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Application of bile acids in drug formulation and

delivery

Maja Stojančević, Nebojša Pavlović, Svetlana Goločorbin-Kon & Momir Mikov

To cite this article: Maja Stojančević, Nebojša Pavlović, Svetlana Goločorbin-Kon & Momir Mikov
(2013) Application of bile acids in drug formulation and delivery, Frontiers in Life Science, 7:3-4,
112-122, DOI: 10.1080/21553769.2013.879925

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© 2014 Taylor & Francis

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Frontiers in Life Science, 2013
Vol. 7, Nos. 3–4, 112–122, http://dx.doi.org/10.1080/21553769.2013.879925

PERSPECTIVE
Application of bile acids in drug formulation and delivery
Maja Stojančevića∗ , Nebojša Pavlovića , Svetlana Goločorbin-Konb and Momir Mikova,c
a Departmentof Pharmacology, Toxicology and Clinical Pharmacology, Faculty of Medicine, University of Novi Sad, Hajduk Veljkova 3,
21000 Novi Sad, Serbia; b Department of Pharmacy, Faculty of Medicine, University of Novi Sad, Hajduk Veljkova 3, 21000 Novi Sad,
Serbia; c School of Pharmacy, Curtin Health Innovation Research Institute, Curtin University, Perth WA, Australia
(Received 16 October 2013; accepted 30 December 2013 )

Bile acids are naturally produced in humans and are known to provide human health benefits through their endocrinological,
microfloral, metabolic and other åffects that are still to be elucidated. In recent years, there has been a growing interest in
using bile acids as absorption enhancers for drug delivery. Bile acids are amphiphilic molecules with a unique ability to
facilitate and promote drug permeation through biological membranes. The role of bile acids in promoting drug permeation
has been experimentally illustrated in various pharmaceutical formulations including oral, nasal, ocular, buccal, pulmonary
and rectal delivery as well as through the blood–brain barrier. Recently, bile acids have drawn attention in the field of drug
delivery due to their ability to act as a drug carrier system in the form of mixed micelles, bilosomes and chemical conjugates
with drug molecules. Bile acids have demonstrated a unique ability to enhance the epithelial transport of hydrophilic drugs
through the paracellular route and that of hydrophobic compounds through both paracellular and transcellular routes. The
aim of this review is to discuss various chemical and pharmaceutical aspects of BAs and their potential applications in drug
formulation and delivery.
Keywords: bile acids; absorption enhancers; drug delivery; drug bioavailability; pharmaceutical formulations

Introduction nucleus carrying 2–4 hydroxyl groups (Sugioka & Moroi

The unique and distinguishable structure and physicochem-
ical properties of bile acids have enabled them to be used
in the development of drugs, as pharmaceutical tools and
potential drug carrier systems that could improve, control
and localize drug delivery. Over the last decades the inter-
est in bile acids has increased considerably, becoming the
focus of intensive research in a variety of different fields
(Monte et al. 2009; Pavlović et al. 2012; Stojančević et al.
2012; Stanimirov et al. 2012). Since the lipid-based drug
delivery system is one of the most popular approaches to
overcome absorption barriers, many lipids, including bile
acids, attract continuous attention in the improvement of
the bioavailability of drugs, as will be discussed (Nanjwade
et al. 2011).
This article aims to provide an overview of the current
knowledge related to use of bile acids, their salts and various
derivatives as drug penetration modifiers and consequently
their potential applications in pharmaceutical dosage forms,
which is summarized in Table 1.
Amphiphilic properties of bile acid molecules
Bile acids have a specific chemical structure, different from
ordinary aliphatic surfactants, due to the presence of a
large, rigid, and planar hydrophobic moiety of a steroid
1998). Bile acids contain structural components that are
hydrophilic on the concave α side and hydrophobic on
the other, convex β side (Mikov & Fawcett 2007). There-
fore, bile acids exhibit a great surface activity and, like all
aliphatic molecules, can self-associate in water, thus form-
ing polymolecular aggregates or micelles, as long as their
concentrations are above a certain concentration, termed the
critical micellar concentration (CMC). Each micelle con-
tains 4–50 molecules, depending on the type and structure,
which can solubilize other lipids as well as hydropho-
bic molecules in the form of mixed micelles (Darkoh
et al. 2010). Bile acids show distinct behavior in terms
of self-association and molecular solubilization, interac-
tion with other substances, the CMC for a given bile salt
being largely determined by its hydrophilic/hydrophobic
balance (Kim et al. 2011). The hydrophobicity of bile
salts and thus their CMC is determined by the number
of hydroxyl groups and their stereospecific position in
the molecule (Wüstner et al. 2000). The hydrophilicity
of the common free and conjugated bile salts decreases
in the order ursodeoxycholic acid (UDC) > cholic acid
(CA) > chenodeoxycholic acid (CDC) > deoxycholic acid
(DC) > lithocholic acid (LC), and taurine-conjugated >
glycine-conjugated > free species (Monte et al. 2009). The
inclusion of the lipophilic components decreases the CMC

∗ Corresponding author. Email: majastojancevic@gmail.com

© 2014 Taylor & Francis

 Frontiers in Life Science 113

Table 1. Applications of bile acids for modification of drug absorption in diverse routes of administration.

Route of
administration Drug Bile acida Delivery system Reference

Oral Ampicillin MKC Micellar Mikov et al. (2005)

Methotrexate MKC Micellar Chen et al. (2009a)
Cefpirom various (DC) Micellar Mrestani et al. (2003)
Octreotide, vasopressin cholylsarcosine Micellar Michael et al. (2000)
lovastatin various (NaDC, NaGC) Micellar Chun (2009); Kim et al. (2011)
Gliclazide MKC Micellar Al-Salami et al. (2008)
Rifaximin various Micellar Darkoh et al. (2010)
Verapamil, lidocaine 7-keto-DC Micellar Poša and Kuhajda (2010)
Ketoconazole, griseofulvin NaTC Micellar Poelma et al. (1990)
Clofazimine NaC Mixed micellar OReilly et al. (1994)
Oxaprozin NaDC Mixed micellar Maestrelli et al. (2011)
Rebamipide NaTC Mixed micellar Mukaizawa et al. (2009)
Tetanus toxoid NaDC Bilosome Mann et al. (2006)
HBsAg NaDC Bilosome Shukla et al. (2008)
Salmon calcitonin various (NaTDC) Bilosome Song et al. (2005)
Fenofibrate NaDC Bilosome Chen et al. (2009b)
Cyclosporine A NaDC Bilosome Guan et al. (2011)
Cafotaxime MKC Bilosome Goločorbin-Kon et al. (2009)
Acyclovir CDC Chemical conjugate Tolle-Sander et al. (2004)
Cis-platinum NaGC Chemical conjugate Criado et al. (1997)
Low molecular weight heparin DC Chemical conjugate Lee et al. (2007)
Insulin DC Chemical conjugate Lee et al. (2005)
Intranasal Acyclovir Various (NaTGC) Micellar Chavanpatil and Vavia (2004)
Dopamine NaDC Micellar Ikeda et al. (1992)
Insulin Various (NaDC) Micellar Gordon et al. (1985)
Insulin NaGC Bilosome Maitani et al. (1992)
Blood–brain barrier Quinine, morphine, pentobarbital MKC Micellar Mikov et al. (2004)
Morphine-6-glucuronide Various (DC) Micellar Yang et al. (2011)
Rhodamine 123 Various (MKC) Micellar Yang et al. (2012)
Transdermal Ketorolac tromethamine NaGC Micellar Fetih et al. (2011)
Cyclosporine A NaC, NaDC Mixed micellar Wu et al. (2001)
Theophylline NaTGC, NaDC Micellar Moghimipour et al. (2012)
Ocular Insulin NaGC Micellar Yamamoto et al. (1989)
β−blockers Various Micellar Saettone et al. (1996)
Rectal Insulin Various (NaDC) Micellar Yamamoto et al. (1992)
Ampicillin Various Micellar Murakami et al. (1984)
Pulmonary Salmon calcitonin TC Micellar Kobayashi et al. (1996)
Insulin NaTC Micellar Johansson et al. (2002)
MKC, 12 monoketocholate; DC, deoxycholate; NaC, sodium cholate; GC, glycocholate; TC, taurocholate; CDC, chenodeoxycholate; TGC, tauro glyco-
cholate.
a The most potent of examined bile acids is emphasized in parentheses.

and an increase the size and solubilization capacity of the
micelles (Nanjwade et al. 2011).
As a consequence of their amphiphilic properties, bile
acids can interact with biological membranes, thus dis-
turbing their functioning. At lower concentrations, bile
salts may cause an increase in membrane permeabil-
ity due to the function of mixed micelles and cell
swelling, whereas at concentrations above their CMC
may associate with phospholipids of cell membranes,
causing a membranolytic effect which is directly related
to the intensity of their enhancer effect (Martin et al.
1992; Garidel et al. 2007).
Despite the fact that hydrophobic bile salts are known
to induce cell membrane damage by their detergent prop-
erties, such effects are not observed under physiological
conditions. The final outcome of bile acids on the cell
membrane depends on many factors including type and
structure of bile acids and membrane characteristics such
as lipid fluidity, charge and hydrophobicity. Binding of bile
acids to cell membrane correlates with their hydrophobic-
ity (Begley et al. 2005). Cytotoxicity of bile salts decreases
with increasing CMC, suggesting that lower cytotoxicity
is the result of a reduced ability to penetrate cell mem-
branes and sequester lipids from them. Minor structural
modifications of natural bile salts have been made in an
attempt to reduce the cytotoxicity without compromising
their ability to enhance membrane permeability (Michael
et al. 2000). Accordingly, it has been shown that replacing
hydroxyl groups in CA with keto groups produces signifi-
cantly less surface active and less lipophilic bile salts with
114 M. Stojančević et al.
higher CMC and consequently with diminished membrane
toxicity (Chen et al. 2012).
Mechanisms of drug absorption enhancement
mediated by bile acids
As previously mentioned, bile acids are one of the most
widely investigated classes of absorption enhancers. The
influence of bile salts on drug absorption appears to be
complex and it depends on the physiochemical properties
of the drug and on the interaction of the micelle with the
physiological environment (Meaney & O’Driscoll 2000).
It has been demonstrated that bile salts enhance the
epithelial transport of hydrophilic drugs through the para-
cellular route and that of hydrophobic compounds through
both paracellular and transcellular routes (Kim et al. 2011).
The possible mechanism of promotory effect on the action
of hydrophilic drugs could be the incorporation of bile
acids into the cell membrane where, at a certain concentra-
tion, they could form reverse micelles that included water
molecules, thus creating hydrophilic pores in the cell mem-
brane (Carey & Small 1972; Poša et al. 2007). Additionally,
bile salts may increase the penetration of drugs via the
paracellular route by binding calcium ions, thereby causing
tight junctions between the cells to open, and by exerting a
mucolytic effect (Mikov & Fawcett 2007).
The underlying mechanisms for improvement of tran-
scellular absorption include increasing the solubility and
dissolution rate of otherwise slightly soluble drugs by
micelle formation (Mithani et al. 1996). Solubilization
of lipophilic drugs within bile salt mixed micelles may
facilitate diffusion through the aqueous diffusion layer,
improving absorption (Nanjwade et al. 2011). Micelles
increase the permeability of the mucosal membrane by over-
coming resistance at the aqueous diffusion layer (Mikov &
Fawcett 2007). Solubilization by bile acids reduces partic-
ulate size and hence may allow the drug to enter the cell
(Darkoh et al. 2010). However, drug solubilization may
decrease the intermicellar free fraction of drug, thus leading
to reduction in absorption rate. Therefore, divergent results
for drug absorption after coadministration with bile salts
are presented in the literature (Nanjwade et al. 2011).
A suggested mechanism for the ability of bile salts to
change the permeability of various cell membranes to drugs
includes interaction of bile salt micelles with membrane
lipid, particularly phospholipid (Murakami et al. 1984). It
has been demonstrated that some bile salts inhibit the active
efflux of P-glycoprotein (Pgp) substrates (Ingels et al. 2004),
probably indirectly by changing the lipid environment of
the Pgp transporter or by interaction with Pgp itself (Chen
et al. 2012). Previous studies reported that Pgp function
was not affected by CA, DC and TC, whereas monoke-
tocholate (MKC), taurolithocholate (TLCA), taurochen-
odeoxycholate (TCDC), glycochenodeoxycholate (GCDC)
and UDC inhibit Pgp mediated drug transport (Roberto et al.
1994; Yang et al. 2012). The absence of a hydroxyl group
at position 12 is the only chemical characteristic which is
common for TLCA, TCDC, GCDC, UDC and MKC, sug-
gesting this is a decisive structural property for bile salts to
inhibit Pgp (Yang et al. 2012).
Some studies reported that hydrophobic bile salts such
as DC and CDC promote absorption more effectively than
CA, which has more polar properties (Mrestani et al. 2003;
Gordon et al. 1985). As a consequence, it has been sug-
gested that transport activity is directly correlated with
the hydrophobicity of bile acid molecules. Increasing the
hydrophilicity of the steroid nucleus by designing glycosy-
lated bile acids has shown that some of these glycosylated
molecules are markedly more effective than TC in enhanc-
ing the intestinal absorption of various drugs, indicating
that hydrophobicity is not a crucial parameter in transport
efficacy, as previously suggested. These compounds were
also less disruptive to membranes than standard bile acid
transport agents (Bowe et al. 1997).
Influence of bile acids on drug bioavailability
The development of technologies that enable poorly mem-
brane permeable drugs to effectively penetrate biological
membranes is one of the greatest challenges in the pharma-
ceutical industry (Fujii et al. 2011). The potential of bile
salts to improve drug absorption is the topic of numerous
studies and will be discussed in this chapter.
Oral route of drug administration
High oral bioavailability is an important consideration
for the development of bioactive molecules as therapeu-
tic agents (Veber et al. 2002). The oral route is the most
convenient route for drug administration and is the most
acceptable for the patient. However, the gastrointestinal
mucosa represents a physical and a biochemical barrier
to the systemic availability of orally ingested, pharmaco-
logically active molecules (Stojančević et al. 2013). When
evaluating absorption, physicochemical properties of the
drug, such as its stereochemistry, partition into membranes,
molecular weight and size, molecular volume, pKa, solu-
bility, chemical stability and charge distribution, as well as
physiological factors such as gastric emptying, gastroin-
testinal motility, intestinal pH, blood flow, lymph flow,
pathological state, nutrient-drug interactions, and mucus
dissolution need to be considered (Le Ferrec et al. 2001).
Since mid-1990s, much attention has been paid to phar-
macological studies of 12-MKC as absorption enhancer
(Mikov et al. 2004). Dosing of ampicillin, whose oral
bioavailability is very low, with MKC resulted in a 38%
increase in the ampicillin maximum plasma concentration
and a 66% increase in the area under the concentration-
time curve (AUC), suggesting that a combinatorial use of
this drug with MKC may be used in order to extend its clin-
ical use and improve its therapeutic efficacy (Mikov et al.
2005).
 Frontiers in Life Science
Chen et al. (2009a) showed that NaC and NaMKC had
concentration dependent effects (0–5 mM) on the apical to
basolateral permeation of methotrexate in Caco 2 cell mono-
layers. At high concentrations they produce a predictable
increase in permeation due to opening the tight junctions.
Conversely, it was found that at low concentrations they pro-
duce a reduction in transcellular permeation of methotrexate
which may be the result of inhibiting influx transporters
directly or by inhibiting them indirectly through disturbing
their lipid environment (Chen et al. 2012).
Using artificial lipid membranes, biological membranes
and the rabbit model, bile salts have been shown to increase
intestinal absorption of cefpirom, a cephalosporin antibiotic
that exhibits highly hydrophilic properties. The absolute
bioavailability of cefpirom was enhanced to 46% through
co-administration with DC, in comparison to cefpirom used
alone (2.8%). Therefore, the role of bile salts in this case
was to increase the lipophilicity of cefpirom (Mrestani et al.
2003).
The nontoxic bile salt derivative, cholylsarcosine, as
well as conventional bile acids TC and CDC, increased
the intestinal absorption of two model peptides, octreotide
and vasopressin, compared to control. The enhancing effect
on peptide permeation across Caco-2 cell monolayers
was ranked in order CDCA > CT > CS, whereas UDC
exhibited no absorption enhancement (Michael et al. 2000).
It has been demonstrated in rats that the addition of
bile salts NaDC and NaGC enhanced oral bioavailability
of the hypolipidemic drug lovastatin 11-fold and 5-fold,
respectively. Since lovastatin is lipophilic, the penetration
enhancement was considered to be the result of increased
transport via both paracellular and transcellular pathways.
Based on the fact that low solubility is the cause of low
bioavailability of lovastatin, the increased AUC by the addi-
tion of bile salts is the result of increased solubility of
lovastatin by micelle formation. They found that the addi-
tion of NaDC and NaGC at 5% to water increased solubility
553-fold and 365-fold, respectively. The additional expla-
nation for enhanced bioavailability could be the inhibition
of Pgp, since lovastatin is known to be a both Pgp substrate
and inhibitor (Chun 2009; Kim et al. 2011).
Furthermore, Al-Salami et al. (2008) investigated the
change in ABC-mediated transport of antidiabetic drug gli-
clazide, alone and in combination with semisynthetic bile
acid 12-MKC, in healthy and diabetic rats, with the propo-
sition that the key transporters involved are Mdr1, Mrp2
and Mrp3. They showed that MKC induced 9-fold reduc-
tion of mucosal to serosal permeation in healthy rats, which
may be the result of the selective inhibition of Mrp3. Con-
versely, in diabetic animals, there was the lack of any net
flux of gliclazide alone or after the addition of MKC, prob-
ably due to lack of action of drug transporters involved or
the suppression of their expression (Al-Salami et al. 2008).
Bile acids solubilize rifaximin, a largely water-insoluble,
nonabsorbable, bacterial RNA synthesis-inhibitory drug, on
a dose-response basis, increasing the drug bioavailability
115
and antimicrobial effect. The aqueous solubility of rifax-
imin increased 70–120-fold in the presence of physiological
concentrations of six bile acids (2.5–20 mM). Besides the
solubilizing effect of bile salts, the possible mechanism for
the increase of the antimicrobial effect may be the weaken-
ing of the bacterial cell membrane, thus increasing the drug
entry into the cytoplasm. These results suggest that rifax-
imin may be more effective in the treatment of infections in
the small intestine, due to the higher concentration of bile in
this region of the intestinal tract than in the colon (Darkoh
et al. 2010).
Examining the influence of CA, CDC, DC and their
keto derivatives on verapamil and lidocaine transport from
an aqueous phase to the rat’s intestine membrane, Poša
and Kuhajda (2010) demonstrated that only 7-keto-DC sig-
nificantly increased the lidocaine transport compared to a
control group, decreasing the concentration of lidocaine in
aqueous phase from 40 to 27.76 mM. Conversely, a signif-
icant increase in verapamil transport was produced only by
CA (concentration change from 40 to 25.8 mM in aqueous
phase). Such effects are explained by chemical proper-
ties and differences in proton donor and proton acceptor
groups of corresponding bile acids and drugs, indicating
the importance of hydrogen bonds in the formation of bile
acid complexes, and thus in the promotive action of bile
acids.
The opposite effect of bile salts on drug absorption was
observed in the study conducted by Poelma et al. (1990)
in which the addition of NaTC at concentrations above
the CMC resulted in the reduction of absorption rate of
the lipophilic drug ketoconazole as well as griseofulvin,
dependent on concentration of bile salt. This result is a con-
sequence of the decrease of the free drug fraction in solution
due to micellar solubilization.
Other routes of drug administration
Intranasal absorption
One of the strategies used to improve drug bioavailability
through the nasal mucosa includes enhancing nasal absorp-
tion using absorption promotors such as bile salts. Even
though some studies indicated that bile salts could cause
nasal irritation when used above a concentration of 0.3%,
it should not be a limiting factor in their further investiga-
tion for nasal formulation, since there is strong evidence for
the positive effect of bile salts in optimization of nasal drug
absorption (Moses et al. 1983).
In vivo studies in rabbits showed that NaTGC increases
the extent of nasal absorption of the antiviral drug acy-
clovir from 10% in the control to 14.5%. Compared
with other absorption enhancers used in this experiment
(hydroxypropyl β cyclodextrin, NaDC, sodium caprate,
and EDTA), sodium caprate showed the greatest increase
of nasal absorption (from 10% to 17.6%) (Chavanpatil &
Vavia 2004). NaDC in concentration of 1% could also
116 M. Stojančević et al.
increase the bioavailability of dopamine for 20% in beagle
dogs via the nasal route (Ikeda et al. 1992).
Furthermore, it has been shown that bile salts promote
the nasal absorption of insulin in human subjects (Moses
et al. 1983) as well as in laboratory animals (Hirai et al.
1981). In the clinical study it was demonstrated that nasal
absorption of insulin correlates positively with hydropho-
bicity of the unconjugated bile salts. The most hydrophobic
bile salt, NaDC, produced marked elevations in serum
insulin concentrations and 50% decrease in blood glucose
concentration (Gordon et al. 1985). Due to the structural
similarity of fusidic acid to bile acids, sodium taurodihydro-
fusidate, was tested as an enhancer of drug permeation, and
demonstrated a significant increase of intranasal absorption
of insulin in sheep (Longenecker et al. 1987).
Bile salts increase insulin bioavailability through the
combination of several suggested mechanisms, such as
alteration of biological membrane integrity and dissocia-
tion of molecular aggregates through micellar solubilization
(Gordon et al. 1985; Donovan et al. 1990). Moreover,
as a possible explanation for promoting the effect of bile
salts, Hirai et al. (1981) have suggested a reduction of
insulin degradation by leucine aminopeptidase, a prote-
olytic enzyme of the nasal mucosa. An additional explana-
tion may be the binding of Ca2+ , which would loosen tight
junctions between cells (Gordon et al. 1985). The synthetic
derivative, 12-MKC, is proved to be an especially good pro-
moter of nasal absorption of insulin in rats (Kuhajda et al.
1997). Additionally, combining β-cyclodextrin with NaDC
had a marked effect of promoting absorption of insulin
through the nasal mucosa, and lowered the serious nasal
ciliotoxicity of NaDC, probably due to ihibition of leucine
aminopeptidase activity (Zhang et al. 2001).
Blood–brain barrier absorption
Overcoming the difficulty of delivering therapeutic agents
to specific regions of the brain presents a major challenge
within the scientific community and drug industry (Misra
et al. 2003). Given that the influence of bile acids on trans-
port through BBB has not been examined enough, they are
becoming an attractive target in this field of research.
Mikov et al. (2004) investigated, for the first time, the
efficacy of a keto derivative of CA, 12-MKC, as a poten-
tial BBB permeator by studying its effect on quinine uptake
into the compartments of the rat central nervous system,
on the analgesic action of morphine, and on the sleep-
ing time induced by pentobarbital, showing a promoting
effect in all three tests applied. Quinine uptake by the left
and right hemispheres was increased about two times ver-
sus the control group, whereas the factor for the brain
stem and cerebellum was about 1.5. The analgesic effect
of morphin was increased from 53% to 97% in different
time intervals compared to the control. In pretreated ani-
mals, pentobarbital-induced sleeping time was statistically
significantly shorter (by about 36% compared to control),
and so was the sleeping time (by about 20% compared to
control).
In permeability studies using rat brain endothelial 4
(RBE4) monolayers, Yang et al. (2011) showed that bile
salts (C, MKC, and TC) at high concentrations (5 mM)
and DC at 1 mM increased paracellular permeability of
morphine-6-glucuronide.
In order to elucidate how bile salts affect transcellu-
lar transport across the BBB, Yang et al. (2012) showed
that bile salts influenced rhodamine 123 (R123) uptake in
RBE4 in a concentration-dependent manner. A possible
mechanism is the increase in membrane fluidity leading
to an increase in the passive diffusion of R123. Another
explanation is ion pair formation between anionic bile salts
and cationic R123, increasing its apparent lipophilicity and
facilitate partitioning of R123 into cell membranes. Of four
examined bile salts (CA, DC, MKC and TC), MKC pro-
duced the greatest increase in uptake and also significantly
reduced efflux of R123, probably by inhibition of Pgp,
affecting the kinetic process of efflux.
Transdermal absorption
Many studies have been performed in order to enhance
permeation through the skin using different substances as
potential enhancers of skin permeability.
Skin permeation of ketorolac tromethamine was greatly
improved by the addition of the trihydroxy bile salt NaGC
(with enhancement ratio of 1.80), suggesting that this
formulation is a promising therapeutic system for the trans-
dermal delivery of ketorolac. This avoids the disadvantages
of parenteral and oral routes, such as the frequent dosing
regimen due to short half-life of this drug, as well as many
adverse effects, such as upper abdominal pain and gastroin-
testinal ulceration. Additionally, high analgesic activity and
low molecular weight make ketorolac a good candidate for
this method of application (Fetih et al. 2011).
In vitro, mixed micelles with bile salts enhanced the
penetration of cyclosporine A (NaC > NaDC) via the trans-
dermal route (Wu et al. 2001). In a recently published study,
Moghimipour et al. (2012) examined the effect of bile salts
NaTGC and NaDC on in vitro theophylline transdermal
absorption through shed snake skin, showing that the bile
salts significantly enhanced the transdermal absorption of
theophylline in comparison to a control group. Their activity
directly correlated with their surface activity.
Ocular absorption
Penetration enhancers in ocular absorption act by increasing
the permeability of the corneal cell membrane and loosening
the tight junctions between the epithelial cells. Penetration
enhancers include different classes of compounds, and bile
salts are one of them (Rupenthal & Alany 2008). They
increase corneal permeability by modifying the integrity
of the corneal epithelium (Sasaki et al. 1995).
 Frontiers in Life Science
Figure 1. Bile acids as drug carrier systems. (a) mixed micellar system (b) bilosome and (c) drug–bile acid chemical conjugate.
Yamamoto et al. (1989) determined that the amount
of insulin that reached the systemic circulation after ocu-
lar administration in rabbits increased from 1% to 5.5%
when it was co-administered with NaGC at a concentration
of 1%.
Bile salts have been shown to increase the permeation of
β-blocking agents through isolated rabbit corneas. The per-
meation of atenolol was significantly increased (5.8-fold) by
0.05% TDC. In the case of timolol the significant enhance-
ments were 5.2-fold by DC, and 2.1-fold by UDC (all at the
0.05% concentration) (Saettone et al. 1996).
Rectal absorption
The rectal route of drug administration is an alternative way
of drug delivery when the oral route is not preferable or to
avoid presystemic metabolism by the liver (de Boer et al.
1982).
As in other mucosal bioavailability studies, insulin is the
most intensive explored polypeptide with respect to rectal
absorption. As is expected, insulin cannot cross the rec-
tal mucosa unless penetration enhancers are added. NaGC
was more effective than NaTC but less effective than NaDC
in improving the rectal absorption of insulin in rabbits
(Yamamoto et al. 1992).
Sodium tauro-24, 25-dihydrofusidate considerably
enhanced the rectal absorption of cefoxitin and vasopres-
sine in rats (van Hoogdalem et al. 1989). Furthermore,
using the in situ rat rectal loop method, Murakami et al.
(1984) found that bile salts markedly promote the rectal
absorption of sodium ampicillin. The proposed mechanism
of action involves increased membrane permeability and
inhibiting some of the proteolytic enzymes at the absorption
site (Mikov & Fawcett 2007).
Regardless of the apparent efficacy in promoting of rec-
tal absorption, several studies indicated severe damage due
to use of bile salts in rectal drug delivery (Murakami et al.
1984).
117
Pulmonary absorption
Due to the relative impermeability of the lungs to many
peptides/macromolecular drugs, many absorption promot-
ers have been examined in order to enhance the systemic
availability of drugs from the lungs (Hussain et al. 2004).
After intratracheal administration of salmon calcitonin
with TC to rats, its pulmonary absorption was markedly
improved. The effect was more pronounced when bile acid
was formulated into dry powder, in comparison with liq-
uid dosage forms (Kobayashi et al. 1996). Furthermore,
the administration of NaTC strongly increases the bioavail-
ability of nebulized insulin in dogs (from 2.6% to 23.2%).
Beside the role in absorption enhancement of inhaled insulin
by NaTC, the possible explanation may be the production
of insulin monomers decreasing the aggregation state of
insulin and widening of tight junctions between adjacent
airway epithelial cells (Johansson et al. 2002).
Although the results are very promising, the use of high
amounts of bile salts may not be feasible for chronic use
since they may damage the epithelial surface (Pilcer &
Amighi 2010).
Bile acids as drug carrier systems
Recently, bile acids have drawn much attention in the field
of drug delivery due to their ability to act as a drug car-
rier system in the form of mixed micelles, bilosomes and
chemical conjugates with drug molecules (Figure 1).
Mixed micellar systems
As mentioned above, drugs that are slightly soluble in water
may be solubilized within bile salt micelles, in order to
improve their absorption. In aqueous solution, bile acid
anions self-associate to form simple micelles (Hofmann
2009). In the gastrointestinal tract, during the lipid diges-
tion, bile salts are found to be associated with phospholipids,
fatty acids and monoglycerides, forming mixed micelles
118 M. Stojančević et al.
(Palin 1985). Some studies reported that the combined use
of bile salts with compounds such as phospholipids, fatty
acids and polyamines may improve their effectiveness as
absorption enhancers and allow a decrease in their con-
centration, thus lowering the risks of membranolytic effect
(Meaney & O’Driscoll 2000; Miyake et al. 2006; Maestrelli
et al. 2011).
Mixed micellar systems, formed by incorporating var-
ious fatty acids into the bile salt NaC, lead to the increase
of the absorption rate of clofazimine in rat gut perfusion
studies, compared to non-micellar and simple micellar sys-
tems, with maximum enhancement (> 800-fold) in the case
when solubilized with NaC/linoleic acid 40:40 mM system
(Oreilly et al. 1994).
In the CaCo-2 cell culture model, mixed micellar sys-
tems of NaTC with linoleic acid significantly increased the
absorption of mannitol, PEG 900 and PEG 4000 relative
to the control, without inducing membrane damage. The
reason for this is probably the combined effect of mixed
micellar systems of NaTC with linoleic acid on both the
paracellular and transcellular routes (Meaney & O’Driscoll
2000).
A recent study by Maestrelli et al. (2011) reported that
the proper combinations of β-cyclodextrin, chitosan, and
NaDC could be exploited to develop effective oral dosage
forms of oxaprozin, as a very poorly water-soluble non-
steroidal anti-inflammatory drug. More than 2-fold increase
in drug permeability and bioavailabilty was observed in
systems containing NaDC compared to oxaprozin applied
alone.
Furthermore, Mukaizawa et al. (2009) proved that com-
binaton of spermine, a polyamine, and NaTC, is a safe
formulation that is able to significantly enhance the trans-
port of a poorly absorbable drug, rebamipide, across Caco-
2 cell monolayers. NaTC increased the permeability of
rebamipide only 14% compared to the control, but six-fold
in combination with spermine, and it was found that absorp-
tion was enhanced via both paracellular and transcellular
routes with a larger contribution of the transcellular route.
The possible mechanisms for the increase of the transcellu-
lar permeability of rebamipide may include an increase in
the membrane fluidity, the opening of the tight junction by
myosin light-chain kinase activated by increased intracel-
lular Ca2+ level and the influence on the distribution of the
proteins comprising tight junctions (Coyne et al. 2003).
Liposomes containing bile salts (bilosomes)
Liposomes were discovered about 50 years ago and since
then have been extensively studied as systems for the deliv-
ery or targeting of drugs to specific sites in the body
(Gregoriadis et al. 2000). Liposomes are nearly spherical,
enclosed vesicles composed of one or more concentric lipid
bilayers arranged around a central aqueous core. Their bio-
chemical structure is very similar to that of normal human
cellular membranes (Bitounis et al. 2012). The size of
liposomes can range from the 20 nm in the smallest vesi-
cle to 1 μm or even more in liposomes that are visible
under a light microscope. The specific structure of lipo-
somes makes it possible to incorporate lipid soluble drugs
into the hydrophobic lipid bilayers, water soluble drugs
into the inner aqueous phase or to carry peptides and small
proteins at the lipid–aqueous interface (Uhumwangho &
Okor 2005). Drug release from liposomes, stability, and
pharmacokinetic profiles depend on composition, size, sur-
face charge, and drug solubility. Liposome formulations of
many different drugs show a significant increase in thera-
peutic activity compared with non-liposomal formulations
(Nanjwade et al. 2011). Soon after their discovery, lipo-
somes were investigated for the oral delivery of peptides and
proteins. Considering their molecular weight, hydrophilic-
ity and susceptibility to degradation by proteases or low pH,
most proteins have low oral bioavailability. Liposomes sta-
bilize proteins in the gastrointestinal tract at some level, but
they also show instabilities after oral application, against
bile salts, pancreatic enzymes, and the acidic conditions in
the stomach (Parmentier et al. 2011).
Although bile salts initially destabilize liposome mem-
branes, they also subsequently lead to stabilization of lipid
vesicles against the further effects of bile acids (Senior
2001). Therefore, it was indicated that liposomes containing
bile salts (bilosomes) may act as more stable carriers than
conventional liposomes and facilitate the transmembrane
transport and absorption of drugs (Guan et al. 2011).
Recently, bilosomes as novel colloidal carriers have
drawn attention in the field of oral immunization, since they
are resistant to the detrimental effects of bile salts in the GI
tract, and thus protect antigens from enzymatic degrada-
tion (Niu et al. 2012). Oral administration of antigens using
bilosomes have showed effective stimulation of both sys-
temic and mucosal immune responses, characterized by IgG
and IgA antibodies, respectively. Mann et al. (2006) have
demonstrated that oral administration of tetanus toxoid by
means of bilosomes successfully induced immunity against
this bacterial protein antigen. In this case, the end-point anti-
body titers by week 3 post-treatment were comparable to
parenterally delivered tetanus toxoid. Likewise, in the study
of Shukla et al. (2008) it has been shown that HBsAg loaded
bilosomes can be used for immunization against hepatitis B,
with five times higher entrapped dose required to produce a
comparable systemic antibody response, with an additional
mucosal immune response.
Song et al. (2005) have demonstrated that liposomes
containing bile salts increase the intestinal absorption of
the model macromolecule salmon calcitonin, which plays
a crucial physiological role in the regulation of calcium
homeostasis and bone remodeling, but has very low oral
bioavailability due to enzymatic degradation and poor per-
meation across intestinal epithelial cells. When compared
to other examined bile salts, anionic (sodium lauryl sulfate),
cationic (lauroyl carnitine chloride, benzalkonium chlo-
ride, benzetonium chloride) and non-ionic (Triton X-100,
 Frontiers in Life Science
Tween 80, Cremophor EL) surfactants in this study, NaTDC
appeared to be the most advantageous when estimated
based on the balance between the permeation enhancement
(10.8-fold increase for 0.1% NaTDC) and damage to the
cells.
The oral bioavailability of the poorly water-soluble drug
fenofibrate, which is a lipid-lowering drug, was signifi-
cantly (5.1-fold) increased by using liposomes containing
a bile salt, NaDC, compared to the fast release formulation
of micronized fenofibrate (Chen et al. 2009b).
Guan et al. (2011) evaluated liposomes containing a
bile salt, NaDC, as oral drug delivery systems to enhance
the oral bioavailability of cyclosporine A. The low oral
bioavailability of this drug is due to its high molecular
weight, poor water solubility, Pgp mediated efflux from
the enterocytes and extensive presystemic metabolism in
the gut wall and liver (Noble & Markham 1995). The
pharmacokinetic study in rats revealed that the relative
oral bioavailabilities of cyclosporine A encapsulated in
bilosomes containing NaDC and conventional cholesterol-
containing liposomes were 120.3% and 98.6%, respec-
tively, compared to Sandimmune Neoral® as the reference,
i.e. a microemulsion-based clinically available commercial
product of cyclosporine A. This improved oral bioavail-
ability is a consequence of enhanced solubilization and
facilitated uptake of intact liposomes through the intesti-
nal epithelia pathway (Guan et al. 2011). However, these
mechanisms need further confirmation based on the results
of in vivo experiments.
Cefotaxime is a third-generation cephalosporin antibi-
otic that, when administered orally, is not appreciably
absorbed from the gastrointestinal tract, thus it must be
given only parenterally. Cefotaxime sodium oral bioavail-
ability in rats was nine-fold increased as a result of encap-
sulation in microvesicles with MKC compared to the same
cefotaxime dose administered orally as saline solution, and
five-fold increased in comparison to cefotaxime microvesi-
cles without MKC, suggesting that this formulation can
extend the application of this drug from parenteral only to
oral application (Goločorbin-Kon et al. 2009).
A comparison of liposome suspension and insulin solu-
tion with or without pretreatment by NaGC showed that
the liposome entrapping insulin at pH 7 had permeated
through the nasal mucosa of rabbit 7.5-fold more effectively
after pretreatment by bile salt, when compared to insulin in
saline solution at the same pH value. The result concluded
that GC remaining in the nasal mucosa led to lysis of the
liposome, causing the release of insulin from the liposome.
Consequently, high activity of insulin on the mucosa surface
resulted in effective permeation (Maitani et al. 1992).
Drug–bile acid chemical conjugates
Bile acids, as natural substrates undergoing an enterohep-
atic circulation involving the liver and the small intestine,
achieve this organotropism by specific sodium-dependent
119
transport systems in the plasma membrane of hepatocytes
and enterocytes (Kramer et al. 1997). Chemical conjugates
of different drugs with bile acids are being investigated
intensively for their ability to target bile acid transporters
in the liver and the small intestines, in order to improve
membrane permeation and oral bioavailability of drugs or
to achieve organ-specific drug action (Kramer 2011).
The interest in bile acids for molecular drug delivery
is explained by their unique combination of rigidity and
chirality, high availability, biocompatibility, and the various
possibilities of functionalization (Verzele et al. 2011). To
use bile acids as vehicles for drugs, the respective drug can
be coupled either to a hydroxyl group at positions 3, 7 or 12
of the bile acid molecule or to the carboxyl group at position
24, and in most initial studies drugs have been attached to
the bile acid side chain, C-24, due to the relatively simple
synthesis process (Kramer 2011).
Some of these studies have shown that conjugates of
gabapentin, as a drug that exhibits low and variable oral
absorption at therapeutic doses, with CDC, coupled at posi-
tion C-24, had affinity towards the apical sodium dependent
bile acid transporter (ASBT) in the terminal ileum (Rais
et al. 2011). Furthermore, it was demonstrated that oral
bioavailability of the valine ester prodrug of acyclovir in
mice was increased remarkably (two-fold) by coupling to
CDC at position C-24, compared to the parent drug itself
(Tolle-Sander et al. 2004). Some authors have investigated
the attachment of a bile acid moiety at the same position to
the cis-platinum molecule, in order to achieve liver selectiv-
ity for the treatment of liver tumors and to reduce undesired
toxic effects (Criado et al. 1997). The experiments with
drug–bile acid conjugates having attached the drug moiety
to the C-24 carboxylic acid function showed that a cer-
tain liver selectivity can be achieved, but their intestinal
absorption, however, was low (Kramer 2011).
It has been demonstrated that the best molecular recogni-
tion by hepatic and ileal bile acid transporters was achieved
with the conjugates having attached the drug at position
3, in line with the prediction from the QSAR transporter
model. A liver selectivity was shown for drugs of diverse
structure and medical indications, coupled with bile acids
at position 3 (HMG-CoA reductase inhibitors, antifibrotic
pyridine 2,4-dicarboxylic acids or oxaprolylpeptides, cyto-
statics such as chlorambucil or 5-fluorouracil, etc.) (Kramer
2011). Kolhatkar and Polli (2012) have tested 19 bile acid
analogs against ASBT and NaTC cotransporting polypep-
tide (NTCP) for binding, as well as translocation. Results
suggested that drug conjugation to the C-3 hydroxyl group,
rather than C-7, had potential to lead to a successful prodrug
targeting ASBT and NTCP.
Recently, a new form of orally active heparin deriva-
tive, a chemical conjugate of low molecular weight heparin
(LMWH) and DC, has been developed, and it has been
shown that DC molecule increases the absolute bioavail-
ability of orally administered LMWH about 25 times by
enhancing its hydrophobic properties. However, a more
120 M. Stojančević et al.
promising approach for improving oral absorption of hep-
arin is the non-covalent, ion-pairing interaction of new oral
bile acid-based carrier, deoxycholylethylamine (DCEA),
with LMWH (Lee et al. 2007).
Bile acids have been considered recently very useful
in the preparation of peptide and protein drugs. Covalent
coupling of a peptide to a ligand which is transported carrier-
mediated, such as bile acids, may improve a most critical
step in peptide delivery, the translocation of the peptide
across the cell membrane (Kramer et al. 1997). Lee et al.
(2005) have demonstrated that, when covalently attached
to DC, recombinant human insulin is orally active, with
high binding affinity to the insulin receptor. Furthermore,
this insulin–bile acid conjugate showed sustained biological
activity. It was shown that C-24 peptide conjugates gener-
ally had mild affinity to the ASBT and slow transport rates,
both decreasing with peptide chain length, although C-3
peptide conjugates retained higher affinity towards hepatic
and intestinal bile acid transporters (Kramer 2011).
It is obvious that chemical conjugates of bile acids with
various drugs may improve drug absorption, but can also
achieve drug organotropism for the liver and the terminal
ileum. Therefore, this represents a promising strategy to
selective and less toxic pharmacotherapy.
Conclusions
The studies presented here appear very attractive from a
pharmaceutical point of view since these findings offer
important pharmacological options for the development of
new drug formulations in combination with bile acids, their
salts and various derivatives. Currently, bile acids as com-
pounds that may facilitate transport of drugs across various
membranes are the topic of extensive research. By under-
standing the relationship between bile acid structure and
the effect induced in the cell membrane, and of course the
mechanism of action, it is possible to improve efficacy and
reduce toxicity profile of bile acids. The available infor-
mation will probably yield, in the near future, new drug
formulations with improved pharmaceutical properties and
enhanced effectiveness of drug-based therapy.
Funding
This work was supported by the Ministry of Education and
Science, Republic of Serbia, under Grant No. 41012.
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