**8. The effect of co-administration of gliclazide on bile acids & probiotics**

Gliclazide is used in Type 2 diabetes (T2D) to stimulate insulin production but it also has beneficial extrapancreatic effects which makes 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. Gliclazide has three main structural features, an aromatic ring, a sulphonylurea group and an azabicyclic ring (Figure 2).

Fig. 2. The chemical structure of gliclazide with three main groups: aromatic ring, sulphonylurea moiety and azabicyclooctyle ring.

In a recent study investigating the applications of bile acids and probiotics in T1D, the bile acid analogue, MKC, was administered i.v. (four groups) and orally (four groups) to healthy, diabetic, probiotic pretreated healthy and probiotic pretreated diabetic rats. The pharmacokinetic parameters of MKC after i.v. administration were found to be similar in all four groups suggesting no significant differences in pharmacokinetic parameters between healthy and diabetic rats irrespective of probiotic pretreatment. Cmax (maximum concentration), AUC (area under the curve) and F (bioavailability) values after oral administration to untreated healthy rats were also found similar to corresponding values in untreated diabetic rats suggesting similar mechanisms of absorption and systemic distribution of MKC. MKC also showed clear evidence of enterohepatic recycling with

Potentials and Limitations of Bile Acids and Probiotics in Diabetes Mellitus 377

alone. This synergistic effect could be due to gliclazide enhancing the production and/or absorption of MKC active metabolites in the gut. The administration of gliclazide+MKC also produced the most significant reduction in blood glucose levels in probiotic pretreated diabetic rats (from 12.6 ± 2.0 to 10 ± 2.0 mmol/l, p<0.01). Overall, pretreatment with probiotics and subsequent oral administration of gliclazide+MKC resulted in the greatest effect in this model of T1D as well as in improved signs and symptoms in the animals. In healthy rats, neither probiotic treatment, nor oral administration of gliclazide, MKC or gliclazide+MKC had any effect on blood glucose levels. More interestingly, the authors hypothesized that the chronic treatment of diabetic rats with probiotics may have stimulated the metabolism of the stable bile acid, MKC, in a similar way as reported between cholic acid and *Lactobacilli* (Pigeon et al. 2002). The hypothesis of direct induction of probiotic treatment to bile acid metabolism may explain the therapeutic efficacy of probiotics in treating various disorders implementing a better role of bile acids in such therapeutic effects. Holding true, this should take us a step closer to understand better how probiotic administration exerted a hypoglycaemic effect, when administered alone, to T1D rats. This should also create a new approach to enhancing probiotic efficacy, through the concurrent

This multidrug therapy shows potential in T1D. This is illustrated by the reduction of blood glucose levels, improvement of diabetic symptoms, and the lower rate of diabetes development by alloxan when injected to rats pretreated with probiotics. Furthermore, the change in PK of gliclazide and MKC after probiotic pretreatment emphasizes the importance of not only investigating the use of probiotics in a disease state, but also investigating the influence of probiotics on drugs that could be used for such a disease. In addition, T1D clearly illustrates different gut biomorphology and response compared with healthy control which should be taken into account when discussing multidrug approach to the disease. Gliclazide has been used for decades to treat T2D and thus future work should include applying the combination of probiotics, gliclazide and MKC on T2D rats then implications of the findings may be extrapolated to human subjects as appropriate. However, these findings should not be overplayed since variation in gliclazide pharmacokinetics is higher in human than rats (Palmer & Brogden 1993) which may limit further the applications of

One of the applications of the findings is the use of gliclazide, MKC and probiotics in T2D. T2D is characterized by hyperglycemia and hypercholesterolemia and thus bile acids have been used to lower cholesterol levels in diabetic patients (Goldfine 2008). Accordingly, the use of gliclazide, MKC and probiotics may improve glucose and

There is a great conclusion regarding the importance of gut microflora, made by Sir Henry Shaw (1818–1885): 'I have finally come to the conclusion that a good reliable set of bowels is

Many autoimmune and inflammatory diseases have shown positive response to probiotic and prebiotic treatments (Sherman et al. 2009; Tlaskalova-Hogenova et al. 2011). These diseases include acute gastroenteritis, antibiotic-associated diarrhoea and colitis, inflammatory bowel disease, type 1 diabetes, irritable bowel syndrome and necrotizing enterocolitis. The composition of the intestinal microflora may also affect mammalian

**9. The effect of gut microflora and diet on inflammation** 

worth more to a man than any quantity of brains'.

administration with stable bile acids.

these findings in human.

cholesterol unbalance in T2D.

probiotic pretreatment delaying its absorption. This suggests different pharmacokinetic properties of the stable bile acid, MKC, in healthy rats compared with diabetic rats. This further supports the authors' previous findings showing that bile acid recirculation in diabetic animals is disturbed compared with healthy ones. When MKC was administered i.v. (to four groups) or orally (to four groups), there was no significant changes in blood glucose in any group of rats after the i.v. dose but, after oral administration to untreated diabetic rats, the elevated blood glucose level was significantly reduced from 23.6 ± 3.1 to 14.1 ± 2.4 mmol/l. Interestingly, diabetic rats pretreated with probiotics showed less weight loss, urine production and water consumption, and improvement in behaviour (curious, active) and survival rate than untreated diabetic rats. In a more recent study, the authors combined bile acid with an antidiabetic drug, gliclazide, and administered that to a rat model of T1D. Interestingly, and through unknown mechanism, the combination of MKC and gliclazide exerted a better hypoglycaemic effect to probiotic pretreated diabetic rats than MKC alone. In this study, pharmacokinetic parameters of i.v. MKC were not affected by the concomitant i.v. administration of gliclazide in either healthy or diabetic rats with and without probiotic pretreatment. Accordingly, even though exact mechanism of interaction, at the molecular level, between MKC and gliclazide is unknown, there is a clear synergistic effect between MKC, gliclazide and probiotic pretreatment in T1D resulting in a profound hypoglycaemic effect and sound reduction in the diabetic complications in those treated diabetic animals.

Overall, the authors confirmed that at the start of experiments, baseline blood glucose levels in each of the four groups (untreated and probiotic treated healthy and diabetic rats) were comparable. the authors also presented initial data supporting the effect of probiotics on the development of T1D. The administration of probiotics to healthy rats had no effect on blood glucose levels but the same treatment of diabetic rats reduced the elevated blood glucose levels by nearly 30% and improved clinical signs and symptoms. These findings present a clear synergistic effect between bile acids, probiotics and gliclazide. More importantly, it shows clearly that intervention by bile acids and probiotics exert a direct and significantly positive effect on glycemic control and the progression of diabetic complications. Even though the details of such effect remains unclear, multitherapeutic approach in treating diabetes showed better efficacy and continue to gain interest worldwide.

Having said that a likely explanation for the effect of probiotics is that they stimulate the GI mucosa to produce insulinotropic polypeptides (Cornell 1985) and glucagon-like peptide-1 (Raymond et al. 1981) and/or induce the gut microflora to release endotoxins which cause an increase in skeletal muscle glucose uptake (Raymond et al. 1980). Probiotic treatment alone was found to influence gliclazide permeation differently in health and diabetic animals (Al-Salami et al. 2008f) while the fact that administration of gliclazide following probiotic pretreatment did not further reduce glucose levels indicates the effect of probiotics is not due to stimulation of insulin release by residual pancreatic cells or to regeneration of functional pancreatic cells. Furthermore, i.v. administration of MKC to healthy and diabetic rats with and without probiotic pretreatment produced little effect. However, oral administration of MKC to diabetic rats produced a significant effect 3 hours after administration suggesting it arises from metabolic activation of MKC in the gut. The effect of oral MKC was not significant in probiotic pretreated diabetic rats that had lower blood glucose levels at the time of MKC administration possibly due to an interaction in the gut. The combination of gliclazide and MKC produced a greater effect in diabetic rats than MKC

probiotic pretreatment delaying its absorption. This suggests different pharmacokinetic properties of the stable bile acid, MKC, in healthy rats compared with diabetic rats. This further supports the authors' previous findings showing that bile acid recirculation in diabetic animals is disturbed compared with healthy ones. When MKC was administered i.v. (to four groups) or orally (to four groups), there was no significant changes in blood glucose in any group of rats after the i.v. dose but, after oral administration to untreated diabetic rats, the elevated blood glucose level was significantly reduced from 23.6 ± 3.1 to 14.1 ± 2.4 mmol/l. Interestingly, diabetic rats pretreated with probiotics showed less weight loss, urine production and water consumption, and improvement in behaviour (curious, active) and survival rate than untreated diabetic rats. In a more recent study, the authors combined bile acid with an antidiabetic drug, gliclazide, and administered that to a rat model of T1D. Interestingly, and through unknown mechanism, the combination of MKC and gliclazide exerted a better hypoglycaemic effect to probiotic pretreated diabetic rats than MKC alone. In this study, pharmacokinetic parameters of i.v. MKC were not affected by the concomitant i.v. administration of gliclazide in either healthy or diabetic rats with and without probiotic pretreatment. Accordingly, even though exact mechanism of interaction, at the molecular level, between MKC and gliclazide is unknown, there is a clear synergistic effect between MKC, gliclazide and probiotic pretreatment in T1D resulting in a profound hypoglycaemic effect and sound reduction in the diabetic complications in those

Overall, the authors confirmed that at the start of experiments, baseline blood glucose levels in each of the four groups (untreated and probiotic treated healthy and diabetic rats) were comparable. the authors also presented initial data supporting the effect of probiotics on the development of T1D. The administration of probiotics to healthy rats had no effect on blood glucose levels but the same treatment of diabetic rats reduced the elevated blood glucose levels by nearly 30% and improved clinical signs and symptoms. These findings present a clear synergistic effect between bile acids, probiotics and gliclazide. More importantly, it shows clearly that intervention by bile acids and probiotics exert a direct and significantly positive effect on glycemic control and the progression of diabetic complications. Even though the details of such effect remains unclear, multitherapeutic approach in treating

Having said that a likely explanation for the effect of probiotics is that they stimulate the GI mucosa to produce insulinotropic polypeptides (Cornell 1985) and glucagon-like peptide-1 (Raymond et al. 1981) and/or induce the gut microflora to release endotoxins which cause an increase in skeletal muscle glucose uptake (Raymond et al. 1980). Probiotic treatment alone was found to influence gliclazide permeation differently in health and diabetic animals (Al-Salami et al. 2008f) while the fact that administration of gliclazide following probiotic pretreatment did not further reduce glucose levels indicates the effect of probiotics is not due to stimulation of insulin release by residual pancreatic cells or to regeneration of functional pancreatic cells. Furthermore, i.v. administration of MKC to healthy and diabetic rats with and without probiotic pretreatment produced little effect. However, oral administration of MKC to diabetic rats produced a significant effect 3 hours after administration suggesting it arises from metabolic activation of MKC in the gut. The effect of oral MKC was not significant in probiotic pretreated diabetic rats that had lower blood glucose levels at the time of MKC administration possibly due to an interaction in the gut. The combination of gliclazide and MKC produced a greater effect in diabetic rats than MKC

diabetes showed better efficacy and continue to gain interest worldwide.

treated diabetic animals.

alone. This synergistic effect could be due to gliclazide enhancing the production and/or absorption of MKC active metabolites in the gut. The administration of gliclazide+MKC also produced the most significant reduction in blood glucose levels in probiotic pretreated diabetic rats (from 12.6 ± 2.0 to 10 ± 2.0 mmol/l, p<0.01). Overall, pretreatment with probiotics and subsequent oral administration of gliclazide+MKC resulted in the greatest effect in this model of T1D as well as in improved signs and symptoms in the animals. In healthy rats, neither probiotic treatment, nor oral administration of gliclazide, MKC or gliclazide+MKC had any effect on blood glucose levels. More interestingly, the authors hypothesized that the chronic treatment of diabetic rats with probiotics may have stimulated the metabolism of the stable bile acid, MKC, in a similar way as reported between cholic acid and *Lactobacilli* (Pigeon et al. 2002). The hypothesis of direct induction of probiotic treatment to bile acid metabolism may explain the therapeutic efficacy of probiotics in treating various disorders implementing a better role of bile acids in such therapeutic effects. Holding true, this should take us a step closer to understand better how probiotic administration exerted a hypoglycaemic effect, when administered alone, to T1D rats. This should also create a new approach to enhancing probiotic efficacy, through the concurrent administration with stable bile acids.

This multidrug therapy shows potential in T1D. This is illustrated by the reduction of blood glucose levels, improvement of diabetic symptoms, and the lower rate of diabetes development by alloxan when injected to rats pretreated with probiotics. Furthermore, the change in PK of gliclazide and MKC after probiotic pretreatment emphasizes the importance of not only investigating the use of probiotics in a disease state, but also investigating the influence of probiotics on drugs that could be used for such a disease. In addition, T1D clearly illustrates different gut biomorphology and response compared with healthy control which should be taken into account when discussing multidrug approach to the disease.

Gliclazide has been used for decades to treat T2D and thus future work should include applying the combination of probiotics, gliclazide and MKC on T2D rats then implications of the findings may be extrapolated to human subjects as appropriate. However, these findings should not be overplayed since variation in gliclazide pharmacokinetics is higher in human than rats (Palmer & Brogden 1993) which may limit further the applications of these findings in human.

One of the applications of the findings is the use of gliclazide, MKC and probiotics in T2D. T2D is characterized by hyperglycemia and hypercholesterolemia and thus bile acids have been used to lower cholesterol levels in diabetic patients (Goldfine 2008). Accordingly, the use of gliclazide, MKC and probiotics may improve glucose and cholesterol unbalance in T2D.
