**12. Oral absorption**

Drug oral administration is the most convenient and popular route of drug delivery. However, some drugs have low bioavailability and slow absorption rate, thus limited efficacy. Bile salts have been shown to increase the absorption of intestinal insulin by masking its hydrophilic surface resulting in higher permeation through the ileal mucosa and into the systemic circulation, thus enhancing insulin bioavailability. In one study, insulin was formulated with different bile salts and administered orally to rabbits. Bile salts enhanced insulin permeation through the ileal mucosa and resulted in a significant effect which varied based on the type of bile salt used (Mesiha et al. 2002a). When insulin was administered with palmitic acid combined with bile salts, in the form of aqueous fatty acid solution, significant hypoglycaemic effects was observed in the treated diabetic animals. In an aqueous environment, insulin's hypoglycaemic effect was improved by the addition of glycocholate and, to a lesser extent, cholate. Accordingly, bile salts improved insulin's hypoglycaemic effect in the following descending order; sodium deoxycholate > sodium cholate > sodium glycocholate > sodium glycodeoxycholate > sodium taurodeoxycholate (Mesiha et al. 2002b). In general, there are few examples of known bile salt derivatives which are known absorption enhancers. Cholylsarcosine (CS) is an absorption enhancer as well as a non-toxic bile salt derivative. It has good stability and safety profile and is resistant to bacterial degradation in the gastrointestinal tract (Mesiha et al. 2002c; Mikov & Fawcett 2006b). Due to its stability, it does not form deoxycholic acid which can cause hepatotoxicity. Chenodeoxycholic acid and cholyltaurine were more effective than CS, but due to their susceptibility to bacterial degradation, they have poor safety profile. The applications of bile salts as absorption enhances is gaining more interest, especially with the ocular, transermal, nasal, buccal and rectal mucosal routes.

### **13. Occular absorption**

Due to the normally high rates of lacrimation and tear wash-out, occular drug delivery has low efficiency and requires the drug to have high diffusibility through the anterior region of the eye Figure 4. However, when a drug is formulated with a suitable abortion enhancer, its permeation can be doubled or even tripled. A good example of bile salts occular applications is the administration of insulin. In one study that investigated the occular permeation of insulin, less than 1% of insulin reached the systemic circulation via the ocular route. The addition of some absorption enhancers may improve the permeation to around

Recent studies suggest a bigger role for Mdr and Mrp transporters in the enterohepatic recirculation of bile acids (Asamoto et al. 2001). Mrp2 and Mrp3 recognize monovalent (those with a single charge) and divalent (those with a double charge) bile acids as their substrates (St-Pierre et al. 2000; St-Pierre et al. 2001; Zollner et al. 2003) while Mdr1 and Mdr3 recognise bile acid taurocholate, glutathione, bile salt glucuronide and sulfate conjugates (Ballatori et al. 2005a; Ballatori et al. 2005b). Mrp2 is located in the apical membrane of the bile canaliculus where it removes newly formed divalent bile acids into the bile duct. Mrp3 is located in the basolateral membrane of the ileal enterocytes where it removes monovalent bile acids from the gut lumen into the portal vein (Houten et al. 2006a). Figure 3 shows the locations of a mucosal and a serosal protein transporters (mucosal

Drug oral administration is the most convenient and popular route of drug delivery. However, some drugs have low bioavailability and slow absorption rate, thus limited efficacy. Bile salts have been shown to increase the absorption of intestinal insulin by masking its hydrophilic surface resulting in higher permeation through the ileal mucosa and into the systemic circulation, thus enhancing insulin bioavailability. In one study, insulin was formulated with different bile salts and administered orally to rabbits. Bile salts enhanced insulin permeation through the ileal mucosa and resulted in a significant effect which varied based on the type of bile salt used (Mesiha et al. 2002a). When insulin was administered with palmitic acid combined with bile salts, in the form of aqueous fatty acid solution, significant hypoglycaemic effects was observed in the treated diabetic animals. In an aqueous environment, insulin's hypoglycaemic effect was improved by the addition of glycocholate and, to a lesser extent, cholate. Accordingly, bile salts improved insulin's hypoglycaemic effect in the following descending order; sodium deoxycholate > sodium cholate > sodium glycocholate > sodium glycodeoxycholate > sodium taurodeoxycholate (Mesiha et al. 2002b). In general, there are few examples of known bile salt derivatives which are known absorption enhancers. Cholylsarcosine (CS) is an absorption enhancer as well as a non-toxic bile salt derivative. It has good stability and safety profile and is resistant to bacterial degradation in the gastrointestinal tract (Mesiha et al. 2002c; Mikov & Fawcett 2006b). Due to its stability, it does not form deoxycholic acid which can cause hepatotoxicity. Chenodeoxycholic acid and cholyltaurine were more effective than CS, but due to their susceptibility to bacterial degradation, they have poor safety profile. The applications of bile salts as absorption enhances is gaining more interest, especially with the ocular, transermal,

Due to the normally high rates of lacrimation and tear wash-out, occular drug delivery has low efficiency and requires the drug to have high diffusibility through the anterior region of the eye Figure 4. However, when a drug is formulated with a suitable abortion enhancer, its permeation can be doubled or even tripled. A good example of bile salts occular applications is the administration of insulin. In one study that investigated the occular permeation of insulin, less than 1% of insulin reached the systemic circulation via the ocular route. The addition of some absorption enhancers may improve the permeation to around

transporter is in green & serosal transporter is in red) expressed in enterocytes.

**12. Oral absorption** 

nasal, buccal and rectal mucosal routes.

**13. Occular absorption** 

4%. This still remains a limiting factor in insulin clinical applications (25). An estimated 80% of administered drug is eliminated through the nasal cavity after occular application (26). Another study (Yamamoto et al. 1989) determined the extent to which absorption promoters could enhance the absorption of insulin via the ocular route. When administered alone, occular insulin serum levels reached Cmax within 15 minutes of occular administration while when formulated with sodium glycocholate, sodium taurocholate and sodium deoxycholate (as absorption enhancers), insulin Cmax was reached within 5 minutes. When insulin was co-administered with sodium glycocholate, the amount of insulin permeating the eyes and reaching the systemic circulation increased from 1% to 5.5%. Sodium deoxycholate was found to be more effective and sodium taurocholate least effective at enhancing the occular absorption of insulin. This implies a good potential of bile acid applications in insulin occular delivery in T1D, when other routes as less desirable.

Fig. 4. The general structure of the eye.

#### **14. Nasal absorption**

The Nasal route is a convenient and popular method of drug administration as it is feasible and it has fast absorption rate. It also provides reasonable bioavailability as it bypasses first pass hepatic metabolism. However, pharmacologically active peptides such as hormones and proteins with molecular weights > 10 kDa do not have the ability to permeate the nasal mucosal layer without being significantly trapped, washed out (through the nasopharyngeal cavity), or degraded before reaching the systemic circulation. In order to optimise nasal drug delivery to drugs such as insulin, suitable permeation enhancers such as bile salts may be appropriate. For insulin to be delivered nasally, it has to permeate the nasal mucosa and

Potentials and Limitations of Bile Acids and Probiotics in Diabetes Mellitus 385

they increased the amount of absorption by a factor ranging from 1 to 5, compared to bile salts alone (Illum et al. 2001). Such maximization of insulin-bile salt mucosal permeation was successful to enhance insulin absorption through the nasal mucosa, and thus shows

The ability of a bile acid to enhance permeation is heavily dependent on its hydroxyl groups and the concentration of bile acid present in solution. Insulin absorption increases when the concentration of bile salt exceeds its aqueous critical mice concentration (CMC). The amount of insulin absorbed also increases with increasing hydrophobicity of the bile salt. The order of bile salts' ability to increase insulin absorption is DCA>CDCA>CA>UDCA (Gordon et al. 1985b). When sodium deoxycholate, the most hydrophobic bile salt, is co-administered with insulin, the absorbed insulin causes more than 30% reduction in blood glucose levels in diabetic subjects (Moses et al. 1983). When a bile salt possess poor hydrophobicity, its efficacy is significantly reduced. When the highly hydrophilic sodium ursodeoxycholate is formulated with insulin then administered to diabetic subjects, the bile salt showed no significant permeation enhancing effect on insulin, and almost no decrease in blood sugar was reported in the treated diabetic subjects. Bile salts may increase the absorption of insulin by forming micelles in which the insulin resides in high concentrations. Another proposed mechanism is that bile acids form reverse micelles which form channels across the nasal membrane through which insulin can move to reach the bloodstream (Gordon et al. 1985b). Bile salts may also bind and trap Ca2+ causing tight junctions to loosen and allowing insulin to pass. In addition, sodium lauryl sulphate (SLS) may enhance drug absorption via the nasal route by lyzing biological membranes. This involves lipid solubilisation and subsequent protein denaturation and dissolution (Donovan et al. 1990). Accordingly, SLS

has a unique ability to enhance absorption efficiently and at low concentration.

great potential in insulin nasal delivery.

Fig. 5. The mucosal layer of the nasal cavity.

into the systemic circulation (nasal vasculature). Large peptides such as insulin are not easily absorbed through the nasal mucosa when administered via a nasal spray (Hirai et al. 1978). Insulin must be transported between or through the apical and basal membranes of columnar cells, basal cells and capillary endothelial cells of blood vessels (Figure 5) (Gordon et al. 1985a; Li et al. 1992). However, it must first cross the mucous layer which varies in thickness averaging between 5 and 20 mm in depth. Mucociliary clearance washes out mucous and entrapped particles from the anterior to the posterior nasal cavity and down the oesophagus. Drugs administered through the nasal route must dissolve rapidly in the mucous before reaching the epithelium. The drug must then move between tight junctions, survive the intercellular matrix and diffuse between the basolateral cells to reach the subepithelial space through which it can enter the nasal vasculature (Junginger 1992). Bile salts exert their permeation enhancing effect through solubilising cellular proteins, membrane phospholipids and through limiting the effect of metabolizing enzymes. Although the exact mechanism by which bile salts solubilise cellular components without necessarily damaging tissues is unknown, bile salts enhance absorption of drugs across membranes. The solubilisation of membrane components may be related to the ability of bile acids to overcome nasal membrane barrier resistance (Shao et al. 1992a). In one study (Shao et al. 1992b), the effect of bile salts on the structure, integrity, configuration and strength of the nasal mucosa, was study. The effect was investigated through administering bile salts to animal's nasal cavity then measuring the levels of cellular proteins (in the cell membrane and the cytoplasm), DNA-metabolizing enzymes and other biomarkers. The study concluded that deoxycholate caused the greatest solubilising effect on the nasal mucosa while taurocholate caused the least effect. Another study (Gordon et al. 1985a) was carried out in human, to investigate the physicochemical properties of bile salts and their relations to the permeation effect in nasal drug delivery. As expected, the rate of absorption of drug molecules was directly correlated to the bile salt's lipophilicity and their permeation effect. The most effective permeation enhancer, through the nasal mucosa, was deoxycholate, followed by, chenodeoxycholate, cholate then finally ursodeoxycholate.

However, large or too frequent doses of bile salts have been found to cause significant damage to the nasal mucosa and subsequent nasal bleeding (Hersey & Jackson 1987a). Moreover, enhancing further the nasal absorption of an insulin-bile salt formulation, through the use of starch microspheres, has been investigated (Illum et al. 2001). Microspheres are non-toxic and biocompatible with rabbit nasal mucosa (Bjork et al. 1991). Illum *et al*. examined the effect of starch microspheres on the absorption enhancing efficiency of bile salts in formulations with insulin, after application in the nasal cavity of sheep. The enhancers were selected on the basis of their perceived or proven mechanism of action and worked predominantly by interacting with the lipid membrane. The microsphere formulation was placed in the anterior part of the nasal cavity where few cilia are present. The bioadhesive properties provide a high drug concentration in close contact with the epithelial surface for an extended time period. Generally, microspheres can assist the passage of small drug molecules but an absorption enhancer is necessary for polypeptides with molecular weights above 6000 Da. Bioadhesive starch microspheres synergistically increase the effect of absorption enhancers on the absorption of insulin across the nasal membrane in sheep. The bioadhesive starch microspheres were shown to increase synergistically the effect of the bile salts on the transport of the insulin across the nasal mucosa. So when bile salts were used in conjunction with bioadhesive starch microspheres,

into the systemic circulation (nasal vasculature). Large peptides such as insulin are not easily absorbed through the nasal mucosa when administered via a nasal spray (Hirai et al. 1978). Insulin must be transported between or through the apical and basal membranes of columnar cells, basal cells and capillary endothelial cells of blood vessels (Figure 5) (Gordon et al. 1985a; Li et al. 1992). However, it must first cross the mucous layer which varies in thickness averaging between 5 and 20 mm in depth. Mucociliary clearance washes out mucous and entrapped particles from the anterior to the posterior nasal cavity and down the oesophagus. Drugs administered through the nasal route must dissolve rapidly in the mucous before reaching the epithelium. The drug must then move between tight junctions, survive the intercellular matrix and diffuse between the basolateral cells to reach the subepithelial space through which it can enter the nasal vasculature (Junginger 1992). Bile salts exert their permeation enhancing effect through solubilising cellular proteins, membrane phospholipids and through limiting the effect of metabolizing enzymes. Although the exact mechanism by which bile salts solubilise cellular components without necessarily damaging tissues is unknown, bile salts enhance absorption of drugs across membranes. The solubilisation of membrane components may be related to the ability of bile acids to overcome nasal membrane barrier resistance (Shao et al. 1992a). In one study (Shao et al. 1992b), the effect of bile salts on the structure, integrity, configuration and strength of the nasal mucosa, was study. The effect was investigated through administering bile salts to animal's nasal cavity then measuring the levels of cellular proteins (in the cell membrane and the cytoplasm), DNA-metabolizing enzymes and other biomarkers. The study concluded that deoxycholate caused the greatest solubilising effect on the nasal mucosa while taurocholate caused the least effect. Another study (Gordon et al. 1985a) was carried out in human, to investigate the physicochemical properties of bile salts and their relations to the permeation effect in nasal drug delivery. As expected, the rate of absorption of drug molecules was directly correlated to the bile salt's lipophilicity and their permeation effect. The most effective permeation enhancer, through the nasal mucosa, was deoxycholate, followed by, chenodeoxycholate, cholate then finally ursodeoxycholate. However, large or too frequent doses of bile salts have been found to cause significant damage to the nasal mucosa and subsequent nasal bleeding (Hersey & Jackson 1987a). Moreover, enhancing further the nasal absorption of an insulin-bile salt formulation, through the use of starch microspheres, has been investigated (Illum et al. 2001). Microspheres are non-toxic and biocompatible with rabbit nasal mucosa (Bjork et al. 1991). Illum *et al*. examined the effect of starch microspheres on the absorption enhancing efficiency of bile salts in formulations with insulin, after application in the nasal cavity of sheep. The enhancers were selected on the basis of their perceived or proven mechanism of action and worked predominantly by interacting with the lipid membrane. The microsphere formulation was placed in the anterior part of the nasal cavity where few cilia are present. The bioadhesive properties provide a high drug concentration in close contact with the epithelial surface for an extended time period. Generally, microspheres can assist the passage of small drug molecules but an absorption enhancer is necessary for polypeptides with molecular weights above 6000 Da. Bioadhesive starch microspheres synergistically increase the effect of absorption enhancers on the absorption of insulin across the nasal membrane in sheep. The bioadhesive starch microspheres were shown to increase synergistically the effect of the bile salts on the transport of the insulin across the nasal mucosa. So when bile salts were used in conjunction with bioadhesive starch microspheres, they increased the amount of absorption by a factor ranging from 1 to 5, compared to bile salts alone (Illum et al. 2001). Such maximization of insulin-bile salt mucosal permeation was successful to enhance insulin absorption through the nasal mucosa, and thus shows great potential in insulin nasal delivery.

The ability of a bile acid to enhance permeation is heavily dependent on its hydroxyl groups and the concentration of bile acid present in solution. Insulin absorption increases when the concentration of bile salt exceeds its aqueous critical mice concentration (CMC). The amount of insulin absorbed also increases with increasing hydrophobicity of the bile salt. The order of bile salts' ability to increase insulin absorption is DCA>CDCA>CA>UDCA (Gordon et al. 1985b). When sodium deoxycholate, the most hydrophobic bile salt, is co-administered with insulin, the absorbed insulin causes more than 30% reduction in blood glucose levels in diabetic subjects (Moses et al. 1983). When a bile salt possess poor hydrophobicity, its efficacy is significantly reduced. When the highly hydrophilic sodium ursodeoxycholate is formulated with insulin then administered to diabetic subjects, the bile salt showed no significant permeation enhancing effect on insulin, and almost no decrease in blood sugar was reported in the treated diabetic subjects. Bile salts may increase the absorption of insulin by forming micelles in which the insulin resides in high concentrations. Another proposed mechanism is that bile acids form reverse micelles which form channels across the nasal membrane through which insulin can move to reach the bloodstream (Gordon et al. 1985b). Bile salts may also bind and trap Ca2+ causing tight junctions to loosen and allowing insulin to pass. In addition, sodium lauryl sulphate (SLS) may enhance drug absorption via the nasal route by lyzing biological membranes. This involves lipid solubilisation and subsequent protein denaturation and dissolution (Donovan et al. 1990). Accordingly, SLS has a unique ability to enhance absorption efficiently and at low concentration.

Fig. 5. The mucosal layer of the nasal cavity.

Potentials and Limitations of Bile Acids and Probiotics in Diabetes Mellitus 387

measured with and without the addition of bile acids. The bioavailability of inhaled insulin was 7.8% but, with the addition of a bile acid, absolute bioavailability reached 10.2% (p < 0.05). This was a small but significant increase which presents bile acids as permeation enhancers in pulmonary drug applications. Bile acids could have enhanced insulin effect through exerting their own hypoglycemic effect causing a further reduction in glucose levels after administration with insulin. The study also reported that the onset of the hypoglycemic effect after insulin inhalation with bile acids was more than ten times faster, then when insulin was injected SC alone. However, interpatient variation was large in terms of hypoglycemia, which was a disadvantage for such a method of insulin delivery. In other studies (Agu et al. 2001a; Agu et al. 2001b), insulin was administered via the lung with and without sodium glycocholate. The addition of 1% sodium glycocholate inhibited insulin degradation within the lung. Although neither the types of proteolytic enzymes involved in insulin hydrolysis nor the specific mode of stabilization by bile acids were investigated, sodium glycocholate was suggested to be an aminopeptidase inhibitor. Bile acids wide use in pulmonary drug formulation is limited by their safety profile. at the dose required to increase absorption, bile acids are non-toxic and relatively safe. However, when aspirated in large amounts, bile acids have been shown to cause pulmonary oedema and haemorrhage

Recent studies have shown that the semisynthetic bile acid analogue, 12-monoketocholic acid (MKC) exerted a significant hypoglycemic effect when administered alone to a rat model of T1D. When administered with insulin, MKC exerted a synergistic effect potentiating the hypoglycemic effect of insulin (Kuhajda et al. 2000; Mikov et al. 2008). MKC hypoglycemic effect was studied using various formulations including the oral, nasal, ocular and rectal applications. Then, the hypoglycemic effect was compared with that of insulin injected subcutaneously. The mixture of MKC and insulin also tested for hypoglycemic activity. Nasal administration of the insulin-MKC mixture resulted in a decrease of blood glucose concentration that reached 54% of that obtained after subcutaneous application of insulin. However, following nasal administration of the MKC, the decrease in blood glucose reached 36% of that obtained after subcutaneous application of insulin. The discovery of a link between bile acids and glucose regulation offers a new perspective in the design of hypoglycaemic drugs in treating diabetes (Miljkovic et al. 2000). The mechanisms by which, bile acids such as MKC exerts its hypoglycemic effect in T1D, was explored further. The hypoglycemic effect of bile acids on T1D rats could be explained through their effect on FXR and PPARs metabolic pathways (Houten et al. 2006b; Trauner et al. 2010). However such

Many studies have been conducted to test the toxicity and safety of primary and secondary bile salts and their derivatives. Some bile salts have excellent safety profiles while others are not safe. Bile salts can be used as therapeutic agents, as absorption enhancers and as formulation excipients. Deoxycholic acid is used in manufacturing steroids and in vaccine production (e.g. influenza vaccine). However, its use is severely limited by its narrow safety profile. In relatively high doses, deoxycholic acid can cause

due to dissolution of pulmonary membranes (Kaneko et al. 1990).

**17. Bile acids as hypoglycemic agents** 

mechanisms remain to be fully characterized.

**18. Safety of bile acids and probiotics** 
