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Advanced Drug Delivery Reviews 57 (2005) 1569 1582

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Thiomers: A new generation of mucoadhesive polymersB


Andreas Bernkop-Schnurch *
Department of Pharmaceutical Technology, Institute of Pharmacy, Leopold-Franzens-University Innsbruck, Innrain 52,
Josef Moller Haus, 6020 Innsbruck, Austria
Received 30 November 2004; accepted 12 July 2005
Available online 19 September 2005

Abstract
Thiolated polymers or designated thiomers are mucoadhesive basis polymers, which display thiol bearing side chains. Based
on thiol/disulfide exchange reactions and/or a simple oxidation process disulfide bonds are formed between such polymers and
cysteine-rich subdomains of mucus glycoproteins building up the mucus gel layer. Thiomers mimic therefore the natural
mechanism of secreted mucus glycoproteins, which are also covalently anchored in the mucus layer by the formation of
disulfide bondsthe bridging structure most commonly encountered in biological systems. So far the cationic thiomers
chitosancysteine, chitosanthiobutylamidine as well as chitosanthioglycolic acid and the anionic thiomers poly(acylic acid)
cysteine, poly(acrylic acid)cysteamine, carboxy-methylcellulosecysteine and alginatecysteine have been generated. Due to
the immobilization of thiol groups on mucoadhesive basis polymers, their mucoadhesive properties are 2- up to 140-fold
improved. The higher efficacy of this new generation of mucoadhesive polymers in comparison to the corresponding
unmodified mucoadhesive basis polymers could be verified via various in vivo studies on various mucosal membranes in
different animal species and in humans. The development of first commercial available products comprising thiomers is in
progress. Within this review an overview of the mechanism of adhesion and the design of thiomers as well as delivery systems
comprising thiomers and their in vivo performance is provided.
D 2005 Elsevier B.V. All rights reserved.
Keywords: Mucoadhesion; Thiolated polymers; Thiomers; Disulfide bonds; Thiolated poly(acrylic acid); Thiolated chitosan

Contents
1.
2.

Introduction . . . . . . .
Synthesis of thiomers . .
2.1. Cationic thiomers
2.2. Anionic thiomers

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B
This review is part of the Advanced Drug Delivery Reviews theme issue on bMucoadhesive Polymers: Strategies, Achievements and
Future ChallengesQ, Vol. 57/11, 2005.
* Tel.: +43 512 507 5371; fax: +43 512 507 2933.
E-mail address: andreas.bernkop@uibk.ac.at.

0169-409X/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2005.07.002

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A. Bernkop-Schnurch / Advanced Drug Delivery Reviews 57 (2005) 15691582

3.

Mechanisms being responsible for improved mucoadhesion .


3.1. Formation of disulfide bonds with the mucus gel layer
3.2. In situ cross-linking process . . . . . . . . . . . . . .
4. Mucoadhesive properties . . . . . . . . . . . . . . . . . . .
5. Dosage forms based on thiomers . . . . . . . . . . . . . . .
5.1. Micro- and nanoparticles . . . . . . . . . . . . . . . .
5.2. Matrix tablets. . . . . . . . . . . . . . . . . . . . . .
5.3. Gels . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4. Liquid formulations . . . . . . . . . . . . . . . . . .
6. In vivo studies: proof of concept . . . . . . . . . . . . . . .
6.1. Oral delivery . . . . . . . . . . . . . . . . . . . . . .
6.1.1. Low molecular weight heparin . . . . . . . .
6.1.2. Insulin . . . . . . . . . . . . . . . . . . . . .
6.1.3. Salmon calcitonin . . . . . . . . . . . . . . .
6.2. Nasal delivery . . . . . . . . . . . . . . . . . . . . .
6.3. Ocular delivery . . . . . . . . . . . . . . . . . . . . .
7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction
Since the concept of mucoadhesion has been pioneered in the 1980s, numerous attempts have been
undertaken in order to improve the adhesive properties of polymers. These attempts include approaches
such as the use of linear poly(ethylene glycol) as
adhesion promoter for hydrogels [1], the neutralization of ionic polymers [2], mucoadhesion by a sustained hydration process [3] and the development of
polymeradhesin conjugates (e.g. [4,5]) providing a
specific binding to epithelia. However, all these systems are based on the formation of non-covalent
bonds such as hydrogen bonds, van der Waals forces,
and ionic interactions. Accordingly, they provide only
relative weak mucoadhesion, in many cases insufficient to guarantee the localization of a drug delivery
system at a given target site. Mucoadhesive polymers
have therefore in many cases not proven to be effective as dpharmaceutical glueT [6,7].
A presumptive new generation of mucoadhesive
polymers are thiolated polymersdesignated thiomers [8]. In contrast to well-established mucoadhesive
polymers these novel polymers are capable of forming
covalent bonds. The bridging structure most commonly encountered in biological systemsthe disulfide bondhas thereby been discovered for the
covalent adhesion of polymers to the mucus gel

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layer of the mucosa. Thiomers are mucoadhesive


basis polymers, which display thiol bearing side
chains (Fig. 1). Based on thiol/disulfide exchange
reactions and/or a simple oxidation process as illustrated in Fig. 2, disulfide bonds are formed between
such polymers and cysteine-rich subdomains of
mucus glycoproteins [9]. Hence, thiomers mimic the
natural mechanism of secreted mucus glycoproteins,
which are also covalently anchored in the mucus layer
by the formation of disulfide bonds. Within this

Polymer

+
SH

thiol bearing
ligand
SH
SH
SH

SH
SH

SH

Thiomer

SH

Fig. 1. Thiolated polymers thiomers.

A. Bernkop-Schnurch / Advanced Drug Delivery Reviews 57 (2005) 15691582

Thiomer

SH

Thiomer

S-S

Thiomer

SH

Mucin

S-S

Mucin

HS

Mucin

Mucin

Mucin

SH

Ox.
Thiomer

S-S

Mucin

Fig. 2. Mechanisam of disulfide bond formation between thiomers


and mucus glycoproteins (mucins) according to Leitner et al. [9].

review the so far gained knowledge on the mucoadhesive properties of thiomers is summarized and discussed. The provided information should represent a
good starting point for further developments in this
promising research field.

2. Synthesis of thiomers
2.1. Cationic thiomers
Cationic thiomers are mainly based on chitosans.
The primary amino group at the 2-position of the
glucosamine subunits of this polymer is the main
target for the immobilization of thiol groups. As outlined in Fig. 3 sulfhydryl bearing agents can be covalently attached to this primary amino group via the
formation of amide or amidine bonds. In case of the
formation of amide bonds the carboxylic acid group
of the ligands cysteine and thioglycolic acid reacts
with the primary amino group of chitosan mediated
for instance by carbodiimides [1012]. An unintended
oxidation of thiol groups during synthesis can be
avoided by performing the reaction under inert conditions. Alternatively the synthesis can be performed

1571

at a pH b 5. At this pH-range the concentration of


thiolate-anions, representing the reactive form for
oxidation of thiol groups, is low, and the formation
of disulfide bonds can almost be excluded. Furthermore, disulfide bonds can be reduced after the synthesis process by the addition of reducing agents such as
dithiotreithol or borohydride.
In case of the formation of amidine bonds 2-iminothiolane is used as coupling reagent [13,14]. It offers
the advantage of a simple one step coupling reaction. In
addition, the thiol group of the reagent is protected
towards oxidation due to its chemical structure. The
amount of immobilized thiol groups in reduced and
oxidized form can be determined via Ellmans reagent
[8] with and without previous quantitative reduction of
disulfide bonds with borohydride [22].
2.2. Anionic thiomers
So far generated anionic thiolated polymers exhibit
all carboxylic acid groups as anionic substructures.
These carboxylic acid groups offer also the advantage
that sulfhydryl moieties can be easily attached to such
polymers via the formation of amide bonds. Appropriate ligands are cysteine, homocysteine and cysteamine [1521]. The formation of amide bonds can be
mediated by carbodiimides. The chemical structure of
so far generated anionic thiolated polymers is shown
in Fig. 3. Thiol oxidation during synthesis can be
avoided as described above. The total amount of
immobilized reduced and oxidized thiol groups can
be determined in the same way as described for
cationic thiomers.

3. Mechanisms being responsible for improved


mucoadhesion
3.1. Formation of disulfide bonds with the mucus gel
layer
The formation of disulfide bonds between the thiomer and the mucus gel layer takes place either via
thiol/disulfide exchange reactions (1) or via a simple
oxidation process of free thiol groups (2). The different types of mucus glycolproteins or designated
mucins exhibiting cysteine-rich subdomains have
been reviewed previously [23]. Generally there are

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A. Bernkop-Schnurch / Advanced Drug Delivery Reviews 57 (2005) 15691582

CH 2 OH

OH

HO

OH

R2
OH

NH
O

NH

NH 2
O

SH

HO

SH
OH

Chitosan-Cysteine [10]

Alginate-Cysteine [15]
CH 2OH
O

CH2 OH

OH

NH

H
O

OH

OH
H

OH
H

SH

O
H

O
CH2 OH

COOH
NH

Chitosan-Thiogylcolic acid [11,12]

SH
O

SH

Carboxymethycellulose-Cysteine [16,17]
NH2+ClH

NH
H

OH
H

CONH

COOH

COOH

HS
COOH

CH2OH

Chitosan-Thiobutylamidine [13,14]
O

CH2OH

HO

CH2OH

H
H
O
H

H
O
H

OH

Poly(acrylic acid)-Cysteine [8, 16]

H
OH

O
H

OH

H
O

H
H
OH

CH3
H

HO

OH

CONH

OH
n

O
R2 =

R1 = OH

OH
SH

Deacetylated Gellan Gum-Cysteine [19]


CH3

COOH

SH

N
H

CH3

COOH

Poly(acrylic acid)-Cysteamine [18]

CH3

COOH
O
CONH

COOH

NH

COOH

COOH

HS
COOH

HS

Poly(methacrylic acid)-Cysteine [20]

Poly(acrylic acid)-Homocysteine [21]

Fig. 3. Structure of thiolated polymers.

A. Bernkop-Schnurch / Advanced Drug Delivery Reviews 57 (2005) 15691582

no mucosal surfaces in which mucins with cysteinerich subdomains are not present. In contrast to noncovalent bonds disulfide bonds are not influenced by
factors such as ionic strength and pH. Velocity and
extent of disulfide bond formation depends on the
concentration of thiolate anions representing the reactive form for thiol/disulfide exchange reactions and
oxidation processes. The concentration of thiolate
anions in turn depends on:
! the pK a value of the thiol group. In dependence on
the polymer backbone and the chemical structure
of the ligand, more or less reactive thiomers can be
designed. Thiol groups of the chitosanthiobutylamidine conjugate (Fig. 3), for instance, exhibit a
pK a value of 9.9 [13], whereas the pK a value of the
thiol groups of poly(acrylate)cysteine conjugates
is 8.35 [24];
! the pH of the thiomer. As only ionic thiomers are
used, they all display a high buffer capacity. The
buffer capacity of a sodium poly(acrylate) matrix
tablet, for instance, can be compared with that of an
at least 25 M acetate buffer. As all charged groups
remain concentrated on the polymeric network a
kind of dmicroclimateT can be established [25].
The reactivity of thiol groups can consequently be
controlled by adjusting the pH of the polymer to a
certain level. The higher the pH is adjusted, the more
reactive are the thiol groups and vice versa; and
! the pH of the surrounding medium. The reactivity of
thiol groups inside the polymeric network is mainly
controlled by the pH of the thiomer, whereas the
reactivity on the surface of the polymer is more
controlled by the pH of the surrounding medium.
As the mucus gel layer being close to the epithelium
has a pH around 7, thiol groups penetrating into the
mucus are always sufficiently reactive.

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disulfide bond breaker dithiothreitol already immobilized mucin could be completely removed from the
thiolated polymer [8].
3.2. In situ cross-linking process
Another likely mechanism being responsible for
the improved mucoadhesive properties of thiomers is
based on their in situ cross-linking properties. During
and after the interpenetration process, which could
be verified for mucoadhesive polymers such as
poly(acrylic acid) recently [26], disulfide bonds are
formed within the thiomer itself leading to additional
anchors via chaining up with the mucus gel layer.
The mechanism is illustrated in Fig. 4. It is similar to
Chain Links:

Polymer

Mucus gel layer

Interpenetration

In situ cross-linking

Evidence for the formation of covalent bonds


between thiomers and the mucus gel layer has been
provided recently. Leitner et al. could show by four
different methods including rheological, diffusion, gel
permeation and certain mucoadhesion studies the formation of disulfide bonds between thiolated polymers
and mucus glycoproteins [9]. In another publication it
was also shown that mucin can be effectively bound
to thiolated polyacrylate, while it is not at all bound to
unmodified polyacrylate. Due to the addition of the

SS
SS

Fig. 4. Schematic presentation of improved mucoadhesion by an insitu cross-linking in comparison to chain links.

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A. Bernkop-Schnurch / Advanced Drug Delivery Reviews 57 (2005) 15691582

the mechanism on which the adhesive properties of


most adhesive is based on, i.e. a penetration of the
adhesive into a certain surface structure followed by
a stabilization process of the adhesive. In case of
superglues, for instance, monomeric cyanoacrylates
penetrate into raw surfaces followed by a polymerization process. Thiolated polymers display in situ
gelling properties due to the oxidation of thiol
groups at physiological pH-values, which results in
the formation of inter- and intramolecular disulfide
bonds.
The in situ gelling behavior of thiomers was characterized in vitro by rheological measurements. The
solgel transition of thiolated chitosans, for instance,
was completed at pH 5.5 after 2 h, when highly crosslinked gels were formed. In parallel, a significant
decrease in the thiol group content of the polymers
was observed, indicating the formation of disulfide
bonds [13,27]. The rheological properties of unmodified chitosan remained constant over the whole observation period. Rheological investigation of thiolated
chitosans furthermore demonstrated a clear correlation
between the total amount of polymer-linked thiol
groups and the increase in elasticity of the formed
gel. The more thiol groups were immobilized on
chitosan, the higher was the increase in elastic mo-

1,E+03

G' (Pa)

1,E+02

1,E+01

1,E+00

1,E-01
0

time (h)
Fig. 5. Increase in the elastic properties ( GV) of a 1.5% (m/v)
chitosanTBA conjugate gel at pH 5.5 and 37 8C as a function of
time. Indicated values are means (FS.D.) of at least three experiments (adopted from Bernkop-Schnurch et al. [13]).

dulus G in solutions of thiolated chitosan (Fig. 5)


[13,27].
These in situ gelling properties are in particular of
interest for liquid or semisolid vaginal, nasal and
ocular formulations, which should stabilize themselves once applied on the site of drug delivery.

4. Mucoadhesive properties
The mucoadhesive properties of thiomers in comparison to well-established polymers are discussed in
detail in this issue by Grabovac et al. Due to the
immobilization of thiol groups on all so far tested
polymers, their mucoadhesive properties were significantly improved irrespectively from the evaluation
method. In case of anionic mucoadhesive polymers
the poly(acrylic acid)cysteine conjugate seems to be
a good example for this observation. Marschutz et al.
could show that the viscosity of poly(acrylic acid)/
mucin mixturesdirectly correlating with the interactions of the polymer with the mucus and consequently
indicating the mucoadhesive propertiescan be more
than 10-fold improved [28]. The same thiolated polymer showed in comparison to the corresponding
unmodified polymer more than 2-fold and 20-fold
improved mucoadhesive properties in tensile studies
and by using the rotating cylinder method, respectively [28]. In addition, it could be shown that the
residence time of poly(acrylic acid) microparticles on
the small intestinal mucosa can be more than 3-fold
prolonged by the immobilization of thiol groups [29].
In case of cationic thiomers, on the other hand, the
chitosanthiobutylamidine conjugate seems to be a
good example, as it has been evaluated by various
mucoadhesion test system. We demonstrated a more
than 100-fold increased viscosity of chitosanthiobutylamidine conjugate in comparison to unmodified
chitosan [13]. Moreover in tensile studies and rotating
cylinder studies the mucoadhesive properties of the
thiolated version were 100-fold and 140-fold
improved, respectively [13,14].
In case of both mentioned thiomers the molecular
mass of the polymer chains had a great impact on their
mucoadhesive properties. For the anionic as well as
for the cationic thiomer the highest mucoadhesive
properties were achieved when they exhibited a medium molecular mass. In case of poly(acrylic acid)

A. Bernkop-Schnurch / Advanced Drug Delivery Reviews 57 (2005) 15691582

cysteine, polymer conjugates exhibiting a molecular


mass of 450 kDa were more mucoadhesive than once
of a molecular mass of 2 kDa, 45 kDA and 1000
3000 kDa [30]. On the other hand, tensile studies
performed with thiolated chitosan exhibiting a molecular mass of 150 kDa, 400 kDa and 600 kDa showed
the relatively highest mucoadhesive properties for the
medium molecular mass thiomer [14]. Utilizing a
medium molecular mass chitosanthiobutylamidine
conjugate displaying 264 AM thiol groups per gram
polymer consequently led to a more than 100-fold
improvement in mucoadhesion in comparison to
unmodified chitosan [14]. Generally, it could be
observed in most performed mucoadhesion studies
with thiomers, that the higher the amount of immobilized thiol groups was, the higher were the mucoadhesive properties. Furthermore, the mucoadhesive
properties of thiomers exhibiting a relative low pH
are always higher [31,32].

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being based on thiolated polymers do not disintegrate. Because of the formation of disulfide bonds
within the polymeric network, the particles are stabilized [29,36]. Consequently, also a controlled drug
release out of thiomer micro- and nanoparticles can
be provided.
Recently, microparticles comprising poly(acrylate)cysteine were generated via the solvent evaporation emulsification method. Particles as illustrated in
Fig. 6 were of spherically and partially porous structure and had a main size in the range of 2060 Am
with a center at 35 Am. Because of the formation of
disulfide bonds within the particles they did not disintegrate under physiological conditions within 48 h.
In addition, a controlled drug release of a model
peptide drug was achieved. Due to the immobilization
of thiol groups on poly(acrylic acid) the mucoadhesive properties of the corresponding microparticles
were 3-fold improved [36].
5.2. Matrix tablets

5. Dosage forms based on thiomers


5.1. Micro- and nanoparticles
Because of their relative small size micro- and
nanoparticles show a prolonged gastrointestinal residence time even without any mucoadhesive properties by diffusing into the mucus gel layer. Coupe et
al., for instance, could demonstrate that particulate
delivery systems display a more prolonged gastrointestinal transit time compared to single-unit dosage
forms [33]. In order to further improve the residence
time of drug delivery systems on mucosal membranes, both approaches: mucoadhesive polymers
(I) and micro-/nanoparticles (II) were consequently
combined. Micro- and nanoparticles based on anionic or cationic mucoadhesive polymers, however,
disintegrate very rapidly, unless multivalent cationic
or anionic compounds such as Ca2+-ions or sulfate
ions are added, respectively, leading to stabilization
via an ionic cross-linking process [34,35]. Due to the
addition of such ionic cross-linkers, on the other
hand, the mucoadhesive properties of these polymers
are strongly reduced. On the contrary, due to the
immobilization of thiol groups on well-established
polymers their mucoadhesive properties are even
further improved, although micro- and nanoparticles

Mucoadhesive matrix tablets are useful for


intraoral, peroral, ocular and vaginallocal or systemic delivery. Due to the in situ cross-linking properties of thiomers the cohesiveness and subsequently
the stability of the swollen carrier matrix can be
guaranteed [37]. Disintegration studies, for instance,
performed with tablets comprising unmodified polycarbophil revealed a stability of less than 2 h,

S
HS

S
S
S

SH

HS
Acc.V Spot Magn WD
20.0 kV 4.0 2567x 17.7

10 m

Fig. 6. SEM image of microspheres based on thiolated poly(acrylic


acid) (adopted from Bernkop-Schnurch et al. [36] and thereafter
modified).

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A. Bernkop-Schnurch / Advanced Drug Delivery Reviews 57 (2005) 15691582

whereas tablets being based on thiolated polycarbophil did not disintegrable at all [17]. Moreover even
no erosion of this swollen drug carrier matrix could
be observed within an observation period of 24 h.
When attached in dry form the mucoadhesion of
matrix tablets is additionally improved by an augmented interpenetration process depending on the
swelling behavior of the delivery system. In order
to make use of this simple adhesion by hydration
process also in case of peroral delivery, matrix
tablets can be enteric coated. If an adhesion shall
be achieved already in the stomach, the coating with
a triglyceride seems to be sufficient in order to avoid
an unintended adhesion in the oral cavity or oesophagus [38]. Additionally, matrix tablets comprising
a thiomer offer the advantage that a controlled drug
release can be easily achieved out of this type of
mucoadhesive dosage forms, which could already be
demonstrated for numerous drugs (e.g. 37,39 40 41).
Hornof et al., for instance, could show that by
simple homogenizing the thiomer with the drug of
choice and compressing tablets out of it results in
many cases in delivery systems, which can guarantee even a zero order release profile for several
hours [41]. The drug release rate is thereby predominately controlled by a hydration and diffusion
process.
5.3. Gels
Mucoadhesive gels are useful in case of intraoral,
vaginal, nasal and ocular delivery. So far, however,
mucoadhesive gel formulations have not reached their
full potential, as the adhesive properties of such delivery systems are often insufficient. The great advantage of the use of thiomers in gel formulations has to be
seen not only in their mucoadhesive but also in their in
situ gelling properties [13,19]. Strong mucoadhesive
properties are senseless, if the adhesive bond fails
rather within the gel formulation itself than between
the gel and the mucosa. Due to the in situ gelling
properties of thiomers, however, this shortcoming can
be overcome.
5.4. Liquid formulations
Thiomers were shown to be stable when stored in
dry form [42]. In aqueous solutions, however, they

were shown to form disulfide bonds in a pH-dependent manner. Because of this instability in aqueous
solutions thiomers have so far not been used in liquid
formulations. Recently, however, Hornof could
demonstrate that thiomers can even be stabilized in
aqueous solutions when the liquid formulations are
produced under inert conditions and the vessels are
packed in an aluminium foil containing an oxygen
scavenger such as iron-oxides inside [43]. Based on
this technology first mucoadhesive liquid formulations comprising thiomers were prepared and tested
in vivo.
In particular in the ophthalmic field thiomers have
already shown potential in form of liquid formulations. In case of the dry eye syndrome the most
prevalent disease in the eye, for instance, liquid
thiomer formulations might be highly beneficial.
One of the most important reasons for this disease
seems to be a defective mucus layer on the ocular
surface. Mucus acts as surfactant, and is therefore
important for the wettability of the epithelial surface
[44]. The main treatment for this disease is the use
of tear substitutes. Most of these formulations contain mucoadhesive hydrophilic polymers such as
carbomer or sodium hyaluronate. The mucoadhesive
properties of these polymers, however, are quite
insufficient making a frequent instillation necessary.
Because of their ability to interact with cysteine-rich
subdomains of mucus glycoproteins on the ocular
surface, eye drops containing a thiomer should be
able to prolong the stability of the precorneal tear
film for a comparable longer time period. A parameter to characterize the quality of the tear film is
the tear film break-up time, which is defined as the
time period after a blink in which the tear film
becomes unstable and dry spots evolve on the cornea. Normally the break-up time exceeds the timespan between blinks, but in patients with dry eye
syndrome the break-up time is decreased to less than
5 s. In Fig. 7 the comparison of the effect of a wellestablished commercial product containing carbomer
and a formulation containing 0.2% (m/v) polyacrylic
acidcysteine and mannitol as tonicity agent on the
tear film break-up time is shown in human volunteers. The eye drops containing thiolated polyacrylic
acid had a positive effect on the tear film stability,
whereas no difference in the tear film break-up time
was observed after application of an isotonic manni-

A. Bernkop-Schnurch / Advanced Drug Delivery Reviews 57 (2005) 15691582

BUT [sec]

4
3
2
1
0
12
0 min

5 min

Time of determination
Fig. 7. Effect of different eye drop formulations on the tear film
break-up time (BUT) of healthy volunteers. The BUT was measured
before eye drop instillation (0 min) and 5 min after eye drop
application in a single eye. The formulations tested were aqueous
eye drops containing an isotonic mannitol solution (grey bars), a
commercially available formulation containing carbomer (white
bars) and aqueous eye drops containing 0.2% (m/v) poly(acrylic
acid)cysteine conjugate and mannitol as tonicity agent (black bars)
(adopted from Hornof [43]). Indicated values are means of at least 5
experiments.

1577

bophil was thereby compared in vivo in rats. The


oral administration of heparin with poly(acrylic
acid)cysteine as carrier matrix resulted in a significantly increased absorption of LMWH compared to
control tablets comprising unmodified poly(acrylic
acid) or to an orally given aqueous heparin solution.
An absolute bioavailability of 19.9 F 9.3% compared
to intravenous application was obtained in case of
the thiomer delivery system. Control tablets with
heparin showed a slight increase in the bioavailability determined to be 5.8 F 1.4% compared to the
oral heparin solution (2.3 F 2.8%). Furthermore, the
thiomer delivery system displayed a prolonged efficacy of heparin compared to the other formulations,
as the maximum with 0.4 F 0.16 IU/ml was reached
after 12 h and the efficacy seemed to maintain for at
least additional 12 h. Results are shown in Fig. 8
[45].
6.1.2. Insulin
The benefit of thiomers for the oral administration
of insulin could meanwhile be shown by various in

tol solution or the commercially available formulation [43].

0,6
0,5

6.1. Oral delivery


6.1.1. Low molecular weight heparin
As the oral administration represents the most
convenient way of dosing for the patients, several
research groups have tried to find suitable strategies
in order to facilitate the gastrointestinal absorption of
orally delivered low molecular weight heparin
(LMWH). Mucoadhesion of the delivery system on
the absorption membrane can thereby provide a comparatively steeper concentration gradient leading in
case of passive drug uptake subsequently to an
improved bioavailability. In addition, a prolonged
residence time of the delivery in the GI tract should
lead to a longer lasting therapeutic effect.
Kast et al. designed oral LMWH delivery systems based on mucoadhesive polymers. The efficacy
of unmodified polycarbophil and thiolated polycar-

heparin (IU/ml)

6. In vivo studies: proof of concept

0,4
0,3
0,2
0,1
0

8 10 12 14 16 18 20 22 24

time [hours]
Fig. 8. Comparison of the concentration profiles of low molecular
weight heparin in plasma obtained after peroral administration of
low molecular heparin incorporated in minitablets comprising thiolated poly(acrylatic acid) (o) and in minitablets comprising the
corresponding unmodified polymer (.) in rats. Data represent the
mean F S.D. of five experiments [45].

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A. Bernkop-Schnurch / Advanced Drug Delivery Reviews 57 (2005) 15691582

vivo studies. Marschutz et al., for instance, could


show a significantly improved decrease in the blood
glucose level of diabetic mice, when the peptide drug
was orally administered in form of matrix tablets
being based on thiolated polycarbophil instead of
unmodified polycarbophil [46].
In another study pegylated insulin was incorporated in thiolated poly(acrylic acid) and orally administered to diabetic mice. When pegylated insulin was
administered orally in aqueous solution no therapeutic
effect could be observed at all. In contrast, when mice
were dosed with tablets comprising the peptide drug
and thiolated poly(acrylic acid), a pronounced
decrease in the blood glucose level was achieved.
This significant effect was maintained for even 24 h.
The results of this study are shown in Fig. 9. The oral
pharmacological efficacy of this mucoadhesive oral
delivery system versus s.c. injection was determined
to be 7% [47].
6.1.3. Salmon calcitonin
In another study salmon calcitonin was used as
model drug. Salmon calcitonin is used for the treatment of chronic bone diseases. It is currently marketed in nasal spray and injectable forms, both
having the drawback of a low patient acceptance.
A higher patient compliance should be achieved by
the application of an oral delivery system for this

120

Glucose level (%)

110
100
90
80
70
60
50

drug. However, so far reached oral bioavailability


was too low to permit therapeutic employment
[48,49]. Therefore, this peptide was regarded as
challenging model drug for testing the potential of
thiomers.
Mucoadhesive tablets comprising overall salmon
calcitonin and chitosanthiobutylamidine conjugate
as substantial polymeric excipient were developed.
In order to avoid an enzymatic degradation of the
peptide drug in the gastrointestinal tract chitosan
enzyme inhibitor conjugates were added. To enteric
coated tablets targeted to the small intestine on the one
hand a chitosanBBI conjugate (BowmanBirk inhibitor) [50] and a chitosanelastatinal conjugate [51]
were added. On the other hand uncoated tablets targeting the stomach contained a chitosanpepstatin
conjugate, which should avoid pepsinic digestion of
salmon calcitonin. The different tablets were orally
given to rats and the plasma calcium level was monitored as a function of time. Studies showed no statistically significant ( p b 0.05) reduction of the plasma
calcium level caused by salmon calcitonin, which was
orally given in solution. Furthermore, no significant
effect was observed after oral administration of tablets
comprising the peptide drug and unmodified chitosan,
although the native polymer is reported to be mucoadhesive [52].
Fig. 10 shows that the presence of the mucoadhesive chitosanthiobutylamidine conjugate is essential
for calcitonin absorption, since only tablets being
based on the thiolated chitosan caused a decrease of
plasma calcium level of more than 5% for several
hours [38,53].
The pharmacological efficacy of the stomach-targeted mucoadhesive calcitonin tablets was determined
to be 1.35% calculated on the basis of the area under
the reduction in plasma calcium levels of the oral
matrix tablets versus i.v.-injection.
6.2. Nasal delivery

40
0

12

16

20

24

28

32

Time (hours)
Fig. 9. Glycemic profiles in diabetic mice after single oral administration of pegylated insulin loaded minitablets comprising thiolated poly(acrylic acid) (.) and of a pegylated insulin solution (o).
Each point represents the mean F S.D. of 10 experiments (adopted
from Caliceti et al. [47]).

Recently, the potential of a thiomer gel formulation could be demonstrated by in vivo studies. Leitner et al. developed a nasal gel formulation for
systemic delivery of hGH. The efficacy of a mucoadhesive gel formulation being based on unmodified
polycarbophil and polycarbophilcysteine was compared in rats. Results as shown in Fig. 11 demon-

1579

have been shown to give an improved bioavailability


of the delivered drugs compared with solutions,
mainly due to their ability to reside longer in the
nasal cavity before being cleared by the mucociliary
clearance system.

100

95

6.3. Ocular delivery


90

85
0

12

16

20

24

Time [h]
Fig. 10. Decrease in plasma calcium level as a biological response
for the salmon calcitonin bioavailability in fasted rats after oral
administration of thiolated chitosan minitablets targeted to the
small intestine (E), of unmodified chitosan minitablets targeted to
the small intestine (o), of thiolated chitosan minitablets targeted to
the stomach (*), and of unmodified chitosan minitablets targeted to
the stomach (n). Indicated values are the mean results of five
experiments (adopted from Guggi et al. [38,53]).

strated a significantly higher and prolonged nasal


bioavailability of hGH, which was incorporated in
the thiomer gel formulation. Utilizing the thiomer gel
formulation an absolute nasal bioavailability of
2.75 F 0.37% was achieved [54]. As thiomers exhibit
also a strong permeation enhancing effect, however,
it is difficult to attribute this improved in vivo
efficacy exclusively to the improved mucoadhesive
properties.
In another study thiolated polyacrylate microparticles were generated for the nasal delivery of hGH.
The intranasal administration of this microparticulate
formulation to rats resulted in a relative bioavailability
of 8.11 F 2.15% that represents a 3-fold improvement
compared to microparticles comprising the corresponding unmodified polymer [55].
When the maximum plasma concentration of hGH
following nasal administration of the thiomer microparticles and the thiomer gel formulation described
above are compared, a 6-fold higher uptake is
achieved with the microparticulate formulation.
These results are in good agreement with reports in
the literature, where nasal particulate dosage forms

Apart from liquid formulations for the treatment of


the dry eye syndrome as described in chapter 5.4., a
bproof of conceptQ has also been provided for ocular
inserts comprising a thiolated polyacrylate. Drugs
administered in traditional topical ophthalmic formulations such as aqueous eye drops have poor bioavailability due to rapid precorneal elimination. To reach
therapeutic levels frequent instillations of the drug are
required, leading to a low patient compliance. Furthermore, the drug level in the tearfilm is pulsed, with an
initial period of overdosing, followed by a longer
period of underdosing [56,57].
Consequently, numerous novel ophthalmic drug
delivery systems were developed to achieve a higher
bioavailability of drugs. Among these formulations
ocular inserts seem promising as they can provide
an effective drug concentration in the eye over an
extended time period because of the prolonged reten-

140

plasma hGH level (IU/ml)

plasma calcium level in %

A. Bernkop-Schnurch / Advanced Drug Delivery Reviews 57 (2005) 15691582

120
100
80
60
40
20
0
0

time (h)
Fig. 11. Concentrationtime profiles of human growth hormone
(hGH) in rat plasma obtained after nasal administration of hGH
incorporated in a thiolated polyacrylate gel (.) and in the corresponding unmodified polyacrylate gel (o). Data represent the
mean F S.D. of 45 experiments (adopted from Leitner et al. [54]).

1580

A. Bernkop-Schnurch / Advanced Drug Delivery Reviews 57 (2005) 15691582

350

7. Conclusion

fluorescein [ng/ml]

300
250
200
150
100
50
0
0 1 2 3 4 5 6 7 8

The chemical modification of well-established


mucoadhesive polymers via derivatisation with various
reagents bearing sulfhydryl functions causes a dramatic
improvement in the polymers properties. Mucoadhesiveness and cohesiveness are strongly improved.
Furthermore, thiolated polymers display in situ-gelling
features. The efficacy of this new generation of
mucoadhesive polymers could already be demonstrated by various in vivo studies in different species
on the gastrointestinal, nasal and ocular mucosa.

time [h]
Fig. 12. Fluorescein concentration in the cornea/tearfilm compartment of a volunteer after application of a thiolated polyacrylate
insert (o) and the corresponding unmodified polyacrylate insert (.)
containing 15% fluorescein as model drug (adopted from Hornof et
al. [41]).

tion of the device on the ocular surface and a controlled release. Dosing of the drugs is also more
accurate and the risk of systemic side-effects is
decreased. Nevertheless, despite all these advantages
ocular inserts were so far not widely used in ocular
therapy. Inserts without appropriate mucoadhesive
properties can move around on the ocular surface
causing irritation and can be easily lost. The erosion
and/or disintegration into smaller pieces of soluble
inserts results in occasional blurring of vision. Ocular
inserts comprising thiomers, however, do not have
these disadvantages.
Hornof et al., for instance, could shown that inserts
based on thiolated poly(acrylic acid) were not soluble
and had good cohesive properties. In addition, a controlled release was achieved for the incorporated
model drug sodium fluorescein. In vivo studies in
human volunteers showed that inserts based on thiolated poly(acrylic acid) provide a fluorescein concentration on the ocular surface for more than 8 h, while
the fluorescein concentration rapidly decreased after
application of aqueous eye drops or inserts based on
unmodified poly(acrylic acid). A representative result
of this study is shown in Fig. 12 [41]. Moreover, these
inserts were well accepted by the volunteers. The
study indicated that ocular inserts based on thiolated
poly(acrylic acid) are promising new solid devices for
ocular drug delivery [58].

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