controlled transdermal delivery of short …...this administration route is the limited permeation...
TRANSCRIPT
GHENT UNIVERSITY
FACULTY OF PHARMACEUTICAL SCIENCES
Department of Pharmaceutics
Laboratory for Pharmaceutical Technology
Master Thesis Performed at:
UNIVERSITY OF HELSINKI - FACULTY OF PHARMACY - Division of Pharmaceutical Technology
Academic year 2011-2012
CONTROLLED TRANSDERMAL DELIVERY
OF SHORT PEPTIDES AND MODEL COMPOUNDS
BY IONTOPHORESIS AND ION-EXCHANGE
Linse MATTE
First Master of Pharmaceutical Care
Promoter
Prof. Dr. C. Vervaet
Co-promoter
Prof. Dr. J. Hirvonen
Commissioners
Prof. Dr. K. Braeckmans
Dr. E. Mehuys
COPYRIGHT
“The author and the promoters give the authorization to consult and to copy parts of this
thesis for personal use only. Any other use is limited by the laws of copyright, especially
concerning the obligation to refer to the source whenever results from this thesis are cited. “
Date
31/05/2012
Promoter Author
Prof. Dr. C. Vervaet Linse Matté
ABSTRACT IN ENGLISH
Because of several advantages over other drug administration routes, transdermal
delivery has gained a lot of interest. The main problem with this administration route is the
low intrinsic permeability of the skin, especially for large and charged molecules such as
peptides. Thus, some enhancement technique is needed in order to deliver these molecules
across the skin. Application of iontophoresis, a non-invasive technique which uses a small and
defined electric current, has been shown effective in literature to facilitate the transdermal
transport of charged and uncharged molecules. In order to have more control in transdermal
permeation during iontophoresis, the drugs can be bound to ion-exchange fibers prior
administration. Ion-exchange materials, consisting of a polymeric framework and ionisable
groups, have the possibility to bind and release charged molecules in a controlled way.
This study investigates the suitability of ion-exchange materials as drug reservoirs for
transdermal iontophoretic delivery of gonadorelin (synthetic LHRH) and its analogues
leuprorelin and nafarelin. Therefore, the behaviour of the peptides concerning the loading
into and the release from the Smopex-101 and Smopex-102 cation-exchange fibers is studied.
Moreover, the advantages and limitations of the ion-exchange fiber/peptide system for
transdermal delivery are identified, analyzed and solutions proposed, where possible. Also,
the protocol combining iontophoresis and ion-exchange fibers for the transdermal delivery of
cationic peptides is validated by using apomorphine as a model compound.
The approach for in vitro transdermal delivery of cationic drugs combining
iontophoresis and ion-exchange fibers is successfully validated with apomorphine.
Transdermal transport is significantly enhanced by iontophoresis. Gradual release from the
fibers and potential for controlling release and following transdermal delivery is seen. With
minor modification the iontophoretic protocol can be used for LHRH and its analogues. The
investigated peptides show good affinity towards both fiber types. The fiber type or amount
of extracting ions in the release medium can control the release from the fibers. Best
potential for controlled release is seen with Smopex-101 cation-exchange fiber combined
with gonadorelin and leuprorelin. Further studies on the behaviour of the peptides loaded
into ion-exchange fibers and their potential in transdermal delivery are still needed but firstly
the limitations and problematic issues identified in our study have to be solved.
ABSTRACT IN DUTCH
De vele voordelen ten opzichte van andere toedieningsroutes zorgt voor stijgende
interesse in transdermale toediening van geneesmiddelen. Om de beperkte doorlaatbaarheid
van de huid, vooral voor grote en geladen moleculen, te verbeteren kan iontoforese
aangewend worden. Deze non-invasieve techniek maakt hierbij gebruik van een kleine en
gedefinieerde elektrische stroom om de toediening van ongeladen en geladen molecules te
vergemakkelijken. Om de doorlaatbaarheid van de huid beter te controleren kunnen de
geneesmiddelen alvorens toediening gebonden worden op ionenuitwisselingsvezels.
Ionenuitwisselingsmaterialen, bestaande uit een polymeer geraamte en ioniseerbare
groepen, zijn in staat om geladen moleculen te binden en deze vrij te stellen op een
gecontroleerde wijze.
Deze studie bestudeert de geschiktheid van ionenuitwisselingsmaterialen als
geneesmiddelenreservoirs voor transdermale afgifte via iontoforese van gonadoreline
(synthetisch LHRH) en de analogen leuproreline en nafareline. Daarvoor wordt hun gedrag
betreffende de binding in en vrijstelling uit Smopex-101 en Smopex-102 ionenuitwisselings-
vezels bestudeerd. Verder worden de voordelen en beperkingen van het vezel/peptide
systeem geïdentificeerd, geanalyseerd en oplossingen voorgesteld, indien mogelijk. Het
protocol dat iontoforese en ionenuitwisselingsvezels combineert voor de transdermale
afgifte van deze peptiden wordt getest met apomorfine als modelgeneesmiddel.
De combinatie van iontoforese en ionenuitwisselingsvezels voor de transdermale
toediening van kationische geneesmiddelen is succesvol gevalideerd met apomorfine.
Iontoforese verbetert het transport doorheen de huid significant. Er is geleidelijke vrijstelling
uit de vezels en potentieel voor gecontroleerde afgifte en opvolging van de transdermale
toediening vastgesteld. Mits kleine wijziging kan het bestudeerde protocol gebruikt worden
voor LHRH en zijn analogen. De onderzochte peptiden vertonen een goede affiniteit voor
beide vezeltypes. Het vezeltype of de hoeveelheid onttrekkende ionen in het medium kan de
vrijstelling controleren. Smopex-101 in de combinatie met gonadoreline of leuproreline
vormt het meeste potentieel voor gecontroleerde vrijstelling. Verdere studies zijn nog
noodzakelijk inzake het vezel/peptide systeem en hun potentieel in transdermale toediening.
Maar eerst moeten de beperkingen en problemen gevonden in deze studie opgelost worden.
Acknowledgements
Firstly, I would like to thank Prof. Dr. C. Vervaet and Prof. Dr. J. Hirvonen
for the assistance, the made Erasmus arrangements and
for generally leading my master thesis.
Moreover, I would like to express my gratitude to Prof. Dr. C. Vervaet
for his critical evaluation of my master thesis.
Sincere thanks to my tutors Ms. K. Malinovskaja and
Dr. T. Laaksonen for their knowledge, good advices,
and help and support throughout this study.
Their great supervision made me possible
to end this master thesis as it is today
I would like to express my gratitude to Dr. H. Santos
for his help with the arrangement of practical things
and administration concerning my Erasmus period.
I would like to thank all the people working
at the Division of Pharmaceutical Technology
for the great lab atmosphere.
Also my friends earn my gratitude
for their interest in what I was doing,
their support and for taking my mind
out of the work during my freetime.
Last but not least, I would like to thank my parents
for their unfailing encouragement and loving support
and to make this whole Erasmus experience possible to me.
TABLE OF CONTENTS
1. INTRODUCTION ........................................................................................................... 1
1.1. IONTOPHORESIS ............................................................................................................ 1
1.1.1. General aspects ............................................................................................. 1
1.1.2. Transport mechanisms and routes ................................................................. 2
1.1.3. Factors affecting iontophoresis ...................................................................... 4
1.1.4. Pharmaceutical applications nowadays .......................................................... 5
1.2. ION-EXCHANGE MATERIALS .......................................................................................... 7
1.2.1. Structure and properties ................................................................................ 7
1.2.2. Theory of ion-exchange .................................................................................. 8
1.2.3. Factors affecting ion-exchange ..................................................................... 10
1.2.4. Pharmaceutical uses nowadays .................................................................... 11
1.3. LHRH AND ITS ANALOGUES ......................................................................................... 13
1.3.1. General aspects and clinical applications in humans ..................................... 13
1.3.2. Iontophoresis of LHRH and its analogues ...................................................... 15
2. AIMS OF THIS STUDY ................................................................................................. 17
3. MATERIALS AND METHODS ....................................................................................... 19
3.1. MATERIALS .................................................................................................................. 19
3.1.1. LHRH and its analogues ................................................................................ 19
3.1.2. Apomorphine............................................................................................... 19
3.1.3. Ion-exchange fibers ...................................................................................... 20
3.1.4. Other chemicals ........................................................................................... 20
3.2. METHODS .................................................................................................................... 21
3.2.1. Loading of the drug into ion-exchange fibers ................................................ 21
3.2.2. Characterization of the ion-exchange fibers .................................................. 21
3.2.3. Release of LHRH and its analogues from the ion-exchange fibers .................. 22
3.2.4. Model drug permeation across porcine skin in vitro ..................................... 22
3.2.5. Analysis of the drugs .................................................................................... 23
4. RESULTS .................................................................................................................... 25
4.1. CHARACTERIZATION OF ION-EXCHANGE FIBERS ........................................................ 25
4.2. LOADING OF LHRH AND ITS ANALOGUES INTO THE ION-EXCHANGE FIBERS ............ 26
4.3. RELEASE OF LHRH AND ITS ANALOGUES FROM THE ION-EXCHANGE FIBERS ............ 27
4.4. MODEL DRUG PERMEATION ACROSS PORCINE SKIN IN VITRO .................................. 30
4.4.1. In vitro permeation of apomorphine solution ............................................... 30
4.4.2. In vitro permeation of apomorphine loaded into Smopex-101 cation-exchange
fibers .................................................................................................................... 32
5. DISCUSSION .............................................................................................................. 34
5.1. EFFICIENCY OF LOADING INTO AND RELEASE OF LHRH AND ITS ANALOGUES FROM
CATION-EXCHANGE FIBERS .................................................................................................... 34
5.2. EFFECT OF IONTOPHORESIS ON TRANSDERMAL DELIVERY ACROSS PORCINE SKIN IN
VITRO ..................................................................................................................................... 38
6. CONCLUSIONS ........................................................................................................... 46
7. REFERENCES .............................................................................................................. 47
LIST WITH USED ABBREVIATIONS
ACN: acetonitrile
Arg: the amino-acid Arginine
D-Leu: the amino-acid D-Leucine
D-Nal: the amino-acid D-Naphtylalanine
FSH: follicle stimulating hormone
Gly: the amino-acid Glycine
GnRH: gonadotropin-releasing hormone
HEPES: 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid
His: the amino-acid Histidine
HPLC: high performance liquid chromatography
IEP: iso-electric point
Ji: iontophoretic flux
Kc: chemical partition coefficient
Ke: electrical partition coefficient
Leu: the amino-acid Leucine
LH: luteinizing hormone
LHRH: luteinizing hormone releasing hormone
MW: molecular weight
NHEt: amino-ethyl
P: partition coefficient
pKa: negative logarithm of the dissociation constant
Pro: the amino-acid Proline
Pyr: the amino-acid L-Pyroglutamic acid
SEM: scanning electron microscope
Ser: the amino-acid Serine
TFA: trifluoric acid
Trp: the amino-acid Thryptophan
1
1. INTRODUCTION
Transdermal drug delivery has generated a lot of interest because it carries along
many advantages over other drug administration routes. Some of the most important ones
are its non-invasive nature, the hepatic bypass, the avoidance of gastro-intestinal degradation
and its possible use for sustained drug release (Berti & Lipsky, 1995). The main problem with
this administration route is the limited permeation of the drugs across the skin membrane.
The skin consists of 3 layers − epidermis, dermis and hypodermis. The outer part of the
epidermis, the stratum corneum, forms the major barrier to drug penetration into the skin
and permeation across the skin. It is a thin multilamellar layer, composed of a lipid-rich
intracellular matrix and densely packed cornified dead cells (Berti & Lipsky, 1995). The poor
permeability across the skin especially concerns drugs which are very lipophilic or hydrophilic,
are charged or of a high molecular weight (Hirvonen, 2005). Prausnitz & Langer (2008) gave
an overview of the techniques that are used to enhance transdermal drug delivery. They
include the use of chemical enhancers, iontophoresis, ultrasound, electroporation, thermal
ablation, microdermabrasion and microneedles.
1.1. IONTOPHORESIS
1.1.1. General aspects
One of the techniques to enhance the transdermal permeation of drugs is
iontophoresis. It is a non-invasive technique which results in a facilitated transport of charged
and uncharged molecules across the skin by applying a small and defined electric current
(typically 0.1-0.5 mA/cm²) (Sieg & Wascotte, 2009). Two electrodes, a positively charged
anode and a negatively charged cathode are placed on the skin and a current is applied (Kalia
et al., 2004). The drug is placed in the same electrode compartment as the charge it carries.
The other electrode is placed elsewhere on the body to complete the electronic circuit. The
major advantages and limitations of iontophoretic drug delivery are shown in Table 1.1
(Shinde et al., 2010; Khan et al., 2011).
2
Table 1.1: Major advantages and limitations of transdermal iontophoretic drug delivery
Advantages Limitations
Bypass of first pass metabolism in the liver Irritation and burns of skin are possible
Avoidance of gastro-intestinal degradation Drug metabolism in the skin is possible
Easy applicable and non-invasive
(minimum risk of infection and trauma)
Molecules with high molecular weight
achieve insufficient drug delivery rates
Drugs with short biological half-lives can be
delivered
Amount of drug that can be delivered is
limited (5-10 mg/h)
Allowance of rapid drug delivery
termination if needed
Ionic form of the drug in sufficient
concentration is needed
Relatively pain free alternative for injections
Feasibility of local and systemic administration
Controlled plasma levels
1.1.2. Transport mechanisms and routes
The Nernst-Planck equation (Equation 1.1) describes the iontophoretic flux, Ji, of drugs
(or more in general ionic species) in a fluid or a membrane (Hirvonen, 2005). In the case of
transdermal iontophoretic drug delivery this membrane is the skin. The equation shows the
contributions of the passive diffusion, electrorepulsion and electro-osmosis, which are the
three processes contributing to the iontophoretic drug flux.
(1.1)
in which: Ji: the flux of ionic species i in a fluid or membrane
Di: the diffusion coefficient of the ionic species i
ci: the concentration of ionic species i
zi: the charge number of the ion i
F: the Faraday constant
R: the gas constant
T: the absolute temperature
φ: the electrical potential
υcci: the solvent velocity
3
The first part of this equation, recognized as Fick’s first law, shows the influence of
passive diffusion across the skin on the total iontophoretic flux. The diffusion constant,
particle size and concentration gradient are affecting the passive diffusion. In most cases, the
fraction of the passive diffusion from the total flux can be considered negligible (Kalia et al.,
2004).
The second part of the Nernst-Planck flux equation shows the contribution of
electrorepulsion (sometimes also called electromigration). This process is the ordered
migration of the ionic species in the presence of an electric field (Kalia et al., 2004). Under the
influence of a current, anions will migrate to the anode, cations to the cathode. The amount
of drug carried by the electromigration is directly proportional to the current density, time of
current application and the area of skin in contact with the electrode compartment (Sieg &
Wascotte, 2009). The transport number of an ion, which is defined as the fraction of current
carried by this ion, is used to describe the efficiency of electromigration of a particular ion
(Kochhar & Imanidis, 2004). This parameter is dependent on the relative mobility of the ion,
its charge and the concentration in relation to the other ions present. Thus, more mobile and
smaller ions (for example Na+ and Cl- of saline, a principal body electrolyte) usually carry the
greatest fraction of the total iontophoretic current.
The last part of the equation is illustrating the electro-osmotic solvent flow, which can
be described as a solvent bulk flow that carries along ions or neutral molecules (Shinde et al.,
2010). This process is believed to be the driving force for the iontophoretic delivery of neutral
molecules and high molecular weight cations (Kalia et al., 2004). A larger amount of
carboxylic groups over amine groups results in the iso-electric point of the skin to be around
4.8 (Marro et al., 2001). Therefore, the skin carries a net negative charge at physiological pH
resulting in a favoured movement of positively charged ions. This leads to a cation
permselectivity of the human skin membrane. The passage of current can cause this solvent
flow in the anode-to-cathode direction.
Physiochemical properties and the structure of the drug determine the contribution of
passive diffusion, electro-osmotic solvent flow and electromigration to the iontophoretic flux
during the drug delivery. As Guy et al. (2000) stated, the increase in molecular weight slows
down the electrical mobility. For smaller ions, electrorepulsion is the dominating transport
4
mechanism. On the other hand, larger and neutral molecules are mainly transported by the
electro-osmotic solvent flow and passive diffusion.
When a current is applied on the skin, ions will use the pathway of the lowest
electrical resistance (Hirvonen, 2005). The ionic bulk flow is favoured along discrete sites or
pores − sweat glands, hair follicles and skin imperfections. Paracellular route can also be
involved, although mostly for water and uncharged polar solutes. Drug delivery can also take
place through pores formed as a result of the applied current (Wang et al., 2005).
1.1.3. Factors affecting iontophoresis
Different factors, which can be divided in four groups, can affect iontophoretic drug
delivery (Khan et al., 2011). The first factor group includes the physicochemical properties of
the drug that needs to be delivered iontophoretically. Properties of the drug formulation
form the second factor group. The third and the fourth group are experimental and biological
factors respectively.
The influencing physicochemical parameters are the molecular weight and size, charge
and concentration of the drug (Khan et al., 2011). The iontophoretic flux is inversely
proportional to the molecular weight and size due to the influence of these factors on the
diffusion constant. The charge of the drug determines the contribution of the different
transport mechanisms to the iontophoretic flux. As expected from Equation 1.1, an increase
in drug concentration would provoke a linear increase in iontophoretic flux. Kalia et al. (2004)
described the complications why this proportionality does not always hold true. Three
reasons are suggested such as increasing the amount of drug does not always increase the
amount of molecules in the membrane, the presence of competing ions and possible drug
interactions with the skin.
Drug formulation parameters like pH, ionic strength and the presence of co-ions have
also their influence. The pH has two mechanisms of action (Kalia et al., 2004). First, altering
the pH can affect the skin charge and thus influence the contribution of the electro-osmotic
solvent flow. Molecules with a smaller charge benefit more from the electro-osmotic solvent
flow. Also, the pH of the formulation can affect the charge and degree of ionisation of the
drug. A higher ionic strength usually results in a decrease of iontophoretic flux (Shinde et al.,
2010). The presence of smaller and more mobile co-ions results in a decrease of transport
5
because of competition for the fraction of current carried and the transport number of the
drug in interest will decrease (Sieg & Wascotte, 2009).
Experimental influencing factors include the density of the applied current, the type of
the current (constant or pulsed), duration of application and the electrode material (Khan et
al., 2011). Increasing the current density does not always result in an increase of the flux
across the membrane. Several reasons have been suggested for the phenomenon such as a
saturation phenomenon in the skin, skin polarisation or neutralisation of the negative charge
of the skin by large cations like peptides and thus inhibition of electro-osmosis (Priya et al.,
2006; Gratieri et al., 2011). The problem of skin polarisation can be avoided by using a pulsed
current (Raiman et al., 2004). Furthermore, for achieving sufficient drug delivery rate the
current density has to be high enough but not too large to avoid harmful effects on the skin
(Khan et al., 2011). Moreover, sufficient amounts of drug delivered by iontophoresis can be
achieved in the most cases by prolonging the application time. Electrode materials can also
influence the iontophoretic process. Ag/AgCl electrodes are preferred over platinum or
zinc/zinc chloride electrodes because of their ability to resist pH changes (Sieg & Wascotte,
2009).
Khan et al. (2011) described the influence of biological factors like intra- and inter-
subject variability, regional blood flow and the condition of the skin. Although transdermal
iontophoresis achieves less intra- and inter- subject variability in comparison with cutaneous
drug delivery, small differences in drug delivery rate are still seen. Regional blood flow does
not affect the drug penetration. On the other hand, it determines systemic and underlying
tissue absorption. A thick stratum corneum or fewer skin appendages decrease the
iontophoretic flux.
1.1.4. Pharmaceutical applications nowadays
At the moment, iontophoretic drug delivery is already clinically used for some
therapies and diagnostic purposes (Hirvonen, 2005; Sieg & Wascotte, 2009). These
applications include local anaesthesia, pain control, treatment of hyperhydrosis, anti-
inflammatory treatment, diagnosis of cystic fibrosis and glucose monitoring.
For the administration of local anaesthetics, an ionized solution of 2% lidocaine and
epinephrine is delivered anodally (Hirvonen, 2005; Sieg & Wascotte, 2009). Epinephrine is
6
added to the formulation for prolonging the anaesthetic effect. Current density and
application time determine the depth and duration of anaesthesia. Rheumatic pain control
can be achieved by iontophoresis of NSAIDs like piroxicam, diclofenac and ketorelac.
Suppression of cancer breakthrough pain is possible with the Ionsys® fentanyl system, which
delivers fentanyl iontophoretically. This system is also used to treat postoperative and
chronic pain. For the treatment of sub-acute superficial local inflammations, iontophoresis of
dexamethasone is used. Hyperhydrosis, the increased production of sweat, can be treated by
the iontophoretic delivery of tap water or anticholinergics in order to obstruct the sweat
ducts.
Diagnosis of cystic fibrosis is performed by the sweat test. Therefore, pilocarpine is
administered iontophorectically (Hirvonen, 2005; Sieg & Wascotte, 2009). Afterwards, the
amount of sweat is measured and levels of sodium chloride are determined. High levels of
sodium chloride are used for the identification of cystic fibrosis.
Reverse iontophoresis is used as a technique to monitor blood glucose levels of
diabetics (Hirvonen, 2005; Sieg & Wascotte, 2009). The electro-osmotic solvent flow leads
glucose to the skin surface. Then, glucose is collected in gel-collection discs. In these discs,
glucose oxidase enzyme generates hydrogen peroxide from the glucose. The reaction in
which hydrogen peroxide is formed, causes electrical signals which are then converted to
blood glucose levels. Glucowatch Biographer® and Biographer® G2 are commercially available
devices for this purpose.
Due to its many advantages transdermal iontophoresis offers several other future
perspectives. Iontophoresis can be used to combine drug delivery and drug monitoring, which
can be a valuable tool for optimizing patient therapy (Hirvonen, 2005). But as a result of the
high cost, time consumption and technological drawbacks, the use of this technique is still
somehow limited. Applications which could benefit from transdermal iontophoresis are the
delivery of drugs with a narrow therapeutic range or poor clinical response. Also
individualized treatment and therapies in vulnerable patient populations such as senior
citizens, neonates and premature neonates could benefit from transdermal iontophoresis.
7
1.2. ION-EXCHANGE MATERIALS
1.2.1. Structure and properties
Ion-exchange materials consist of a polymeric framework with ionisable functional
groups attached and thus, have the possibility to bind and exchange charged molecules.
(Kankkunen, 2002; Hänninen, 2008). The functional groups attached determine whether it is a
cation- or an anion-exchanger. Negatively charged groups like –SO3-, –COO- or –PO3- can
exchange cationic counter-ions, so they are called cation-exchangers. On the other hand
groups like –NH3+, –NH2
+, –S+ can bind anions and thus form the functional groups of anion-
exchangers. Bifunctional or polyfunctional ion-exchangers are the ones with respectively two
or more ionic groups of the same charge. Ion-exchange materials that carry ion-exchange
groups with opposite charges are called amphoteric ion-exchangers. Furthermore, these
materials can be divided into weak and strong ion-exchangers depending on the weak or
strong acidic or basic nature of the functional groups.
Due to their ability to bind and exchange charged molecules, ion-exchange materials
can be used as drug reservoirs for controlled or sustained delivery (Kankkunen, 2002).
Therefore the charged drug can be loaded into the ion-exchange materials and afterwards
released by mobile ions with the same charge as the drug (see Section 1.2.2).
Several types of ion-exchange materials are available, such as resins, gels, membranes
and fibers. Among them, the most important group is formed by ion-exchange resins
(Hänninen, 2008). These resins are prepared as spherical beads and consist of a three-
dimensional polymeric framework, mostly polystyrene or polymethacrylic based, and ion-
exchange functional groups. Cross-linking, which can be achieved by using cross-linking
agents like divinylbenzene, connects the various hydrocarbon chains (Elder, 2005).
Ion-exchange fibers are made of hydrophobic carbon chains with the difference to the
resins that the chains are not cross-linked (Hänninen, 2008). They are chemically inert and
have good thermal and mechanical strength. As a result of the non-cross-linked nature of ion-
exchange fibers, higher surface area to unit volume ratio and smaller thickness, the ion-
exchange process is more rapid and efficient in comparison with resins (Hänninen, 2008). This
leads to more efficient drug loading and release, because cross-linking can hinder the
8
movement of molecules. Furthermore, easier incorporation of large molecules and more
controllability of the loading or release of the drug is seen with the ion-exchange fibers.
The binding capacity, which is given by how many functional groups are present in a
certain amount of ion-exchanger, is an important property for ion-exchange materials
(Kankkunen, 2002; Hänninen, 2008). The functional groups present need to be ionized for
having the ion-exchange property. When the functional groups are weak acids or bases, the
binding capacity is pH dependent.
1.2.2. Theory of ion-exchange
The ion-exchange reaction is the process in which mobile ions are exchanged between
two phases – the ion-exchanger and the external liquid (Hänninen, 2008). It is a completely
stoichiometric, selective and reversible process. Due to the electroneutrality requirements an
equivalent amount of ions of the same charge (sign and valence) have to be exchanged. The
ionic species determine whether the process is anionic or cationic. A general representation
of a cationic ion-exchange process is given below (Hänninen, 2008).
Ion-exchanger-A+ + B+ ↔ Ion-exchanger-B+ + A+
The ion-exchange process is a diffusion process driven by the Donnan potential. The
Donnan potential can be defined as the electrical potential difference between the ion-
exchange phase and the external solution (Hänninen, 2008). It arises as the first ions bound to
the ion-exchanger start to migrate into external solution or ions from the solution into the
ion-exchanger. The mobile ions with the same charge as the ion-exchange material are called
co-ions, whereas the mobile ions with the opposite charge are called counter-ions (Hänninen,
2008). A high absolute value of the Donnan potential means a high co-ion exclusion from the
ion-exchange material and a strong attraction of counter-ions. High rates of ion-exchange are
achieved by using a concentrated external solution. The selectivity and capacity of the ion-
exchanger, and the charge of the ions also have an influence on Donnan potential. The
diffusion process continues till the Donnan equilibrium is reached, which is the equality of
electrochemical potential for each mobile ion between the ion-exchanger phase and the
external solution (Hänninen, 2008).
9
The ion-exchange process is described by Abdekhodaie & Wu (2006) and Hänninen
(2008) in a succession of steps. The first step is the diffusion of the counter-ion through the
solution to the surface of the ion-exchange material (film diffusion). Secondly, the counter-
ion has to migrate within the ion-exchanger to the functional groups (particle diffusion).
Afterwards, the actual ion-exchange reaction occurs at the ionic binding site of the ion-
exchanger. Finally, the bounded counter-ion is exchanged by the mobile one. The released
counter-ion has to undergo the diffusion through the ion-exchange material and afterwards
through the external solution. The rate-determining step is usually migration of drug within
the ion-exchanger or in the diffusion boundary layer (Kankkunen, 2002).
The process is influenced by two interactions − the hydrophobic and the electrostatic
interactions. Thus, two partition coefficients will affect the ion-exchange reaction. The
general equilibrium constant is given as the product of the electrical and chemical partition
coefficient (Equation 1.2) (Jaskari et al., 2001; Kankkunen, 2002).
(1.2)
in which: K: general equilibrium constant
Ke: electrical partition coefficient
Kc: chemical partition coefficient
The hydrophobic interactions are determined by the chemical partition coefficient, Kc.
This coefficient can be described as in Equation 1.3 (Jaskari et al., 2001). The coefficient is an
expression of the tendency of the drug to get into the ion-exchanger due to hydrophobic
interactions. The more hydrophobic the drug is, the larger the value of Kc (> 1), a
phenomenon due to a larger decrease in free energy associated with the interaction.
(1.3)
in which: Kc: chemical partition coefficient
: standard chemical potential of the drug ions in the external phase
: standard chemical potential of the ions in the ion-exchanger phase
R: gas constant
T: absolute temperature
10
The electrical partition coefficient, Ke, can be expressed as in Equation 1.4, given
below (Jaskari et al., 2001). In the case of cationic drugs exchanged in cation-exchangers, Ke
will be bigger than one, because of the positive charge number and a negative Donnan
potential.
(1.4)
in which: Ke: electrical partition coefficient
z: charge number
F: Faraday constant
φD: Donnan potential
R: gas constant
T: absolute temperature
1.2.3. Factors affecting ion-exchange
The factors influencing the ion-exchange process can be classified in ion-exchange
material dependent factors, physicochemical properties of the drug and external conditions.
Their effect on the ion-exchange process will be described below.
First, there are some ion-exchanger dependent factors. Only ionized groups can
behave as functional groups for ion-exchanging. This results in pH dependence when the
functional groups are weak acids or bases (Hänninen, 2008). Cross-linking, as in resins, can
hide some ionized groups, which can decrease the effective ion-exchange capacity. Cross-
linking can also slow down the rate of the ion-exchange reaction by influencing the diffusion
within the ion-exchanger. The ion-exchange capacity influences the Donnan potential
(Hänninen, 2008). The drug loading is more efficient with a higher effective capacity ion-
exchanger (Jaskari et al., 2001). On the other hand the drug release from the exchanger is
less efficient. Also the chemical nature of the fixed ion-exchange groups influences the
strength of interaction between the mobile counter-ion and the ion-exchanger (Jaskari et al.,
2001). Lipophilic drugs tend to bind stronger to strong acidic or basic functional groups,
hydrophilic ones can bind stronger to weaker groups. Moreover, an effect of the preloaded
counter-ion is seen. The selectivity of the ion-exchange material towards one ion to another
is influenced by the nature of the fixed groups in the ion-exchanger and the valence,
11
lipophilicity and radius of the mobile counter-ions (Hänninen, 2008). A higher valence and
ionic radius of the counter-ion results in a higher selectivity towards the exchanger.
Secondly, drug dependent factors have an impact on the ion-exchange process. More
lipophilic drugs tend to have a more efficient loading into the ion-exchanger, but a reduced
release due to the contribution of the chemical partition coefficient in the ion-exchange
mechanism (Jaskari et al., 2001). The pKa of the drug determines the moiety of the drug
ionized at a certain pH when the drug is a weak base or acid (Hänninen, 2008). Only ionized
drugs can be exchanged. Kankkunen et al. (2002) found out that ampholytic drugs can form
an intrinsic molecular salt, which is able to reduce the loading into the ion-exchanger. A larger
molecular size of the drug can lead to lower drug loading by influencing the diffusion rate. On
the other hand it can increase the strength of interactions between the drug and ion-
exchanger (Hänninen, 2008).
Thirdly, the impact of external conditions is described. As mentioned above, the
Donnan potential is affected by the concentration of the external solution. Also the valence of
the counter-ions has an effect, as ions with higher valence are more strongly attracted
towards the ion-exchange material (Jaskari et al., 2001). Multivalent ions have the feasibility
to bind simultaneous to several ion-exchange groups, which may result in cross-linking. This
results in a negative influence on the drug release (Hänninen, 2008). Strongly associated
multivalent ions can also affect the effective charge, which results in an alteration of the
Donnan potential (Jaskari et al., 2001). The pH of the external solution has an impact on the
ionization of the drug and the ion-exchanger (Kankkunen et al., 2002). Also the use of higher
temperature or agitation increases the drug loading or release (Hänninen, 2008).
1.2.4. Pharmaceutical uses nowadays
Ion-exchange materials can be used as a pharmacologically active drug ingredient. For
example anion-exchange resins like colestipol and cholestyramine are available on the market
for the treatment of hypercholesterolemia (Elder, 2005). Ion-exchangers also have a place in
the treatment of hyperkalemia, hypertension, cardiac oedema and toxaemia of pregnancy.
Ion-exchangers are also applicable for the management of drug overdoses because they are
able to bind the poisoning agent (Anand et al., 2001).
12
The application of ion-exchangers in drug stabilization for drugs like vitamin B12,
omeprazole and levodopa has been studied (Kankkunen et al., 2002; Elder, 2005). The
mechanism of stabilization is the protection of drugs from hydrolysis, oxidation or enzymatic
degradation during drug storage or delivery (Hänninen, 2008). Ion-exchange materials can
also behave like buffers and thus improve the stability of drugs in aqueous solutions
(Kankkunen et al., 2002). Such formulation of misoprostol in a suspension is already available
on the market (Anand et al., 2001).
Moreover, Elder (2005) has demonstrated that ion-exchangers can be used for taste
masking of drugs, as many drugs have an unpleasant taste. By complexation of drug with ion-
exchange materials, an insoluble complex is formed which minimizes the odour and taste.
This drug-resin complex dissociates in gastric pH and makes the drug thereby available for
absorption.
The advantages of the use of ion-exchange materials as a controlled release system
has also been demonstrated (Elder, 2005). These include dose interval reduction and
improved drug safety and drug stability. Ion-exchangers have been studied for oral,
transdermal, nasal and ocular controlled release systems (Hänninen, 2008). Several
commercial products are already using ion-exchangers such as Nicorette® (chewing gum for
easier smoking cessation), Delsym® (suspension against cough containing dextromethorphan)
or Betopic S® (ophthalmic suspension with betaxolol for the treatment of glaucoma).
The combination of ion-exchange materials with iontophoresis has been investigated
(Conaghey et al., 1998; Jaskari et al., 2000; Anand et al., 2001; Kankkunen et al., 2002). The
main purpose of this combination is the improved drug stability and precisely controlled drug
delivery rate. The principle of the combination of iontophoresis with ion-exchange materials
can be seen in Figure 1.1. Conaghey et al. (1998) investigated the use of an ion-exchange
resin containing hydrogel for the iontophoretic transport of nicotine through the skin. They
found that an enhanced and controlled release could be achieved. Jaskari et al. (2000)
demonstrated the use of Smopex-102 (cation-exchange fiber) and Smopex-108 (anion-
exchange fiber) for the delivery of tacrine, nalodol, propranolol and sodium salicylate.
Kankkunen et al. (2002) showed that an improved stability and release control of levodopa
and metaraminol can be achieved with the use of ion-exchange fibers.
13
Figure 1.1: The principle of the combination of a cation-exchanger with iontophoresis for transdermal drug delivery. The drug is first loaded to the ion-exchanger, then released by mobile co-ions. The drug is then delivered through the skin by an iontophoretic current. SC means stratum corneum, which is the barrier for the transdermal drug delivery. ED stands for dermis and epidermis (Hirvonen, 2005).
1.3. LHRH AND ITS ANALOGUES
1.3.1. General aspects and clinical applications in humans
Luteinizing hormone releasing hormone (LHRH) or gonadotropin-releasing hormone
(GnRH) is synthesized in the body by the hypothalamus and released in a pulsatile way. It
induces the release of pituitary follicle stimulating hormone (FSH) and luteinizing hormone
(LH). These two hormones are together also called gonadotropins. In the body these
hormones are responsible for ovulation, spermatogenesis and steroid secretion which occurs
in the gonads. LHRH is a peptide consisting of ten amino-acids.
The synthetic drug of LHRH is called gonadorelin. This decapeptide is available on the
market in the form of an acetate (Barbieri, 1992). It is approved by the Food and Drug
Administration for the treatment of anovulatory infertility due to a GnRH deficiency. The
system is called Lutrepulse® and delivers a pulse of gonadorelin acetate by an intravenous
injection. The pulses are repeated with a certain interpulse interval.
As a result of the short half-life of gonadorelin, which is between 2 to 8 minutes
initially and in a terminal stadium 10 to 40 minutes, more stable analogues such as nafarelin
and leuprorelin have been synthesized (Barbieri, 1992). The peptide bonds between amino-
acid 5-6 and 6-7 in LHRH are easily degraded by endopeptidases. Thus, altering amino-acid 6
can form more stable LHRH analogues. Nafarelin is formed by switching the glycine in
14
gonadorelin by D-2-naftylalanine (D-Nal). Leuprorelin carries at position 6 D-leucine (D-Leu)
instead of the glycine amino-acid. The amino-acid sequences and molecular weights of LHRH,
leuprorelin and nafarelin are presented in Table 1.2.
Table 1.2: Amino-acid sequence and molecular weight of LHRH and its analogues
Peptide Molecular weight (g/mol) Sequence
gonadorelin 1182.3 Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2
leuprorelin 1209.4 Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt
nafarelin 1322.5 Pyr-His-Trp-Ser-Tyr-D-2-Nal-Leu-Arg-Pro-Gly-NH2
Zilberstein et al. (1983) demonstrated that the GnRH-receptors are down-regulated
after continuous administration of LHRH. When LHRH or its agonist is administered, short-
time stimulation due to the release of the reserve pool of the gonadotropins occurs.
Afterwards, the body goes through a state of suppression of this hormone activity. This
mechanism is the basis for therapeutical uses of LHRH and its analogues, as described below.
Tolis et al. (1982) demonstrated the feasibility of using LHRH analogues in the
treatment of prostate cancer. The results were varying between clinical improvement, less
pain or decrease in the tumour size. Continuous administration of these analogues led to
failure of the androgen production. Thus, the administration of LHRH analogues forms the
basis for one of the techniques of hormone ablation therapy against prostate carcinoma.
The meta-analysis of Klijn et al. (2001) showed that the treatment with LHRH agonists
is useful in the case of advanced breast cancer in pre-menopausal women. This treatment
causes a medical castration. As a result a fall in gonadotropin release and afterwards a drop in
serum estradiol is seen. The investigators showed the advantage of the combination with
tamoxifen, the nowadays standard therapy, over LHRH alone. Furthermore, the
hypoestrogenic state caused by administration of LHRH agonists results in amenorrhea and
atrophy of endometriotic tissue and is thus useful in the treatment of endometriosis (Olive &
Pritts, 2001). Even for the preoperative treatment of uterine leiomyomas, this hypoestrogenic
state is favourable (Stein & Ascher-Walsh, 2009). Central precocious puberty, a disease which
stands for the early onset of secondary sexual characteristics in male or female, can also be
treated by LHRH analogues (Partsch et al., 2002). The pituitary-gonadal suppression stops the
further sexual development.
15
LHRH agonists are also used in in vitro fertilization. They reduce premature
luteinisation by inhibiting the incidence of a premature LH surge and have the ability to
decrease the androgen concentrations in the ovarian (Casper, 1991). The combination of
these two mechanisms leads to improved oocytes. A better response to in vitro fertilization is
also seen when through the administration of LHRH agonists the gonadotropin release is
suppressed and afterwards controlled stimulation is achieved by administering gonadotropins.
1.3.2. Iontophoresis of LHRH and its analogues
Clinically relevant concentrations of LHRH and its analogues can be achieved when
delivered iontophoretically across the skin. The feasibility of achieving therapeutic plasma
concentrations of LHRH by transdermal iontophoresis has been demonstrated in in vitro
studies (Bhatia & Singh, 1998; Raiman et al., 2004). The in vitro study of Kochhar & Imanidis
(2004) showed the feasibility for the skin iontophoresis of therapeutically effective doses of
leuprorelin. Administration of leuprorelin in human was also demonstrated to be successful in
the in vivo study of Meyer et al. (1988). Moreover, sufficient transdermal fluxes were also
achieved with nafarelin during the in vitro transdermal iontophoresis (Delgado-Charro & Guy,
1995).
LHRH and its analogues, which only differ in the sixth amino-acid as seen in Section
1.3.1 and Table 1.2, show an anomalous behaviour concerning the iontophoretic flux and its
relation with the concentration and current applied during their transdermal iontophoresis.
The reason for this behaviour is caused by the electro-osmotic solvent flow. Electro-osmosis
is considered to be the most important transport mechanism for non-charged or larger
molecules like peptides as described in Section 1.1.2. Nafarelin delivered with anodal
iontophoresis shows the ability to reverse the direction of the electro-osmotic solvent flow
(Delgado-Charro & Guy, 1994). It has been shown that this cationic peptide associates
strongly with the negatively charged skin. As a result, the charge of the skin is neutralized and
the peptide transport is inhibited. This occurred especially when the applied concentration of
nafarelin was increased (Delgado-Charro & Guy, 1995). The same was proved for leuprorelin
by Hoogstrate et al. (1994). LHRH itself has not been shown this property (Delgado-Charro &
Guy, 1994). An increase of iontophoretic flux as expected from the Nernst-Planck equation
(Equation 1.1) as a result of the appliance of a higher current is not seen for nafarelin.
16
Although a small increase of the flux was seen up to 0.63 mA/cm² according to the research
of Delgado-Charro et al. (1995), the dependence on the current density was very weak.
Hirvonen et al. (1996) investigated the influence of the core peptide structure of these
three peptides. The core peptide structure is formed by the amino-acids from 6 to 8. The
amino-acids 6 (D-Nal in nafarelin and D-leu in leuprorelin instead of glycine in gonadorelin)
and 7 (leucine) in nafarelin and leuprorelin form a hydrophobic surface. In combination with
the positively charged arginine in position 8, it creates a oligopeptide motif (hydrophobe-
hydrophobe-cation) which was the reason for their peculiar influence on the electro-osmotic
solvent flow. This has been proven by the synthesis and transdermal iontophoresis of a set of
tripeptides similar to the core peptide structure of the three peptides nafarelin, leuprorelin
and LHRH.
Another aspect to consider is the degradation of nafarelin and gonadorelin during the
iontophoretic delivery. Enzymatic degradation of LHRH in the skin was proven by Bi & Singh
(1998). Raiman et al. (2004) demonstrated in vitro that LHRH and nafarelin do not degrade in
the buffer or under the influence of a current. The degradation was about 20 % in the case of
LHRH in contact with the stratum corneum. Total degradation of LHRH was seen in 72 h in
contact with the (epi)dermal side of the skin. Five major hydrolytic degradation products
were identified. Nafarelin was stable in contact with stratum corneum but the drug was
clearly degrading during the first hours in contact with the (epi)dermal skin side. Rodriguez
Bayon & Guy (1996) saw an increase in number and amount of metabolites of nafarelin
during transdermal iontophoresis in vitro when the pH was altered from pH 5 to 7.
Raiman et al. (2004) showed that the delivery of LHRH and nafarelin across the skin
can be improved by pulsed iontophoresis. They also demonstrated that pulsed iontophoresis
offers important advantages over constant current iontophoresis. Firstly, the accumulation of
the drug on the skin surface can be avoided and thus it can lead to a more controllable
peptide delivery. Secondly, the process has been shown to be more cost-effective because
less peptide is necessary for achieving therapeutic plasma levels.
17
2. AIMS OF THIS STUDY
As mentioned in the introduction, transdermal delivery has several advantages over
other drug administration routes of which the most important advantage is the avoidance of
gastrointestinal and hepatic metabolism. The main problem with the transdermal
administration route is the intrinsic low permeability for drugs and other molecules. For large
and charged molecules such as LHRH and its analogues a technique to enhance transdermal
drug delivery is needed. The application of iontophoresis has been shown effective in
literature to facilitate drug delivery across the skin. In order to have more control in
transdermal permeation during iontophoretic peptide delivery the drugs can be bound to ion-
exchange fibers prior administration. The main aim of this study is to test the suitability of
ion-exchange materials as drug reservoirs for the transdermal iontophoretic delivery of LHRH
and its analogues. Furthermore, the advantages and limitations of the ion-exchange
fiber/peptide system will be identified, analyzed and solutions proposed, where possible.
To determine whether the iontophoretic protocol developed during previous work in
the Division of Pharmaceutical Technology (Faculty of Pharmacy, University of Helsinki) can
be used for the investigation of iontophoretic delivery of gonadorelin, leuprorelin and
nafarelin (LHRH and its analogues) both from solutions and cation-exchange fibers, the
protocol is first validated with a suitable model compound. Apomorphine is chosen for the
model for the anodic iontophoretic delivery because of several practical reasons. It is a small
and lipophilic molecule that possesses a positive charge at the pH used in the experiment and
has been successfully delivered across the skin iontophoretically in the same laboratory in the
past. Iontophoretic experiments of apomorphine solutions and of the model compound
loaded into Smopex-101 cation-exchange fibers will thus be performed. From the experiment
it can be derived whether a similar iontophoretic protocol with minor modifications can be
used for the transdermal delivery in vitro for the three mentioned peptides.
To test the affinity of gonadorelin, leuprorelin and nafarelin towards the two cation-
exchange fiber types a loading experiment during 48 h will be carried out. Therefore, fiber
bundles are prepared by wrapping a defined amount of ion-exchange fibers into cotton gauze
and immersing them into a concentrated peptide solution.
18
After the three compounds (gonadorelin, leuprorelin and nafarelin) are loaded into
the fibers, the objective is to determine the release profiles and the efficiency of release of
the three peptides from the Smopex-101 and Smopex-102 cation-exchange fibers. The
possible suitable and less suitable combinations of fiber types and peptides for controlled
release can be identified. Furthermore, the influence of the amount of extracting counter-
ions in the release medium is investigated.
From the combination of the results of testing the iontophoretic protocol on the
model compound apomorphine, the affinity of the peptides towards the cation-exchange
fibers and the release efficiency and profile, the potential and possible problems for future
experiments can be identified. In the case of problems, suggestions for solutions are
proposed.
19
3. MATERIALS AND METHODS
3.1. MATERIALS
3.1.1. LHRH and its analogues
The three investigated drugs – leuprorelin, nafarelin and gonadorelin (synthetic LHRH)
are used in the acetate salt forms and are all achieved from ChemPep (Wellington, USA).
Their physicochemical properties are shown in Table 3.1.
Table 3.1: Physicochemical properties of the investigated peptides
Drug MWa (g/mol) pKab LogPc IEPd
gonadorelin 1182.3 10.63e -3.6f 9.1g
leuprorelin 1209.4 − 0.7e −
nafarelin 1322.5 10.98e 1.21e −
a MW: molecular weight
b pKa: negative logarithm of the dissociation constant
c P: partition coefficient
d IEP: iso-electric point
e computed variables (http://www.drugbank.ca)
f experimental variable (http://www.drugbank.ca)
g (King & Millar, 1982)
3.1.2. Apomorphine
Apomorphine is a non-selective dopamine agonist and is potentially useful in the
treatment of Parkinson’s disease, especially for counteracting the ‘on-off’ oscillations
(Junginger, 2002). The molecular structure is presented in Figure 3.1. The drug is purchased
as R-(−)-apomorphine hydrochloride hemihydrate (MW=312.79 g/mol) from Sigma Life
Science (St. Louis, USA).
Figure 3.1: The molecular structure of apomorphine (Junginger, 2002).
20
3.1.3. Ion-exchange fibers
The cation-exchange fibers used in these experiments are purchased from Johnson
Matthey (Turku, Finland). The characteristics of the fibers are shown in Table 3.2.
Table 3.2: Characteristics of cation-exchange fibers
Fiber Framework Ion-
exchange groups
Dry
content
(w/w %)
Loading
capacity
(mmol/g)
pKa
values
Smopex-101 Polyethylene Sulphonic acid 43 % 3.9 0-1
Smopex-102 Polyethylene Carboxylic acid 66 % 5.6 3-5
3.1.4. Other chemicals
An overview of the other used chemicals is given in Table 3.3. Deionized Milli-Q water
with a resistivity ≥ 18 MΩ/cm achieved with a Millipore device (Molsheim, France) is used in
all the experiments to prepare solutions.
Table 3.3: Overview of other used chemicals
Product Company City Country
Acetonitrile (ACN)a VWR International Leuven Belgium
Agarose Biokar Diagnostics Beauvais France
Citric acid anhydrous Hawkins, Inc. Minneapolis USA
HEPESb buffer Sigma Life Sciences St. Louis USA
L-(+)-Ascorbic acid Sigma-Aldrich St. Louis USA
Potassium chloride Sigma-Aldrich St. Louis USA
Sodium chloride Riedel-de Haën Seelze Germany
Trifluoric acid (TFA) Sigma-Aldrich St. Louis USA
Tri-Sodium citrate dihydrate Merck Darmstadt Germany
a HPLC grade
b HEPES is the abbreviation for 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid
21
3.2. METHODS
3.2.1. Loading of the drug into ion-exchange fibers
Fiber bundles of Smopex-101 and Smopex-102 are prepared by weighing 100 mg of
dry fiber mass (actual mass: 232.56 mg of Smopex-101, 151.52 mg of Smopex-102) and
wrapping them into porous cotton gauze, obtained from Mölnlycke Health Care (Gothenburg,
Sweden). Before loading the fiber bundles are pretreated with 50 ml 100 mM NaCl for one
hour. After one hour this solution is changed for a fresh 50 ml 100 mM NaCl solution. The
Smopex cation-exchange fiber bundles are then washed with 50 ml Milli-Q water for 15
minutes. This washing step is repeated six times and thus altogether the duration of the
washing is 1.5 h. After the washing period the fiber bundles are squeezed a bit for removing
the washing solution from the fiber bundles.
The loading of gonadorelin, leuprorelin and nafarelin into the fibers is performed by
immersion of 10 bundles of either Smopex-101 and Smopex-102 into 50 ml of 0.5 mM
peptide solution (pH=7.4) and keeping them on a shaker (IKA Labortechnik, Staufen, Germany)
(180 movements /min) at ambient room temperature for 48 h. The loading of apomorphine
into 10 bundles of Smopex-101 has been done in two phases. The same settings as during the
loading of the peptides are used, except that the loading solution has been renewed after 24
h. The used loading solution is 50 ml of a 0.25 % (w/V) apomorphine solution (pH=3.0). To
remove the unattached drugs, the fiber bundles are washed 5 times with 50 ml of Milli-Q
water for 10 minutes. The amount of absorbed drug in the fiber bundles is determined by
high performance liquid chromatography (HPLC) from the difference between the amount in
the initial loading solution and the combined post-loading and washing solutions.
3.2.2. Characterization of the ion-exchange fibers
Micrographs of Smopex-101 and Smopex-102 cation-exchange fibers unloaded and
loaded with gonadorelin are obtained using FEI Quanta™ FEG scanning electron microscope
(SEM) (FEI Company, Hillsboro, USA). The samples are fixed onto a two-sided carbon tape
with silicone adhesive and sputtered with platinum for 25 seconds with an Agar sputter
device (Agar Scientific Ltd., Essex, UK) prior to imagining.
22
3.2.3. Release of LHRH and its analogues from the ion-exchange fibers
Drug release from the cation-exchange fiber bundles is tested in vitro in amber glass
bottles (Schott AG, Mainz, Germany) at ambient room temperature. Drug containing fiber
bundles are individually placed into 15 ml of extracting solution containing either 15.4 mM,
154 mM or 1540 mM NaCl in 25 mM HEPES (pH=7.4). Bottles are placed on the shaker with a
motility of 180 movements/min. 300 µl samples are taken at fixed time intervals for 24 h (0.5
h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h and 24 h) and replaced by the same volume of fresh buffer.
Peptide concentrations in the samples are determined by HPLC.
3.2.4. Model drug permeation across porcine skin in vitro
Full-thickness porcine skin is separated from the inner side of pig ears with scalpel and
forceps and kept in the freezer at -20 °C until further use. In vitro permeation studies are
performed in 3 ml Side-by-side®-diffusion cells (Laborexin, Helsinki, Finland). Full-thickness
porcine skin is clamped between two identical halves of diffusion cells (volume 3 ml). The
area of exposed skin is 0.785 cm2. To mimic physiological conditions, the cells are
thermostated at 37 °C by a surrounding socket. 25 mM HEPES-buffered physiological NaCl
(pH=7.4) is placed in the receiver compartment of the diffusion cells. Drug solution (1 mg/ml)
or drug containing cation-exchange fiber is placed in the donor compartment in 5 mM citric
acid buffer including 154 mM NaCl (pH=5.0). Both the donor and receiver buffer contain 0.1 %
(w/V) ascorbic acid to prevent apomorphine from auto-oxidative breakdown. As
apomorphine is a positively charged drug it is iontophorised from the anodic compartment.
300 µl samples for HPLC analysis are collected from the receiver (in experiments with cation-
exchange fibers also from donor) compartment and replaced by fresh buffer at 0.5 h, 1 h, 2 h,
3 h, 4 h, 5 h, 6 h, 8 h (current off) and 24 h.
Platinum electrodes are used in all the iontophoretic experiments. During the
experiments the electrodes are separated from the donor and receiver compartments by salt-
bridges, which consist of 1 M KCl gelled with 2 % (w/w) agarose inside silicone tubing (inner
diameter 2 mm, length circa 15 cm). Salt-bridges prevent direct contact and possible reaction
of the drug with the electrodes. The electrolyte that surrounds the electrodes is 2 M KCl. A
constant current (Ministat potentiostat, Sycopel Scientific Ltd., Boldon, United Kingdom) of
0.5 mA/cm2 is applied for 8 h and the next 16 h the passive flux is monitored. The
current/voltage is monitored throughout each experiment (Fluke 8808A multimeter, Everett,
23
USA). Pictures from the set-up during the in vitro iontophoretic experiment from Smopex-101
cation-exchange fibers loaded with apomorphine are shown in Figure 3.2.
Figure 3.2: Set-up of an iontophoretic experiment from Smopex-101 cation-exchange fibers loaded with apomorphine. Left: general overview. Right: one Side-by-side® diffusion cell in close-up. Between the two halves of the cells full-thickness porcine skin is clamped. Apomorphine solution or Smopex-101 cation-exchange fibers loaded with apomorphine in 5 mM citric acid buffer including 154 mM NaCl is placed in the donor compartment. The receiver compartment is filled with 25 mM HEPES buffered saline. Receiver and donor compartment include also 0.1 % ascorbic acid. Platinum electrodes are separated from the donor and receiver compartments with salt-bridges. The surrounding electrolyte solution of the electrodes is 2 M KCl.
3.2.5. Analysis of the drugs
Apomorphine is quantified by a high performance liquid chromatography (1100
Infinity, Agilent Technologies, Santa Clara, USA) based method, whose parameters are
described in Table 3.4. A linear relationship between the drug concentration and the peak
area is established in the concentration range from 0.1 µg/ml to 100 µg/ml with a correlation
of 0.999. The limit of detection of this method is 0.1 µg/ml.
The concentrations of LHRH and its analogues in all the experiments are analyzed with
high performance liquid chromatography (1260 Infinity, Agilent Technologies, Santa Clara,
USA). The parameters of these HPLC methods are also shown in Table 3.4. A linear correlation
between drug concentration and peak area holds true for gonadorelin and leuprorelin in the
range of 0.1 – 25 µg/ml with a correlation of 0.998 and 0.996 respectively. Nafarelin has a
24
correlation of 0.980 in the range 0.1 – 1 µg/ml and 0.993 in the range 1 µg/ml – 25 µg/ml. The
limit of detection is 0.1 µg/ml for the three investigated peptides.
Table 3.4: HPLC method parameters for the used drugs. The used column, mobile phase, wavelength λ, flow rate, temperature of the column (column T), injection volume (Inj. V) and retention time (RT) are shown.
Drug Column Eluent λ
(nm)
Flow rate
(ml/min)
Column
T (°C)
Inj. V
(µl)
RT
(min)
apomorphine Geminia C18 3µm
100 x 4.6 mm
85%:15%
00.1%TFAc:ACN 273 1.2 30 20 4.3
gonadorelin Vydacb C4 5 µm
250 x 4.6 mm
80%:20%
0.1%TFAc:ACN 223 1.0 22 60 4.4
leuprorelin Vydacb C4 5 µm
250 x 4.6 mm
75%:25%
0.1%TFAc:ACN 220 1.0 22 100 5.6
nafarelin Vydacb C4 5 µm
250 x 4.6 mm
70%:30%
0.1%TFAc:ACN 223 1.0 22 80 5.2
a The column is achieved from Phenomenex Inc. (Torrance, USA)
b The column is obtained from Grace Davison Discovery Science (Deerfield, USA)
c The 0.1 % TFA solution is adjusted to pH 2.0
25
4. RESULTS
4.1. CHARACTERIZATION OF ION-EXCHANGE FIBERS
The SEM micrographs of unloaded Smopex-101 cation-exchange fibers and Smopex-
101 fibers loaded with gonadorelin in different magnifications are presented in Figure 4.1.
Similar micrographs of unloaded Smopex-102 cation-exchange fibers and Smopex-102 loaded
with gonadorelin are presented in Figure 4.2. Both cation-exchange fiber types have a
cylindrical shape. The surface of the fibers looks quite flat on larger magnification but with a
closer look, grooves are easily seen. According to the micrographs all the fibers are similar in
length and diameter (estimated length around 300 µm and a diameter of 30 µm). Based on
the images no significant differences in the appearance can be detected between either
loaded and unloaded Smopex-101 or loaded and unloaded Smopex-102 fibers. Furthermore,
both fiber types do not differ remarkably in their appearance.
Figure 4.1: SEM micrographs of Smopex-101 cation-exchange fibers loaded with gonadorelin (A and B) and unloaded (C and D). Note the difference in scaling. The ion-exchange fibers are deposited on a metal plate with special tape, sputtered for 20 s with platinum and then analysed with SEM.
26
Figure 4.2: SEM micrographs of Smopex-102 cation-exchange fibers loaded with gonadorelin (A and B) and unloaded (C and D). Note the difference in scaling. The ion-exchange fibers are deposited on a metal plate with special tape, sputtered for 20 s with platinum and then analysed with SEM.
4.2. LOADING OF LHRH AND ITS ANALOGUES INTO THE ION-EXCHANGE FIBERS
The amount of drug (mmol/g fiber) loaded into the Smopex-101 and Smopex-102
cation-exchange fibers after a loading period of 48 h with a 0.5 mM peptide solution is shown
in Table 4.1. For all three peptides no difference in the extent of loading is seen between both
cation-exchange fiber types. Furthermore, the tendency of the peptide to bind to the fibers
increases slightly with increasing molecular weight of the peptides (molecular weight of the
peptides see Table 1.2).
Table 4.1 shows also the stability of the peptide drugs in the loading solution as the
percentage of intact peptide in the loading solution after 48 h. Since this value is over 98 %
for all three investigated peptides, we can conclude that the drugs are stable throughout the
48-hour loading period. Thus, the decrease of the drug concentration in the solution during
loading can only be because of the binding to the cation-exchange fibers.
27
Table 4.1: Drug content of cation-exchange fibers after a 48-hour loading period with a 0.5 mM peptide solution and stability of the loading solutions (percentage of intact peptide in the loading solution after 48 h).
Drug Smopex-101 (mmol/g) Smopex-102 (mmol/g) Stability (%)
gonadorelin 0.0245 0.0240 99.04
leuprorelin 0.0260 0.0261 98.49
nafarelin 0.0277 0.0276 100.35
4.3. RELEASE OF LHRH AND ITS ANALOGUES FROM THE ION-EXCHANGE FIBERS
The release profiles of gonadorelin (A, B), leuprorelin (C, D) and nafarelin (E, F) from
Smopex-101 (A, C, E) and Smopex-102 (B, D, F) cation-exchange fibers during 24 h are
presented in Figure 4.3. The fiber bundles used for determination of the release profiles are
the ones achieved during the loading of the drug into the ion-exchange fibers (results
presented in Section 4.2). One bundle of Smopex-101 cation-exchange fibers contains 2.89
mg gonadorelin, 3.16 mg leuprorelin or 3.67 mg nafarelin. 2.84 mg gonadorelin, 3.16 mg
leuprorelin or 3.65 mg nafarelin is loaded into one bundle of Smopex-102 cation-exchange
fibers. 15 ml of 25 mM HEPES buffer (pH=7.4) including either 15.4 mM NaCl, 154 mM NaCl or
1540 mM NaCl is used as a releasing medium.
As seen in Figure 4.3, a larger fraction of the drugs tend to be released from the fibers
with an increasing electrolyte concentration. A 10-fold higher salt concencentration in the
HEPES-buffer leads to a proportional higher amount of drug released from the cation-
exchange fiber bundles. For example in the case of gonadorelin released from Smopex-101
cation-exchange fibers, the average percentage released from the fibers is around 4 % with
the buffer including 15.4 mM NaCl, around 12 % with the buffer including 154 mM NaCl and it
increases to around 20 % with the buffer including the highest salt concentration.
The difference between the two fiber types in the amount of drug released (different
scales of the ordinate axes in Figure 4.3) should be noted. Smopex-102 ion-exchange fibers
release higher amounts of drug compared to Smopex-101 cation-exchange fibers. In the case
of gonadorelin around 33 % of the loaded drug can be released from Smopex-102 fibers with
highest electrolyte concentration in comparison with around 20 % from Smopex-101 fibers.
The same hold true for leuprorelin and nafarelin (around 20 % and 17 % respectively with
Smopex-102 in comparison with around 13 % and 2 % respecitvely with Smopex-101).
28
Figure 4.3: The release profiles of gonadorelin (A, B), leuprorelin (C, D) and nafarelin (E, F) from Smopex-101 (A, C, E) and Smopex-102 (B, D, F) cation-exchange fibers during 24 hours (mean ± standard error of the mean, n = 3). The extracting solutions are 25 mM HEPES combined with 15.4 mM NaCl, 154 mM NaCl or 1540 mM NaCl.
29
Gonadorelin (Figure 4.3 A and B) achieves the highest amount of drug released from
both fiber types (around 20 and 33 % with Smopex-101 and Smopex-102 cation-exchange
fibers respectively with the HEPES buffer including 1540 mM NaCl). The second and third
highest amount released are seen with leuprorelin (approximately 13 % and 20 % from
Smopex-101 and Smopex-102 fibers respectively with 25 mM HEPES including 1540 mM NaCl
used as extracting solution (Figure 4.3 C and D) and nafarelin (approximately 2 % and 17 %
released from Smopex-101 and Smopex-102 cation-exchange fibers respectively with the
highest electrolyte concentration, Figure 4.3 E and F). In all cases the amount of peptide
released remains still less than 50 % of the amount of drug loaded into the fibers.
Gonadorelin and leuprorelin are gradually released from Smopex-101 cation-exchange
fibers for up to 12 hours (Figure 4.3 A and C). Afterwards the amount of drug in the release
medium starts to decrease. Therefore, the combination of gonadorelin and leuprorelin with
Smopex-101 cation-exchange fibers shows potential for controlled release for up to 12 hours.
Potential for controlled release is also shown in the combination of Smopex-102 cation-
exchange fibers with gonadorelin since a gradual release of gonadorelin is achieved for up to
4 hours (Figure 4.3 B). Afterwards a decrease of the drug in the release medium is also seen.
Gradual release is not seen in the case of leuprorelin released from Smopex-102 cation-
exchange fibers (Figure 4.3 D). This peptide comes out of the fibers fast (maximum achieved
in approximately 2 hours) and afterwards the amount of drug in the release medium starts to
decrease.
Nafarelin does not seem to be a suitable drug candidate for using the ion-exchange
fibers as drug reservoirs (Figure 4.3 E and F). Firstly, nafarelin is not gradually released from
both fiber types. The peptide is coming out of the fibers very fast or irregularly. The amounts
released from Smopex-101 cation-exchange fibers are very small (approximately 2 % of
nafarelin released with the highest electrolyte concentration). Moreover, increasing the
electrolyte concentration of the release medium is not resulting in higher amounts of
nafarelin released from both cation-exchange fiber types. Approximately 2.1 % from Smopex-
101 and 21 % from Smopex-102 is released with the release medium including 154 mM NaCl.
The extent of release is approximately 2 % from Smopex-101 and 17 % from Smopex-102
cation-exchange fibers with the highest electrolyte concentration used as release medium.
Furthermore, the amount of nafarelin in the release medium starts to decrease quickly.
30
4.4. MODEL DRUG PERMEATION ACROSS PORCINE SKIN IN VITRO
4.4.1. In vitro permeation of apomorphine solution
The permeation curves of the model drug apomorphine across porcine skin in vitro
from a 1 mg/ml solution during an iontophoretic experiment with a current density of 0.5
mA/cm² and a passive permeation experiment (current density of 0.0 mA/cm²) is presented
in Figure 4.4. During the experiment, the current is on for 8 h, afterwards the passive drug
permeation through porcine skin is followed for up to 24 h.
Figure 4.4: Amount of apomorphine permeated (µg/cm²) through porcine skin in vitro (mean ± standard error of the mean, n= 5-6). Initial concentration of the apomorphine solution in donor compartment is 1 mg/ml. The current is on for 8 h, whereafter passive drug permeation is followed for up to 24 h.
As shown in Figure 4.4 the application of iontophoresis significantly enhances the
transdermal permeation of apomorphine. The passive flux of apomorphine is 0.838 ± 0.074
nmol/h×cm² (mean ± standard error of the mean). The iontophoretic steady-state flux at
current density of 0.5 mA/cm² calculated from the linear slope of the permeation curve is
27.304 ± 3.904 nmol/h×cm². By passive permeation 8.501 ± 0.727 µg of apomorphine is
permeated across the skin in 24 h per cm² of skin. With a iontophoretic steady-state flux
obtained with a current of 0.5 mA/cm² 204.970 µg of apomorphine can be delivered in 24 h
31
per cm² of skin. The iontophoretic enhancement factor of the flux, which shows the
enhancement of transport across porcine skin by the application of current in comparison
with the passive transdermal flux, is 33.
The amount of drug permeated passively through porcine skin is increasing
proportionally with the time. When iontophoretic current of 0.5 mA/cm² is applied the steady
state flux is achieved in approximately 3 hours, corresponding with a lag time of 142 minutes.
The lag time is calculated as the intersection of the linear slope with the x-axis. The
reversibility of skin permeability to normal state is tested by measuring the passive flux after
switching off the current at 8 h. The post iontophoretic flux is significantly higher compared
to the normal passive flux of apomorphine (11.249 ± 0.425 nmol/h×cm² compared to 0.838 ±
0.074 nmol/h×cm²).
The transference number (transport number), which shows the fraction of current
carried by the drug ions can be calculated from Equation 4.1 (Kochhar & Imanidis, 2004). This
value is 0.146 ± 0.021 %. The apparent permeability coefficient represents a measurement of
the skin permeability to the drug under iontophoretic conditions and can be calculated as in
Equation 4.2. The value is 3.146×10-³ cm/h.
(4.1)
in which: TN: transference number
z: valence of the drug
F: Faraday constant
dQ/dt: amount of drug permeated (mass units per time)
I: total current passed through the skin
(4.2)
in which: Kp: apparent permeability coefficient
J: flux of the drug
Ci: initial concentration of the drug
The pH control of the donor and receiver solutions is obtained by using buffers (5 mM
citric acid pH=5 and 25 mM HEPES buffer pH=7.4 in donor and receiver compartment,
32
respectively). The pH values of the donor and receiver solutions measured after the
experiments do not differ significantly from the pH values of the initial solutions even though
a slight decrease is seen in both compartments during the experiments.
4.4.2. In vitro permeation of apomorphine loaded into Smopex-101 cation-
exchange fibers
Figure 4.5 shows the amount of model drug apomorphine in the donor and receiver
compartment of the diffusion cells during iontophoretic delivery across porcine skin using
Smopex-101 cation-exchange fibers as drug reservoirs. As with the solution, the current of 0.5
mA/cm² is applied for 8 h, whereafter the passive permeation of apomorphine is monitored
during the next 16 h. The Smopex-101 cation-exchange fibers used in this experiment contain
237.238 mg apomorphine in 1 g of fiber. This amount is achieved after two loading periods of
24 h with a 0.25 % (w/V) apomorphine solution and sufficient washing to remove the
unbound drug from the fibers. Each donor compartment contains Smopex-101 cation-
exchange fibers loaded with 23.72 mg apomorphine. During the loading period the drug is
stable (stability > 99 % calculated as the amount of intact drug after each 24-hour loading
period).
As seen in Figure 4.5 apomorphine is gradually released from the Smopex-101 cation-
exchange fibers for up to 8 h. Afterwards, the increase in amount of drug in the donor
compartment is slowed down. The gradual release is coincident with the time of the current
applied. The iontophoretic permeation curve of apomorphine from Smopex-101 cation-
exchange fibers resembles the one obtained from the solution. Similarly, the steady-state flux
is obtained within 3 hours and after switching off the current, the flux decreases. Although, as
only 2.40 % on average of the amount of apomorphine loaded into the fiber is released into
the donor compartment in 24 hours, the drug flux from Smopex-101 cation-exchange fibers
remains smaller compared to that from drug solution. The steady state flux of the drug
through porcine skin is 4.376 ± 0.324 nmol/h×cm² (mean ± standard error of the mean). The
transference number as calculated in Equation 4.1 is 2.346×10-3 ± 1.736×10-3 %. The apparent
permeability coefficient is 7.222×10-3 cm/h. The measured pH values of the solutions in the
donor and receiver compartment (data not shown) do not differ significantly from the initial
values however a slight decrease of the pH in both compartments can be seen.
33
Figure 4.5: Amount of apomorphine (µg) in the donor and receiver compartment during iontophoretic delivery across porcine skin from Smopex-101 cation-exchange fiber loaded with 23.72 mg apomorphine (mean ± standard error of the mean, n= 6). The used current density is 0.5 mA/cm². Current is on for 8 h, whereafter the passive drug permeation is monitored for up to 24 h.
34
5. DISCUSSION
5.1. EFFICIENCY OF LOADING INTO AND RELEASE OF LHRH AND ITS ANALOGUES
FROM CATION-EXCHANGE FIBERS
First, it should be noted that the three peptide drugs gonadorelin, leuprorelin and
nafarelin can achieve efficient loading into the investigated cation-exchange fiber types.
These investigated cation-exchange fiber types are Smopex-101 (containing sulphonic acid as
ion-exchange groups) and Smopex-102 (containing carboxylic acid as ion-exchange groups).
The amount of peptide loaded into the fibers remains low because only small amounts of the
drugs are used in the loading solution (50 ml of a 0.5 mM peptide solution). As almost all of
the drug in the initial loading solution is taken up by the fibers during the 48-hour loading
period, only a very small fraction of the drug is left in the combined post-loading and washing
solution. With these small amounts of drug used for the loading, there is no room for
achieving higher amounts of drug loaded into the fibers. The loading efficiency is close to
100 %. The aim of the loading experiments is not to completely occupy all the ion-exchange
functional groups in the ion-exchanger with the drug, but to test the affinity of these three
peptides towards the cation-exchangers with different ion-exchange groups. Furthermore, it
might be that as a result of the low amount of peptide loaded into the fibers, no difference in
structure on the SEM micrographs presented in Figure 4.1 is seen between unloaded Smopex-
101 cation-exchange fibers and loaded ones with gonadorelin. The same holds true for
Smopex-102 cation-exchange fibers (Figure 4.2). Thus, it can be concluded that the drug
loading does not change the structure of the ion-exchange fibers.
Although there is a significant difference in binding capacity between Smopex-101
cation-exchange fibers (3.9 mmol/g) and Smopex-102 cation-exchange fibers (5.6 mmol/g)
(Table 3.2), the drug content in the two fiber types after loading with a 0.5 mM peptide
solution is similar (Table 4.1). The reason again for that might be the low amounts of total
peptide used for loading that could not bring out the differences. It is possible that
experiments with more concentrated loading solutions could lead to different results but due
to the high price of the used compound it is impractical. Hänninen et al. (2005) have
demonstrated that a higher loading of the anionic drug can be achieved with anion-exchange
fibers with a higher binding capacity compared to anion-exchange fibers with a lower binding
35
capacity. Therefore, the investigators used 0.5 % (w/V) loading solutions of 5-hydroxysalicylic
acid, salicylic acid or 5-chlorosalicylic acid for 3 hours, whereafter the loading solution is
replaced by a new one with the same concentration for 18 more hours. These concentrations
used were higher than the concentration of peptides used during our loading period and thus
Hänninen et al. (2005) observed differences which are not detected in this study. According
to the Henderson-Hasselbalch equation at the pH value of the loading solution used (pH=7.4)
100 % of the sulphonic acid groups of the Smopex-101 fibers and 99.60 % to 100 % of the
carboxylic acid groups of Smopex-102 cation-exchange fibers are ionized. As the degree of
the ionization is similar in both fiber types during loading, similar extent of ion-exchange
process and binding of the drug can also be expected.
As the molecular weight of the peptide increases (Table 1.2), slightly higher binding of
the drug is obtained (Table 4.1). This can be due to stronger interactions between the drug
and the ion-exchange fiber as their size is bigger with increasing molecular weight (Hänninen,
2008). Furthermore, the lipophilicity of these three peptides also increases significantly with
increasing molecular weight. The most lipophilic peptide out of the three peptides used is
nafarelin (logP=1.21) followed by leuprorelin (logP=0.7) and then gonadorelin (logP=-3.6).
Because the chemical equilibrium coefficient contributing to the ion-exchange process is
higher in the case of lipophilic drugs (Jaskari et al., 2001), the ion-exchange process during
the loading is more efficient in this case. Thus, with more lipophilic drugs, it leads to a higher
amount of drug loaded into the cation-exchange fibers. This is completely according to our
findings. Jaskari et al. (2001) saw clearly that drug content after loading of nadolol,
propranolol and tacrine who are presented in order of increasing lipophilicity (and thus
increasing logP) into several types of cation-exchange fibers was increasing with increasing
logP value.
Gonadorelin and leuprorelin loaded on Smopex-101 (Figure 4.3 A and C) might present
a suitable approach for controlled delivery of the drug since these combinations lead to a
gradual release profile of the drug for up to 12 h. The combination of gonadorelin loaded into
Smopex-102 cation-exchange fibers (Figure 4.3 B) might not be favourable as a drug reservoir
because gradual release of the drug from the cation-exchange fibers is only seen for up to
approximately 4 h. The combination of leuprorelin and Smopex-102 cation-exchange fibers
might not be suitable as a drug reservoir since no gradual release is seen. But in this case, the
36
release profile shows a quite large variation between the replicates. Therefore, more
replicates have to be included in the results. Unfortunately, the aim for achieving gradual
release and thus probably the potential for controlled release with the combination of
nafarelin with Smopex-101 and Smopex-102 (Figure 4.3 E and F) has failed.
The drug release process from the cation-exchange fibers can be understood as
follows. The ion-exchange materials tend to equilibrate with the external solution (HEPES
buffered saline). A slight amount of chloride ions start to migrate from the external solution
into the ion-exchange fibers according to the concentration gradient (Hänninen et al., 2003).
The same holds true for the drug ions that start to diffuse from the ion-exchange fibers into
the external solution. This concentration difference of the ions in the two phases and the
immobile charges of the fibers creates an electrical potential difference between the phases,
the Donnan potential. The Donnan potential pulls back the chloride ions into the solution and
the sodium ions into the ion-exchanger and thus it accomplishes the interchange of sodium
ions and peptide cations between the two phases. Each peptide cation is interchanged by one
sodium ion because of the electroneutrality requirement of the ion-exchange process. The
ion-exchange process continues till the Donnan equilibrium is reached, which is the equality
of the electrochemical potential for the sodium ions, chloride ions and peptide cations. This
achievement of the Donnan equilibrium is indicated by a plateau in the release profiles
(Figure 4.3).
As seen in Figure 4.3, a 10-fold higher NaCl concentration combined with the 25 mM
HEPES buffer used as extracting solution and thus a 10-fold higher ionic strength of the
extracting solution results generally in a proportional increase of drug amount released from
the cation-exchange fibers. The same is described by Jaskari et al. (2001). The higher the salt
concentration in the external solution is, the lower the absolute value of the Donnan
potential is. A lower absolute value of the Donnan potential results in a weak interaction
between the cation-exchange fibers and the cationic peptides. Also the probability of
interchange of sodium and drug ions increases with a more concentrated external solution.
The higher probability of interchange and the lower the absolute value of the Donnan
potential when a higher NaCl concentration is used in the release medium results in a higher
amount of drug released from the cation-exchange fibers. A higher concentration of an
extracting cation in the extracting solution can be used as an approach for achieving higher
37
amounts of drug released from the cation-exchange fibers. Thus, it can be concluded that via
the composition of the external solution the extent and rate of the release of the peptide
drugs from the cation-exchange fibers can be controlled.
In general, gonadorelin loaded into both Smopex fiber types (Figure 4.3 A and B)
achieves the highest amounts of drug released from the fibers followed by leuprorelin and
nafarelin. One exception to this rule is mentioned before (the release of nafarelin from both
fiber types with 25 mM HEPES combined with 154 mM NaCl). The peculiar behaviour of
nafarelin can be due to several reasons, which will be discussed later. The molecular weight
of the peptides (Table 3.1) increases with decreasing extent of release from the cation-
exchange fibers. One reason for that might be that the increasing molecular size can hinder
the molecules from moving out of the fibers. Abdekhodaie & Wu (2006) stated a retarded
ion-exchange process for larger drug ions as a result of their smaller diffusion rate. Hänninen
et al. (2003) described the decreasing extent of release with increasing lipophilicity of the
investigated salicylates due to stronger interactions between the drug and the ion-exchange
fibers. Since the peptides only differ in the sixth amino-acid, which is a more hydrophobic
amino-acid in the sequence of gonadorelin over leuprorelin to nafarelin (logP of -3.6 over 0.7
to 1.21), the finding of these investigators could also explain our results.
Smopex-101 and Smopex-102 cation-exchange fibers consist of the same polyethylene
framework and differ only in the functional ion-exchange groups which are sulphonic acid and
carboxylic acid groups in the case of Smopex-101 and Smopex-102, respectively. As a result of
the same polymeric framework, no significant difference between Smopex-101 and Smopex-
102 cation-exchangers are seen when the fibers are analysed by SEM micrographs (Figure 4.1
and Figure 4.2). Because the extent of release of the peptides is higher with the Smopex-102
cation-exchange fibers, the only difference between the two fibers types, the ion-exchange
functional groups, might be the reason. This phenomenon can be due to the weaker
interactions between the carboxylic acid groups and the drugs in comparison with the
interactions of the peptides with the sulphonic acid groups. Jaskari et al. (2001) found out
that more lipophilic drugs are released more easily from weak acidic or basic ion-exchange
groups then from strong acidic or basis ion-exchange groups. In our case a higher extent of
release of the three peptides from the Smopex-102 cation-exchange fibers (with weak acidic
functional groups) is seen in comparison with Smopex-101. Thus, the explanation of the
38
investigators does not hold true only for the most lipophilic peptide nafarelin (logP=1.21) but
for the three peptides according to our results.
Gradual release and thus potential for controlled release is only seen in the cases
described above for up to a certain time point. In most of the cases after this time point the
concentration of the peptide usually decreases in the surrounding buffer solution. There
might be several reasons for this phenomenon. Firstly, it could be due to degradation of the
peptides in the release medium because several degradation pathways for peptides are
known (http://www.sigmaaldrich.com/life-science/custom-oligos/custom-peptides/learning-
center/peptide-stability.html). This is in contradiction with Raiman et al. (2004) who saw no
degradation of LHRH and nafarelin in a buffer solution composed of 25 mM HEPES and 150
mM NaCl, which is quite similar to the extracting solution used in this study. Another
possibility is that there might be some adsorption of the released peptides onto the glass
surface of the bottles used for the release experiments because the adsorption of nafarelin
onto glass and other surface has been discussed in literature (Anik & Hwang, 1983; Anik &
Johnson, 1991). 59 % of nafarelin solution at a concentration of 1 µg/ml was adsorbed at
equilibrium onto borosilicate solid glass beads (circa 100 cm²). Onto other surfaces the
percent adsorbed differs from 0.6 % onto a 1-ml Glaspak syringe with an initial concentration
of 100 µg/ml to 93.2 % onto a Millex-GS filter with an initial concentration of 20 µg/ml. The
adsorption of nafarelin onto the glass surface is ascribed to ionic amine-silanol bonding. Since
the two other peptides also have amine groups, this explanation could be possible. Moreover,
it has been demonstrated that the solubility of nafarelin drops rapidly with increasing ionic
strength of the solution (Anik & Johnson, 1991). This is a process which is also known as
salting out of peptides. At higher NaCl concentrations, the solution even starts to form an
opaque gel. Thus, with an increasing concentration of NaCl in the 25 mM HEPES buffer, the
peptide can form precipitates or a gel in the ion-exchange phase which hinders the
movement out of the ion-exchange fiber bundles. Another possibility is that nafarelin or the
other peptides might aggregate after being released from the ion-exchange fibers.
5.2. EFFECT OF IONTOPHORESIS ON TRANSDERMAL DELIVERY ACROSS PORCINE
SKIN IN VITRO
The system for the investigation of the in vitro transdermal delivery of LHRH and its
analogues across porcine skin both from the solutions and cation-exchange fibers is tested
39
with the model drug apomorphine. For that purpose, transdermal in vitro iontophoretic
experiments are carried out both from apomorphine solution and from cation-exchange
fibers loaded with the drug. As mentioned before, apomorphine is chosen for a model drug
since the transdermal delivery of the drug has been studied extensively in the Division of
Pharmaceutical Technology (Faculty of Pharmacy, University of Helsinki) and it possesses
suitable phsyicochemical characteristics needed for the anodal iontophoresis across the skin.
As the pKa of apomorphine is 7.2, the drug has a valence of +1 at the pH used in our
iontophoretic experiments (Van der Geest et al., 1997; Li et al., 2001). The logP value of
apomorphine of 2.15 (Van der Geest et al., 1997) and a smaller molecular mass compared to
those of the peptides makes apomorphine a suitable drug candidate to be delivered
transdermally. Furthermore, as apomorphine is a less expensive compound and easier to
handle in laboratory setting compared to the peptides (that may aggregate or adsorb onto
surfaces) the choice of apomorphine for testing the iontophoretic protocol has also practical
justifications. The only limitation of apomorphine is the fast oxidation of the drug in aqueous
conditions. In order to warrant the stability of the drug during iontophoretic experiments
antioxidant (such as ascorbic acid) should be included in the formulation and the pH of the
solution kept as low as possible. Smopex-101 fibers are chosen for investigating the potential
of the ion-exchange materials as drug reservoir for apomorphine because previous studies in
this lab have demonstrated a significant degradation of the drug when loaded into Smopex-
102 cation-exchange fibers (not published yet).
The pH of the donor solution in all the experiments is set at 5. The choice of this donor
solution pH takes the following factors into consideration – the avoidance of a pH where
degradation of the drug can occur, the fixed charge of the skin, the solubility of the drug at
certain pH values and the biocompatibility. For avoiding skin irritation and chemical burns,
the pH of an iontophoretic formulation has to be between 5 and 7.4 (Gratieri et al., 2011).
The degree of drug ionization will determine if the drug is mainly transported across the skin
by electrorepulsion or electro-osmotic solvent flow (Gratieri et al., 2011). The latter is the
dominating mechanism for larger and neutral molecules. Furthermore, for favouring the
movement of positively charged ions through the skin, the pH used has to be higher than 4.8
since this value is the iso-electric point of the skin (Marro et al., 2001). The degree of fixed
negative charge within the skin increases with increasing pH and thus the transport of
40
positively charged drugs such as apomorphine will increase as a result of the electro-osmotic
force. The change of the fixed charge of the skin was the reason why Li et al. (2001) saw a
significantly increase of the transdermal flux of apomorphine when the pH of the donor
solution was increased from pH 3 to 6. The use of higher pH values than pH 5 in our
experiments are not reasonable. This is due to an increase in the degradation rate of
apomorphine with higher pH values and a possible change of the charge of the drug.
The platinum electrodes used in the experiments cause an alteration in the pH of the
solution in which they are placed since the reactions described beneath take place on the
surface of the electrodes (Banga, 1998). The oxidation at the anode causes the production of
hydrogen ions which leads to a pH drop in the solution containing the anode. On the other
hand, the production of hydroxyl ions at the cathode causes a rise of the pH in the solution
containing the cathode:
H2O 2 H+ + ½ O2 + 2e- (at anode)
2H2O + 2e- H2 + 2OH- (at cathode)
For avoiding the alteration in pH of the donor and receiver solutions, the electrodes
are placed in 2 M KCl and connected to the donor and receiver compartment with salt-
bridges. A slight decrease in the pH values of the donor and receiver compartment before
and after every experiment is still seen (data not shown). The decrease of the receiver
compartment pH value might be due to the acidic nature of the donor solution. Since the
hydrogen ions are also cationic, they can be transported across the skin by the iontophoretic
current. Also, there might be a small possibility that hydrogen ions produced on the anodes
can pass through the salt-bridges to the donor solution and cause a pH drop there. However,
the alterations of the pH values are not significant.
As seen in Figure 4.4 and described above, the application of the iontophoresis
significantly enhances the transdermal permeation of apomorphine (flux increases from
0.838 ± 0.074 nmol/h×cm² to 27.304 ± 3.904 nmol/h×cm², iontophoretic enhancement factor
of 33). The application of iontophoresis can enhance the transport of drugs such as
apomorphine across the skin 50 to 1000 times in comparison to their passive transdermal flux
and intestinal absorption (Junginger, 2002). Reversibility of the membrane permeability to
normal state is tested by measuring the passive flux after switching off the current at 8 h. The
41
post-iontophoretic flux is significantly higher compared to the normal passive flux of
apomorphine. Van der Geest et al. (1997) saw the same phenomenon of the higher post-
iontophoretic flux in comparison to the passive flux of apomorphine for which they suggested
two explanations. Firstly, it could be ascribed to an alteration of the skin barrier as a result of
current application. A second reason might be increased water content of the skin induced by
iontophoresis, which may lead to increased flux of apomorphine through the skin.
The permeation curve of apomorphine in the iontophoretic experiment of the
Smopex-101 cation-exchange fibers loaded with apomorphine (Figure 4.5 right) resembles
the curve which shows the permeation of apomorphine from a 1 mg/ml solution (Figure 4.4).
The smaller amount of drug permeated across porcine skin from Smopex-101 cation-
exchange fibers is most likely due to the smaller amount of the drug in the donor
compartment with the experiment from the fibers. While during the iontophoresis
experiment from the apomorphine solution the donor contains 3 mg of apomorphine, the
fiber only releases on average 2.40 % out of the 23.72 mg it contains. This is only 0.569 mg.
The efficiency of apomorphine transport can also be described by the transport
number (calculated as in Equation 4.1) that shows the ratio of the current carried by
apomorphine ions from the total applied current (Kochhar & Imanidis, 2004). In both
iontophoretic experiments these numbers are small (0.146 ± 0.021 % in the case of
apomorphine solution and 2.346×10-3 ± 1.736×10-3 % in the case of the ion-exchange fibers).
This can be due to the presence of sodium ions in the buffer (154 mM), which are smaller and
more mobile ions than apomorphine. Therefore, they have the ability to carry a bigger
fraction of the current.
The apparent permeation coefficient of the drug can be calculated by Equation 4.2
and represents a measurement of the skin permeability to the drug under iontophoretic
conditions. The value is slightly higher with the iontophoretic experiment from the Smopex-
101 cation-exchange fibers than from the apomorphine solution (7.222×10-3 cm/h compared
to 3.146×10-3 cm/h). Van der Geest et al. (1997) and Jaskari et al. (2000) saw a decreasing
permeability with increasing drug concentration in the donor compartment. Again, the lower
concentration in the donor compartment has to be stated as an explanation for the higher
42
value of the apparent permeation coefficient in our iontophoretic experiments when the
fibers are used as drug reservoir.
Gradual release of apomorphine from Smopex-101 cation-exchange fibers is also seen
when the fibers are used as a drug reservoir in the iontophoretic experiment. The release is
extensive for up to 8 h in parallel with the time of current applied. Xin et al. (2012) saw a
significantly higher release of diclofenac from ion-exchange fibers across a 0.22 µm cellulose
acetate microporous membrane when a 0.5 mA/cm² is applied in comparison without the
applied current. In their experiment donor and receiver compartment were filled with
deionized water. Thus, in our iontophoretic experiments both the applied current and the
Donnan potential can contribute to the release of apomorphine from the ion-exchange fibers.
In the receiver compartment the variation between the replicates of the Smopex-101
cation-exchange fibers loaded with apomorphine as a drug reservoir for the in vitro
permeation experiment is smaller compared to the experiments from the apomorphine
solutions. This can again be a result of the lower concentration of the drug in the donor
compartment. Another explanation might be that the fibers control the transdermal delivery
by gradually releasing the drug and cushioning the variability of drug permeation between
the different skin samples. The possibility that fibers can control transdermal delivery could
form a motivation for more extensively studying the use of ion-exchange materials as a drug
reservoir in the iontophoretic drug delivery across the skin.
As a result of the gradual release of apomorphine from the Smopex-101 fibers and the
control of the transdermal flux by the ion-exchange fibers, the use of cation-exchange fibers
shows potential in the transdermal delivery of apomorphine. Because of the potential of the
model drug, iontophoretic delivery of LHRH and its analogues from Smopex cation-exchange
fibers should be studied next. But first, several factors have to be considered.
The first thing to suggest is to develop more sensitive and precise analytical methods
for the analysis of the peptides from receiver samples. There are several factors that have to
be taken in consideration when quantifying peptide amounts from the receiver compartment
with HPLC. Firstly, the receiver samples are highly complex biological samples that contain
many macromolecules that may interfere with the drug peak in the HPLC chromatogram.
Secondly, the skin also releases cationic compounds that are transported to the receiver
43
compartment with the electric current in a similar manner as the peptides. As a result false
chromatogram peaks can be considered as peptide peaks by mistake. Thirdly, skin peptidases
can degrade the peptides (Priya et al., 2006). The resulting degradation products may
interfere with the peaks of the drug. Fourthly, sensitivity of the methods might not be
adequate since very small amounts are transported across the skin as small concentrations
are used in the donor compartment and the high molecular mass of the investigated peptides.
Combined liquid chromatography and electrospray ionization tandem mass spectrometry can
be suggested for the quantification of these peptides since as used by Raiman et al. (2004).
Another option is the use of radiolabeled peptides, which are then quantified by liquid
scintillation counting (Bhatia & Singh, 1998).
Moreover, the skin is known to be a metabolically active organ although it has less
proteolytic enzymes than other mucosa (Priya et al., 2006). Thus, metabolism of peptides
during iontophoretic delivery has to be considered. For nafarelin, degradation during
iontophoretic delivery is observed by Delgado-Charro & Guy (1995). For overcoming this
problem they tested the use of aminopeptidase inhibitors in the formulation, but this
approach was ineffective. Another approach is using a pulsed current profile during the
iontophoretic delivery (Raiman et al., 2004). For nafarelin, these investigators saw no
degradation, while LHRH degradation was still seen in contact with the dermis. But no
degradation was observed during the 12-hour current application process. The pulsed current
protocol might prevent the peptides from adsorption processes with structures along the
iontophoretic pathway and thus protect the peptide from hydrolytic degradation. Another
advantage of this protocol is the improved drug permeability.
Furthermore, all the possible problems mentioned in Section 1.3.2 have to be kept in
mind such as the ability of nafarelin and leuprorelin to inverse the electro-osmotic solvent
flow (Delgado-Charro & Guy, 1994; Hoogstrate et al., 1994). Moreover, it has been proven
that nafarelin easily adsorbs onto glass and other surfaces as discussed above (Anik & Hwang,
1983; Anik & Johnson, 1991). Since the adsorption onto glass was ascribed to silanol-amine
bonding, it can easily occur with the other peptides as well. The salt-bridges can also form a
surface where onto the peptides can adsorb since nafarelin showed adsorption onto various
surfaces other than glass. This might lower the concentration measured and thus
underestimate the efficiency of the transdermal transport. As a result of the larger molecular
44
mass the cationic peptides also might have lower mobilities than the other ions present in the
solutions such as sodium ions (Gratieri et al., 2011). Thus, the peptide cations might have a
smaller transference number than in the iontophoretic delivery of apomorphine. In order to
improve the transdermal flux of the peptides, some formulation parameters such as the drug
concentration or the pH can be modified. The pH can be changed in order to increase the
ionized fraction of the drug. However, these changes are not always possible and other
factors have to be taken in account such as the stability of the drug or the skin tolerance to
the used pH value.
When using Smopex-101 and Smopex-102 cation-exchange fibers a few things has to
be kept in mind. From the investigated peptide release from the fibers it can be derived that
the combination of Smopex-102 cation-exchange fibers and gonadorelin might not be a good
combination for forming a drug reservoir since gradually release is only seen during a short
period (for up to 4 h). Since there might be no potential for controlled release, the use of
cation-exchange fibers as a drug reservoir might have no advantage in this case. This might
hold also true for the combination of leuprorelin with Smopex-102 cation-exchange fibers
and nafarelin with Smopex-101 and Smopex-102 cation-exchange fibers. Moreover, as
discussed above, nafarelin shows incompatibility with solutions of high ionic strength because
it forms precipitates or even a gel in these media (Anik & Johnson, 1991). Thus, this peptide
cannot be combined with NaCl. Because the cations of a salt are needed as the counter-ions
in the ion-exchange process, the combination of nafarelin and ion-exchange materials is not a
suitable approach for forming a drug reservoir that can control the iontophoretic delivery.
The iontophoretic transdermal flux of apomorphine from cation-exchange fibers is
lower compared to the one from the solutions. The smaller amount of the drug present in the
donor compartment when the drug was loaded into the fibers might be the reason, as
discussed above. As a result of the possible smaller iontophoretic flux from the fibers, it is
important to determine whether the drug flux of LHRH and its analogues is still sufficient
enough for reaching therapeutically relevant concentrations in the receiver compartment.
Due to the high price of the peptides it might be impractical to try to load higher amounts of
the peptides into the fibers in order to solve the problem. A study to optimize the
iontophoretic flux when the fibers are used as drug reservoir might be needed since the
iontophoretic patch developed later in the process has to be dose-efficient for keeping the
45
total patch cost under control (Green, 1996). Dose efficiency is defined as the fraction of the
drug delivered to the systemic circulation divided by the initial amount loaded into the drug
reservoir of the patch.
Firstly, the behaviour of LHRH and its analogues peptides during some iontophoretic
experiments across porcine skin in vitro has to be tested. The suggested current densities for
investigation should be in a range of 0.1 mA/cm² and 0.5 mA/cm² since the latter is the
maximal applicable current density onto the skin. Higher current densities can cause skin
burns and irritation (Sieg & Wascotte, 2009). Whereas a passive permeation experiment is
usually carried out in iontophoretic studies to determine how the application of iontophoresis
can enhance the transdermal permeation, it is impractical to do with the peptides because
they have a larger molecular weight than the studied model compound apomorphine. Thus,
the peptides would probably not show a detectable passive permeation. Afterwards,
preliminary tests of gonadorelin and leuprorelin loaded onto either Smopex-101 and Smopex-
102 cation-exchange fibers in the donor compartment of the iontophoretic experiment across
porcine skin in vitro can be executed. Nafarelin cannot be investigated more deeply in this
approach because of the many issues discussed above. The iontophoretic protocol has to be
modified when studying the peptides. The pH of the donor compartment can be changed to
pH 7.4, taking degradation of the drug, the fixed charge of the skin, the solubility of the drug
and the biocompatibility into account. This pH value results in a sufficient degree of ionization
of the investigated peptides. As mentioned above, charged molecules can benefit from the
electrorepulsion driving force and thus have a bigger flux across the skin in comparison with
uncharged large molecules. Moreover, this pH is high enough for the skin to carry the fixed
negative charge as discussed above. By using pH 7.4, the buffer in the donor compartment is
changed to a 25 mM HEPES buffer. The addition of anti-oxidant is not needed anymore
because auto-oxidation of the peptides is not occurring. Moreover, the current has to be
applied for 12 hours due to the larger molecular size of the peptides and thus the lower
intrinsic permeability of the peptides across the skin. The transport of the peptides across the
skin can be increased by a longer application time of the current (Khan et al., 2011).
46
6. CONCLUSIONS
The approach for in vitro transdermal delivery of cationic drugs combining
iontophoresis and ion-exchange fibers is successfully validated with the model drug
apomorphine. Iontophoresis enhances the in vitro transdermal transport of apomorphine
compared to passive permeation. When the positively charged drug apomorphine is loaded
into cation-exchange fibers from where it is gradually released during iontophoresis, only
small fraction of the total amount in the donor compartment is transported across the skin
during the application of iontophoresis compared to that of the apomorphine solution.
Overall, the approach of combining ion-exchange fibers and iontophoresis has great potential
in controlling the release and following transdermal delivery of therapeutically active
molecules. With minor modification the same iontophoretic protocol can be used to test the
permeation of gonadorelin (synthetic LHRH) and its analogues (leuprorelin and nafarelin)
both from the solution and from cation-exchange fibers.
Gonadorelin, leuprorelin and nafarelin (LHRH and its analogues) show good affinity
towards cation-exchange fibers during loading. At the same time only partial amounts of
peptides loaded into the fibers are released during 24 h. Higher fractions of drugs are
released from cation-exchange fibers with carboxylic acid as ion-exchange groups (Smopex-
102 fibers) compared to cation-exchange fibers with sulphonic acid as ion-exchange groups
(Smopex-101 fibers). The release of the peptides from the cation-exchange fibers can be
controlled by modifying either the fiber type or the amount of the extracting counter-ions in
the release medium. Overall, the combination of gonadorelin and leuprorelin loaded into
Smopex-101 fibers show the best potential for gradual release of the peptides from the fibers.
Nafarelin, the most lipophilic one of the three peptides, might not be a suitable drug for use
in the combination with ion-exchange materials due to the peculiar release profiles from
cation-exchange fibers.
Further studies on the behaviour of LHRH and its analogues loaded into cation-
exchange fibers and their potential in transdermal delivery are still needed. Also several
limitations and problematic issues have to be solved in order to successfully transport LHRH
peptides across the skin in vitro using an approach that combines ion-exchange fibers and
iontophoresis. Most importantly, precise and sensitive analytical methods for the
quantification of the three investigated peptides have to be developed.
47
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