Type 1 diabetes (T1D) is a metabolic disorder characterized by destruction of the pancreatic beta-islet cells leading to complete loss of insulin production. Gliclazide is used in Type 2 diabetes (T2D) to stimulate insulin production but it also has beneficial extrapancreatic effects which make it potentially useful in T1D. In fact, some T2D patients continue to use gliclazide even after their diabetes progresses to T1D since it provides better glycemic control than insulin alone. About 30% of a gliclazide dose undergoes enterohepatic recirculation which may contribute to the observed high interindividual variability in its pharmacokinetics. This may limit its efficacy in T1D especially since diabetes can disturb the gut microbiota and give rise to changes in bile composition and enterohepatic recirculation. Improving the absorption of gliclazide through the use of bile acids and probiotics may reduce this variability and improve the efficacy of gliclazide in T1D. The aim of this thesis was to investigate the interaction between the semisynthetic bile acid, 12-monoketocholic acid (MKC) and gliclazide in terms of pharmacokinetics and hypoglycemic effects in a rat model of T1D with and without probiotic pretreatment. A parallel ex vivo (Ussing chamber) study was carried out to investigate the mechanism of the interaction.
Sensitive LC-MS and HPLC methods (Chapter 2) were developed to determine the concentrations of gliclazide and MKC in Ringer's solution and rat serum. Diabetes was induced in male Wistar rats by intravenous (i.v.) alloxan (30 mg/kg). Rats with blood glucose concentration > 18 mmol/l and serum insulin concentration < 0.04 [mu]g/l, 2-3 days after alloxan injection were considered diabetic. A total of 280 male Wistar rats (Chapter 3) were randomly allocated into 28 groups (n=10) of which 14 were made diabetic. Then 7 groups of healthy and 7 groups of diabetic rats were gavaged with probiotics (10⁸ CFU/mg, 75 mg/kg) every 12 hours for three days after which single doses of gliclazide (20 mg/kg), MKC (4 mg/kg) or the combination were administered either by tail vein injection (i.v.) or by gavage. The other 14 groups (7 healthy and 7 diabetic) were gavaged with saline every 12 hours for three days and then treated in the same way. Blood samples were collected from the tail vein for 10 hours after the dose and analyzed for blood glucose, serum gliclazide & serum MKC concentrations. Serum concentration-time curves for gliclazide and MKC were used to determine pharmacokinetic parameters.
In the parallel ex vivo study (Chapter 4), 88 rats were randomly divided into 22 groups (n=4 rats per group, 8 chambers per rat), of which 11 groups were made diabetic. Of the 22 groups, 8 groups (4 healthy and 4 diabetic) were pretreated with probiotics as described above to study their influence on gliclazide and MKC flux, 8 groups (4 healthy and 4 diabetic) were used to investigate the interaction between gliclazide and MKC during transport, and 6 groups (3 healthy and 3 diabetic) were used to study the influence of selective inhibitors of the drug transporters Mrp2, Mrp3 and Mdr1 on gliclazide flux. 10 cm piece of the ileum was removed from each rat, the underlying muscle layer and connective tissue removed and the epithelial sheets mounted into Ussing chambers. Gliclazide, MKC or a combination were added to either the mucosal or serosal side and samples collected from both sides for 3 h to determine mucosal-to-serosal absorptive flux (Jss[MtoS]) and serosal-to-mucosal secretory flux (Jss[StoM]) of gliclazide and MKC as appropriate.
In diabetic rats, gliclazide alone had no effect on blood glucose levels (Ch3, exp2) whereas MKC reduced it from 23 � 3 to 18 � 3 mmol/l (Ch3, exp3) and the combination of gliclazide and MKC reduced it even further from 24 � 4 to 16 � 3 mmol/l (Ch3, exp4). In diabetic rats, probiotic treatment reduced blood glucose by 2-fold (Ch3, exp1) and enhanced the hypoglycemic effect of the combination of gliclazide and MKC (blood glucose decreased from 24 � 3 to 10 � 2 mmol/l).
The bioavailability of gliclazide was higher in healthy rats (53.2 � 6.2%) than in diabetic rats (39.9 � 6.0%) (Ch3, exp2). In healthy rats, MKC enhanced gliclazide bioavailability (82.7 � 8.2%) but in diabetic rats MKC had no effect on gliclazide bioavailability (Ch3, exp4). In healthy rats, probiotic pretreatment significantly reduced gliclazide and MKC bioavailabilities (p<0.01) while in diabetic rats, probiotic pretreatment significantly increased the low bioavailability of gliclazide to a level similar to that in healthy rats (Ch3, exp2 & 3). MKC showed clear evidence of enterohepatic recycling and probiotics delayed and reduced its systemic absorption (Ch3, exp3). In ileal tissues from healthy rats, Ussing chamber studies showed gliclazide is most likely a substrate of Mrp2 and Mrp3 (Ch4, exp5) and MKC significantly reduced gliclazide Jss[MtoS] probably through Mrp3 inhibition (Ch4, exp1). In ileal tissue from diabetic rats, MKC had no effect on gliclazide Jss[MtoS] and Jss[StoM] (Ch4, exp2) and none of the inhibitors had any effect of gliclazide flux (Ch4, exp6). This suggests that these transporters are dysfunctional in this model of T1D.
Probiotics and MKC have hypoglycemic effects that appear to be enhanced by gliclazide and all appear to interact at the level of ileal drug transporters. The combination of probiotic treatment, gliclazide and MKC exerted the greatest hypoglycemic effect in T1D rats. Accordingly, the application of this combination may have potential in improving the treatment of T1D.
| Identifer | oai:union.ndltd.org:ADTP/256948 |
| Date | January 2009 |
| Creators | Al-Salami, Hani, n/a |
| Publisher | University of Otago. School of Pharmacy |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Hani Al-Salami |
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