Thiomers A new generation of mucoadhesive polymers.pdf

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Thiomers: A new generation of mucoadhesive polymersB

Andreas Bernkop-Schnürch *

Department of Pharmaceutical Technology, Institute of Pharmacy, Leopold-Franzens-University Innsbruck, Innrain 52,

Josef Möller 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 bonds—the bridging structure most commonly encountered in biological systems. So far the cationic thiomers

chitosan–cysteine, chitosan–thiobutylamidine as well as chitosan–thioglycolic acid and the anionic thiomers poly(acylic acid)–

cysteine, poly(acrylic acid)–cysteamine, carboxy-methylcellulose–cysteine and alginate–cysteine have been generated. Due to

the immobilization of thiol groups on mucoadhesive basis polymers, their mucoadhesive properties are 2- up to 140-foldimproved. 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. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15702. Synthesis of thiomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1571

2.1. Cationic thiomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1571

2.2. Anionic thiomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1571

0169-409X/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.addr.2005.07.002

B This review is part of the Advanced Drug Delivery Reviews theme issue on bMucoadhesive Polymers: Strategies, Achievements and

Future Challenges Q , Vol. 57/11, 2005.

* Tel.: +43 512 507 5371; fax: +43 512 507 2933.

E-mail address: [email protected].

Advanced Drug Delivery Reviews 57 (2005) 1569–1582

www.elsevier.com/locate/addr


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3. Mechanisms being responsible for improved mucoadhesion . . . . . . . . . . . . . . . . . . . . . . . . . . 1571

3.1. Formation of disulfide bonds with the mucus gel layer . . . . . . . . . . . . . . . . . . . . . . . . . 1571

3.2. In situ cross-linking process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1573

4. Mucoadhesive properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15745. Dosage forms based on thiomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1575

5.1. Micro- and nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1575

5.2. Matrix tablets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1575

5.3. Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1576

5.4. Liquid formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1576

6. In vivo studies: proof of concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1577

6.1. Oral delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1577

6.1.1. Low molecular weight heparin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1577

6.1.2. Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1577

6.1.3. Salmon calcitonin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1578

6.2. Nasal delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1578

6.3. Ocular delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1579

7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1580

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1580

1. Introduction

Since the concept of mucoadhesion has been pio-

neered in the 1980s, numerous attempts have been

undertaken in order to improve the adhesive proper-

ties of polymers. These attempts include approaches

such as the use of linear poly(ethylene glycol) as

adhesion promoter for hydrogels [1], the neutraliza-

tion of ionic polymers [2], mucoadhesion by a sus-

tained hydration process [3] and the development of

polymer–adhesin conjugates (e.g. [4,5]) providing a

specific binding to epithelia. However, all these sys-

tems are based on the formation of non-covalent

bonds such as hydrogen bonds, van der Waal’s forces,

and ionic interactions. Accordingly, they provide only

relative weak mucoadhesion, in many cases insuffi-

cient to guarantee the localization of a drug delivery

system at a given target site. Mucoadhesive polymershave therefore in many cases not proven to be effec-

tive as d pharmaceutical glueT [6,7].

A presumptive new generation of mucoadhesive

polymers are thiolated polymers—designated thio-

mers [8]. In contrast to well-established mucoadhesive

polymers these novel polymers are capable of forming

covalent bonds. The bridging structure most com-

monly encountered in biological systems–the disul-

fide bond–has thereby been discovered for the

covalent adhesion of polymers to the mucus gel

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 illu-

strated 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

SH

SHSH

SH

SH

SH

SH

SH

Polymer

Thiomer

thiol bearingligand+

Fig. 1. Thiolated polymers thiomers.

A. Bernkop-Schnürch / Advanced Drug Delivery Reviews 57 (2005) 1569–15821570


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review the so far gained knowledge on the mucoad-

hesive properties of thiomers is summarized and dis-

cussed. 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 out-lined in Fig. 3 sulfhydryl bearing agents can be cova-

lently 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 [10–12]. An unintended

oxidation of thiol groups during synthesis can be

avoided by performing the reaction under inert con-

ditions. Alternatively the synthesis can be performed

at a pHb5. 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. Further-more, disulfide bonds can be reduced after the synth-

esis process by the addition of reducing agents such as

dithiotreithol or borohydride.

In case of the formation of amidine bonds 2-imi-

nothiolane 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 Ellman’s reagent

[8] with and without previous quantitat ive 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. Appro-

priate ligands are cysteine, homocysteine and cystea-

mine [15–21]. 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 thio-

mer 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 differ-

ent types of mucus glycolproteins or designated

mucins exhibiting cysteine-rich subdomains have

been reviewed previously [23]. Generally there are

Thiomer SH Mucin+ S-S Mucin

Thiomer SHMucin+S-S Mucin

Thiomer SH + MucinHS

Thiomer S-S Mucin

Ox.

Fig. 2. Mechanisam of disulfide bond formation between thiomers

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

A. Bernkop-Schnürch / Advanced Drug Delivery Reviews 57 (2005) 1569–1582 1571


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OH

H

H NH

H

O

OH

CH2OH

H

SH

NH2+Cl-

SH

O

OH HH

HH

H

OHCH

O

2

NH

O NH2

O

NH

2

O

CH OH

H

HH

HHHO

O

SH

OH

HH

H

H

H

O

H

O

O

H

HH

H

O

2

2

CH

CH

OH

OH

OH

OHO

NHCOOH

SHO

SH

COOH COOHCONH

HSCOOH

CONH COOHCOOH

NH

SHO

OH

O

OO

OH

O

OO

O

O

OH

H

H

R2

Chitosan-Cysteine [10]

Carboxymethycellulose-Cysteine [16,17]

Alginate-Cysteine [15]

Chitosan-Thiogylcolic acid [11,12]

Chitosan-Thiobutylamidine [13,14] Poly(acrylic acid)-Cysteine [8, 16]

Poly(acrylic acid)-Cysteamine [18]

O NH

COOH

COOH

SH

Poly(acrylic acid)-Homocysteine [21]Poly(methacrylic acid)-Cysteine [20]

Deacetylated Gellan Gum-Cysteine [19]

HSCOOH

CONH COOHCOOH

CH3 CH3 CH3

OH

CH2OH

O

H

H

H

HH

O

OH

OH

O

H

H

H

HH

O

O

OH

OH

O

H

H

H

HH

CH2OH

O

OH

O

HH

O

n

HO

A

H

H

CH3

H

HO

B C D

R

OOH

NH

SHR2 =R1 = OH

Fig. 3. Structure of thiolated polymers.



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no mucosal surfaces in which mucins with cysteine-

rich subdomains are not present. In contrast to non-

covalent bonds disulfide bonds are not influenced by

factors such as ionic strength and pH. Velocity andextent of disulfide bond formation depends on the

concentration of thiolate anions representing the reac-

tive 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 chitosan–thiobutyla-

midine conjugat e (Fig. 3), for instance, exhibit a p K a value of 9.9 [13], whereas the p K a value of the

thiol gr oups 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.

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 for-

mation 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

disulfide bond breaker dithiothreitol already immobi-

lized 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 t he mucus gel layer.

The mechanism is illustrated in Fig. 4. It is similar to

Interpenetration

In situ cross-linking

Mucus gel layer

Polymer

SS

SS

Chain Links:

Fig. 4. Schematic presentation of improved mucoadhesion by an in-

situ cross-linking in comparison to chain links.



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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 polymer-

ization 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 char-

acterized in vitro by rheological measurements. The

sol–gel transition of thiolated chitosans, for instance,

was completed at pH 5.5 after 2 h, when highly cross-linked gels were formed. In parallel, a significant

decrease in the thiol group content of the polymers

was o bserved, indicating the formation of disulfide

bonds [13,27]. The rheological properties of unmodi-

fied chitosan remained constant over the whole obser-

vation 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-

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 andocular formulations, which should stabilize them-

selves once applied on the site of drug delivery.

4. Mucoadhesive properties

The mucoadhesive properties of thiomers in com-

parison 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 signifi-cantly 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. Marschütz et al.

could show that the viscosity of poly(acrylic acid)/

mucin mixtures–directly correlating with the interac-

tions of the polymer with the mucus and consequently

indicating the mucoadhesive properties–can be more

than 10-fold improved [28]. The same thiolated poly-

mer 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, respec-

tively [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

chitosan–thiobutylamidine 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 chitosan–thiobu-

tylamidine conjugate in comparison to unmodifiedchitosan [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 med-

ium molecular mass. In case of poly(acrylic acid)–

1,E-01

1,E+00

1,E+01

1,E+02

1,E+03

0 1 2 4 6

time (h)

G ' ( P a

)

Fig. 5. Increase in the elastic properties ( G V) of a 1.5% (m/v)

chitosan–TBA conjugate gel at pH 5.5 and 37 8C as a function of

time. Indicated values are means (FS.D.) of at least three experi-

ments (adopted from Bernkop-Schnürch et al. [13]).



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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 mole-

cular 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 chitosan–thiobutylamidine

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 immobi-lized thiol groups was, the higher were the mucoad-

hesive properties. Furthermore, the mucoadhesive

properties of thiomers exhibiting a relative low pH

are always higher [31,32].

5. Dosage forms based on thiomers

5.1. Micro- and nanoparticles

Because of their relative small size micro- and

nanoparticles show a prolonged gastrointestinal resi-

dence time even without any mucoadhesive proper-

ties by diffusing into the mucus gel layer. Coupe et

al., for instance, could demonstrate that particulate

delivery systems display a more prolonged gastro-

intestinal transit time compared to single-unit dosage

forms [33]. In order to further improve the residence

time of drug delivery systems on mucosal mem-

branes, both approaches: mucoadhesive polymers

(I) and micro-/nanoparticles (II) were consequently

combined. Micro- and nanoparticles based on anio-

nic 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

being based on thiolated polymers do not disinte-

grate. Because of the formation of disulfide bonds

within t he polymeric network, the particles are sta-

bilized [29,36]. Consequently, also a controlled drugrelease out of thiomer micro- and nanoparticles can

be provided.

Recently, microparticles comprising poly(acry-

late)–cysteine were generated via the solvent evapora-

t ion emulsification method. Particles as illustrated in

Fig. 6 were of spherically and partially porous struc-

ture and had a main size in the range of 20–60 Am

with a center at 35 Am. Because of the formation of

disulfide bonds within the particles they did not dis-

integrate 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 mucoadhe-

sive properties of the cor responding microparticles

were 3-fold improved [36].

5.2. Matrix tablets

Mucoadhesive matrix tablets are useful for

intraoral, peroral, ocular and vaginal—local or sys-

temic delivery. Due to the in situ cross-linking prop-

erties 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 poly-

carbophil revealed a stability of less than 2 h,

SS SH

HS

S

S

HS

Acc.V Spo t Magn WD

20.0 kV4.0 2567x 17.7

10 µm

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

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

modified).



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whereas tablets being based on thiolated polycarbo-

phil 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 aug-

mented 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 oeso-

phagus [38]. Additionally, matrix tablets comprisinga 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 guaran-

tee even a zero order release profile for several

hours [41]. The drug release rate is thereby predo-

minately 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 deli-

very systems are often insufficient. The great advan-

tage 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-depen-

dent manner. Because of this instability in aqueous

solutions thiomers have so far not been used in liquid

formulations. Recently, however, Hornof coulddemonstrate 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 formula-

tions comprising thiomers were prepared and tested

in vivo.

In particular in the ophthalmic field thiomers have

already shown potential in form of liquid formula-

tions. 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 con-

tain 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 para-

meter 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 cor-

nea. Normally the break-up time exceeds the time-

span between blinks, but in patients with dry eyesyndrome the break-up time is decreased to less than

5 s. In Fig. 7 the comparison of the effect of a well-

established commercial product containing carbomer

and a formulation containing 0.2% (m/v) polyacrylic

acid–cysteine and mannitol as tonicity agent on the

tear film break-up time is shown in human volun-

teers. 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-



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tol solution or the commercially available formula-

tion [43].

6. In vivo studies: proof of concept

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 com-

paratively 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 sys-

tems based on mucoadhesive polymers. The efficacy

of unmodified polycarbophil and thiolated polycar-

bophil was thereby compared in vivo in rats. The

oral administration of heparin with poly(acrylic

acid)–cysteine as carrier matrix resulted in a signi-

ficantly increased absorption of LMWH compared tocontrol tablets comprising unmodified poly(acrylic

acid) or to an orally given aqueous heparin solution.

An absolute bioavailability of 19.9F9.3% compared

to intravenous application was obtained in case of

the thiomer delivery system. Control tablets with

heparin showed a slight increase in the bioavailabi-

lity determined to be 5.8F1.4% compared to the

oral heparin solution (2.3F2.8%). Furthermore, the

thiomer delivery system displayed a prolonged effi-

cacy of heparin compared to the other formulations,

as the maximum with 0.4F0.16 IU/ml was reachedafter 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

0

1

2

3

4

5

12

Time of determination

B U T [ s e c ]

0 min 5 min

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.

0

0,1

0,2

0,3

0,4

0,5

0,6

0 2 4 6 8 10 12 14 16 18 20 22 24

time [hours]

h e p a r i n

( I U / m l )

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 thio-

lated poly(acrylatic acid) (o) and in minitablets comprising the

corresponding unmodified polymer (.) in rats. Data represent themeanFS.D. of five experiments [45].



10/14

vivo studies. Marschütz 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 incorpo-

rated in thiolated poly(acrylic acid) and orally admi-

nistered 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 treat-

ment of chronic bone diseases. It is currently mar-

keted 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

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 chitosan–thiobutylamidine 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 chit osan–BBI conjugate (Bowman–Birk inhi-

bitor) [50] and a chitosan–elastatinal conjugate [51]

were added. On the other hand uncoated tablets tar-geting the stomach contained a chitosan–pepstatin

conjugate, which should avoid pepsinic digestion of

salmon calcitonin. The different tablets were orally

given to rats and the plasma calcium level was mon-

itored as a function of time. Studies showed no sta-

tistically significant ( pb0.05) 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 mucoad-

hesive [52].

Fig. 10 shows that the presence of the mucoadhe-

sive chitosan–thiobutylamidine 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-tar-

geted 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

Recently, the potential of a thiomer gel formula-

tion could be demonstrated by in vivo studies. Leit-

ner et al. developed a nasal gel formulation for

systemic delivery of hGH. The efficacy of a mucoad-

hesive gel formulation being based on unmodified

polycarbophil and polycarbophil–cysteine was com-

pared in rats. Results as shown in Fig. 11 demon-

40

50

60

70

80

90

100

110

120

0 4 8 12 16 20 24 28 32

Time (hours)

G l u c o s e

l e v e

l ( % )

Fig. 9. Glycemic profiles in diabetic mice after single oral admin-

istration of pegylated insulin loaded minitablets comprising thio-

lated poly(acrylic acid) (.) and of a pegylated insulin solution (o).Each point represents the meanFS.D. of 10 experiments (adopted

from Caliceti et al. [47]).



11/14

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

2.75F0.37% 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 microparti-

cles were generated for the nasal delivery of hGH.

The intranasal administration of this microparticulate

formulation to rats resulted in a relative bioavailabilityof 8.11F2.15% that represents a 3-fold improvement

compared to microparticles comprising the corre-

sponding unmodified polymer [55].

When the maximum plasma concentration of hGH

following nasal administration of the thiomer micro-

particles 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

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 mucociliaryclearance system.

6.3. Ocular delivery

Apart from liquid formulations for the treatment of

the dry eye syndrome as described in chapter 5.4., a

b proof of concept Q has also been provided for ocular

inserts comprising a thiolated polyacrylate. Drugs

administered in traditional topical ophthalmic formu-

lations such as aqueous eye drops have poor bioavail-

ability due to rapid precorneal elimination. To reachtherapeutic levels frequent instillations of the drug are

required, leading to a low patient compliance. Further-

more, 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-

0

20

40

60

80

100

120

140

0 1 2 3 4 5 6

time (h)

p l a s m

a h G H l e v e

l ( µ I U / m l )

Fig. 11. Concentration–time profiles of human growth hormone

(hGH) in rat plasma obtained after nasal administration of hGH

incorporated in a thiolated polyacrylate gel (.) and in the corre-sponding unmodified polyacrylate gel (o). Data represent the

meanFS.D. of 4–5 experiments (adopted from Leitner et al. [54]).

85

90

95

100

0 4 8 12 16 20 24

Time [h]

p l a s m a c a

l c i u m

l e v e l

i n %

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]).



12/14

tion of the device on the ocular surface and a con-

trolled 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 con-

trolled release was achieved for the incorporated

model drug sodium fluorescein. In vivo studies in

human volunteers showed that inserts based on thio-lated poly(acrylic acid) provide a fluorescein concen-

tration 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].

7. Conclusion

The chemical modification of well-established

mucoadhesive polymers via derivatisation with variousreagents bearing sulfhydryl functions causes a dramatic

improvement in the polymer’s properties. Mucoadhe-

siveness 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 demon-

strated by various in vivo studies in different species

on the gastrointestinal, nasal and ocular mucosa.

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