<p>U ovoj disertaciji su ispitivani efekti žučnih kiselina na transportne procese kod<br />kojih se ispoljava efekat građenja molekulskih agregata (micele, me&scaron;ovite micele,<br />kompleks sa vodoničnim vezama itd). Ispitan je uticaj temperature na kritičnu micelarnu<br />koncnentraciju holne, deoksiholne i henodeoksiholnekiseline i njihovih keto derivata,<br />određena je entropija formiranja micele, koja je važan parametar ne samo u<br />samoasocijaciji žučnih kiselina već i u njihovoj interakciji sa hidrofobnim molekulima.<br />Određen je kompleks sa vodoničnim vezama izmeđ u lidokaina i žučnih kiselina,<br />regresiona jednačina koja povezuje strukturne parametre žučnih kiselina i ravnotežnu<br />konstantu formiranja tog kompleksa. Zatim je ispitivano delovanje žučnih kiselina u<br />hloroformu na kinetiku prelaza lidokaina i verapamila iz vodene faze u hloroform (model<br />za predtretman sa žučnim kiselinama) U ovom radu je određena i solubilizacija lecitina i<br />holesterola sa žučnim kiselinama.</p> / <p>In this work,&nbsp; effects of bile acids which form molecular&nbsp; aggregates (micelles,<br />mixed micelles, hydrogen complex etc.) on transportation processes were investigated.<br />Influence of temperature on critical micellar concentration of cholic, deoxyholic and<br />henodeoxycholic acids and its keto derivatives was&nbsp; examined. Also, micelle formation<br />entropy was determined. This is very important parameter for self-association of bile<br />acids and their interactions with hydrophobic molecules.<br />Hydrogen complex of lidocain and bile acids was investigated and regression equation<br />which connects structural parameters of bile acids&nbsp; and equilibrium constant of forming<br />this complex was established. After that, effects of bile acids on transfer kinetics of<br />lidocaine and verapamil from aqueous phase to chlorophorm was investigated. Also,<br />micellar solubilization of lecithin and cholesterolby bile acids was determined.</p>

Cholestasis is an important liver pathology. During cholestasis bile acids accumulate in the bile canaliculus affecting hepatocyte viability. The actions of bile acids require changes in the release of Ca2+ from intracellular stores and in Ca2+ entry. The target(s) of the Ca2+ entry pathway affected by bile acids is, however, not known. The overall objective of the work described in this thesis was to elucidate the target(s) and mechanism(s) of bile acids-induced modulation of hepatocytes calcium homeostasis. First, it was shown that a 12 h pre-incubation with cholestatic bile acids (to mimic cholestasis conditions) induced the inhibition of Ca2+ entry through store-operated Ca2+ channels (SOCs), while the addition of choleretic bile acids to the incubation medium caused the reversible activation of Ca2+ entry through SOCs. Moreover, it was shown that incubation of liver cells with choleretic bile acids counteracts the inhibition of Ca2+ entry caused by pre-incubation with cholestatic bile acids. Thus, it was concluded that SOCs are the target of bile acids action in liver cells. Surprisingly, despite the effect of choleretic bile acids in activating SOCs, the Ca2+ dye fura-2 failed to detect choleretic bile acid-induced Ca2+ release from intracellular stores in the absence of extracellular Ca2+. However, under the same conditions, when the sub-plasma membrane Ca2+ levels were measured using FFP-18 Ca2+ dye, choleretic bile acid induced a transient increase in FFP-18 fluorescence. This evidence suggested that choleretic bile acids-induced activation of Ca2+ entry through SOCs, involving the release of Ca2+ from a region of the endoplasmic reticulum (ER) located in the vicinity of the plasma membrane.

Chapter 1. Bile acid derived adaptive dendrons Bile acids are naturally occurring rigid, chiral molecules with unique facial amphiphilicity making it an attractive build block for designing supramolecular systems. Synthesis of bile acid derived chiral dendrimers with acetates protecting the peripheral hydroxyl groups has already been reported by our group (Figure 1). These dendrons did not survive an attempted deprotection of the acetates, as the dendritic linkages were ester linkages. To keep the facial amphiphilicity of bile acid fragments intact, we have worked on two different synthetic strategies. Bile acid derived dendritic components having chloroacetate functional group were synthesized and the α-halo ketone was reacted with a bile acid carboxylate to generate a dendritic species with free hydroxyl group having a glyocolate spacer (Figure 2). At the same time we also were able to protect bile acid hydroxyl group as its corresponding benzyl ether and after dendron synthesis, benzyl groups were removed by hydrogenolysis to give bile acid derived dendritic components with free hydroxyl groups and simple ester linkages (Figure 2). Dye solubilization ability of these dendrons was tested. We observed that some of these structures had the ability to solubilize both a polar dye in a nonpolar solvent and/or a nonpolar dye in a polar solvent. We carried out different extraction techniques (liquid-liquid, solid-liquid) and transport experiments to establish that these dendrons can act as both as normal and inverse micellar mimics. Depending upon the polarity of the medium, this dendron (Figure 2, right) can adopt different conformation and hence this is described as an “adaptive dendron” (Figure 3). Chapter 2. Bile acid derived anion receptors We discovered that the self-condensation of 3α-chloroacetyloxy cholic acid produced a “cholaphane” with free hydroxyl groups in just two step from naturally occurring bile acid. This cyclic dimer (Figure 4) is an inside-out cyclodextrin analog having a polar interior and nonpolar outer surface. The structure of this molecule was confirmed by X-ray crystallography (Figure 5). This molecule showed a remarkable ability to bind two fluoride ions in its cavity (K1 = 1900 M-1 and K2 = 250 M-1 in CHCl3). The pair of doublets from the glycolate methylene hydrogen spacers were found to collapse to a singlet and they again reappear as a pair of doublets with increase in the concentration of fluoride. This anomalous behaviour of gylcolate methylene spacers were rationalized by MP2 calculation at the 6-31+G* level which showed that upon interaction with fluoride, electron density on C-H hydrogen decreased while that on the other geminal hydrogen increased. Detailed NMR study and interaction of fluoride with different acyclic compounds enabled us to determine the mode of fluoride binding. Based on the NMR data and calculation results, fluoride binding models were proposed involving O-H…F- and C-H…F- interactions. When the binding affinity of cyclic dimer was examined for other anions, this molecule showed weak affinity to chloride ions (K ~ 100 M-1) whereas for other bigger anion (HSO4-, H2PO4-) it showed no binding. Similar interactions were utilized to generate bile acid based tripodal geometry where those receptors were able to bind anions weakly (K ~ 100-200 M-1 for fluoride, chloride and bisulphate).

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 142-175). Also available in print.

Deoxycholic acid (DCA) is a secondary bile acid postulated to be involved in the etiology and the progression of colorectal cancer, but its specific mechanisms are not fully understood. DCA has been shown to induce apoptosis allowing selection for apoptosis-resistant cells, which highlights the importance of understanding the mechanisms of action of DCA. Previously, it has been demonstrated that DCA perturbs the plasma membrane, leading to the activation of receptor tyrosine kinases. Because the insulin-like growth factor-1 receptor (IGF-IR), a receptor tyrosine kinase, is demonstrated to play a significant role in protecting colorectal cancer cells from apoptosis, we hypothesized that DCA modulates IGF-IR functions in colorectal cancer cells. We demonstrated that DCA induced the dynamin-dependent endocytosis of IGF-IR through both clathrin-mediated and caveolin-1-dependent mechanisms. Endocytosis of IGF-IR sensitized cells to DCA-induced apoptosis, which demonstrated that IGF-IR played a role in protecting cells against DCA-induced apoptosis. Since DCA-induced endocytosis of IGF-IR was determined to be a caveolin-1 dependent process, caveolin-1 knockdown in HCT116 (HCT116-Cav1-AS) prevented the DCA-mediated endocytosis of IGF-IR. However, we observed an increased sensitivity of DCA-induced apoptosis in the Cav1-AS cells. This suggested that caveolin-1 knockdown altered the plasma membrane dynamics such that although IGF-IR was maintained at the plasma membrane, it facilitated a pro-apoptotic signal. We demonstrated that DCA induced the activation of the pro-apoptotic p38 signaling pathway in HCT116-Cav1-AS, but not in HCT116-Mock, via IGF-IR. Inhibition of both the IGF-IR and p38 independently in HCT116-Cav1-AS significantly decreased their sensitivity to DCA-induced apoptosis. These observations demonstrated that, in a caveolin-1 dependent manner, IGF-IR played a dynamic role in the DCA-mediated apoptosis. Finally, we provided preliminary evidence demonstrating that autophagy played a central role in protecting DCA-resistant cells from DCA-induced apoptosis.Since resistance to DCA also confers apoptosis-resistance, understanding the mechanisms that lead to or prevent DCA-induced cell death is significant, since they can lead to the development of novel therapeutic strategies to sensitize apoptosis-resistant colorectal cancer cells to undergo cell death.

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.

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.

Diss. (sammanfattning) Stockholm : Karol. inst., 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.