model group was 7.53±1.32μg/ml, compared with the control group 0.63±0.12μg/ml, a significant difference was achieved with P<0.01. (ii) Clinical trail.2ml blood was withdrawn from the patients who were receiving GTM 80mg bladder irrigation in a frequency of twice a day. The tested GTM serum concentrations were all<0.5μg/ml, revealing that GTM bladder irrigation in bacterial cystitis was safe. In conclusion, GTM bladder irrigation was safe in bacterial cystitis, but may have potentiality to cause ototoxicity and nephrotoxicity when used in the patients who were suffered from hemorrhagic cystitis.4. Mechanism study of GTM oral administraion.The aim of this study was to investigate the probable mechanism of GTM across intestinal tract. As P-glycoprotein (P-gp), multidrug resistance protein 2 (MRP2), and breast cancer resistance protein (BCRP) can express in AP side and play an important role in drug transportation, act as efflux pumps. So efflux pump inhibitors were added to evaluate the effect of these efflux pumps on the AP to BL transport of GTM. To assess the importance of the paracellular passage,2.5mM EGTA were incubated with Caco-2 cells to disrupt the tight junction of Caco-2 cells. The results revealed that GTM across the intestinal mucosa mainly through paracellular pathway, and may also be affected by P-gp and MRP2. As 2.5mM EGTA,20μM verapamil (VRA) and 100μM probenecid (PRO) could enhance the Papp of GTM to about 4.97,3.5 and 2.44 fold, resprctively, with P< 0.01. Meanwhile, it did not affected by BCRP, as when incubated with 25μM dipyridamole (DIP), the Papp of GTM was 0.807±0.103×10-6 cm/s, P>0.05.