diva portal161065/fulltext01.pdfmucoadhesive properties of the polymer in the gel and by the...

Dissertation for the Degree of Doctor of Philosophy (Faculty of Pharmacy) inPharmaceutics presented at Uppsala University in 2001

ABSTRACT

Paulsson, M., 2001. Controlled Release Gel Formulations for Mucosal Drug Delivery.Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 259. 52 pp. Uppsala. ISBN 91-554-5173-X.

Drug delivery to nasal or ocular mucosa for either local or systemic action faces manyobstacles – these routes are protected by effective mechanisms. Gel formulations withsuitable rheological and mucoadhesive properties increase the contact time at the site ofabsorption. However, drug release from the gel must be sustained if benefits are to begained from the prolonged contact time. The work presented here is the characterization of gels and the determination of themucoadhesive properties of polymers using rheology. Gelrite gels were formed insimulated tear fluid at concentrations of polymer as low as 0.1%, and it was shown thatsodium was the most important gel-promoting ion in vivo. Rheology, although it may bea questionable technique for evaluating mucoadhesive properties of polymers, showedthat interactions between mucin and polymers were most likely to be seen with weakgels. It was possible to control the release of uncharged drug substances by includingsurfactants that form micelles in the gel. This release depended on lipophilicinteractions between the drug and the polymer and/or the micelles. Controlled-releaseformulations of charged drugs could be designed by mixing the drugs with oppositelycharged surfactants in certain ratios. In this way, vesicles in which the drug andsurfactant constituted the bilayer formed spontaneously. The vesicle formation wasaffected by the presence of polymer, and very small vesicles that gave a slow releaserate were formed when a lipophilically modified polymer was used. The gels were also evaluated in the Ussing chamber using porcine nasal mucosa. Therate of transport of drugs through the mucosa could be controlled by the rate of releasefrom the formulation. Furthermore, the Ussing chamber could be used to evaluate thepotential toxicity of formulations.

Mattias Paulsson, Department of Pharmacy, Uppsala Biomedical Centre, Box 580,SE-751 23 Uppsala, Sweden

© Mattias Paulsson 2001

ISSN 0282-7484ISBN 91-554-5173-XPrinted in Sweden by Uppsala University, Tryck & Medier, Uppsala 2001

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Contents

PAPERS DISCUSSED 7

ABBREVIATIONS 8

1. INTRODUCTION 9

2. AIMS 11

3. MATERIALS AND METHODS 12

3.1 Materials 123.1.1 Drugs, model substances and radiolabelled markers 12

3.1.2 Polymers 12

3.1.3 Surfactants 12

3.1.4 Other chemicals 12

3.2 Preparation of samples 133.2.1 Preparation of Carbopol gels 14

3.2.2 Preparation of Gelrite gels 14

3.2.3 Mucin mixtures 14

3.3 Rheology 153.3.1 Temperature scans 15

3.3.2 Evaluation of mucoadhesion 15

3.4 Determination of micellar partition coefficient 16

3.5 Drug release measurements and diffusion coefficient calculation 16

3.6 Cryo-TEM and surface tension measurements 18

3.7 The horizontal Ussing chamber 183.7.1 Tissue preparation 18

3.7.2 Viability measurement 18

3.7.3 Drug transport 19

3.7.4 Histology 19

3.8 Statistical analysis 19

6

4. GELATION OF GELRITE GELS 20

4.1 Effect of ionic content 21

4.2 Temperature scans 23

5. MUCOADHESION 25

5.1 Mechanism and theory of bioadhesion 25

5.2 Methods of characterizing mucoadhesion 26

5.3 Mucin interactions with Carbopol 934 and Gelrite 27

6. INTERACTIONS BETWEEN POLYMER AND DRUG 30

6.1 Polymers used in the formulations 30

6.2 Effects on gel strength 30

6.3 Effects on drug release 32

7. INTERACTIONS BETWEEN POLYMER, DRUG AND NON-IONIC SURFACTANTS 34

7.1 Interactions between drug and surfactant 34


8. INTERACTIONS BETWEEN POLYMER, DRUG AND IONIC SURFACTANTS 36

8.1 Interaction between drug and surfactant 36


9. EVALUATION OF GELS IN THE USSING CHAMBER 40

9.1 The evaluated formulations 40

9.2 Transport over porcine nasal mucosa 41

10. CONCLUSIONS 43

ACKNOWLEDGEMENTS 44

REFERENCES 46

7

Papers discussedThis thesis is based on the following papers, which will be referred to by roman numeralsin the text:

I Paulsson, M., Hägerström, H., Edsman, K. Rheological Studies of theGelation of Deacetylated Gellan Gum (Gelrite®) in Physiological Conditions.Eur. J. Pharm. Sci. 1999, 9, 99-105. Reproduced with permission. © 1999 Elsevier Science.

II Hägerström, H., Paulsson, M., Edsman, K. Evaluation of Mucoadhesion forTwo Polyelectrolyte gels in Simulted Physiological Conditions Using aRheological Method. Eur. J. Pharm. Sci. 2000, 9, 301-309. Reproduced withpermission. © 2000 Elsevier Science.

III Paulsson, M., Edsman, K. Controlled drug release from gels using surfactantaggregates: I. Effect of lipophilic interactions for a series of unchargedsubstances. J. Pharm. Sci. 2001, 9, 1216-1225. Reproduced with permission. © 2001John Wiley & Sons

IV Paulsson, M., Edsman, K. Controlled drug release from gels using surfactantaggregates: II. Vesicles formed from mixtures of amphiphilic drugs andoppositely charged surfactants. Pharm. Res. 2001, 18, 1585-1591. Reproducedwith permission. © 2001 Plenum Publishing Corp. Kluwer Academic.

V Paulsson, M., Edsman, K. Lipophilic interactions of amphiphilic drugs withsurfactants and polymers for controlled drug release from gels. Submitted.

VI Östh, K., Paulsson, M., Björk, E., Edsman, K. Evaluation of controlled drugrelease from gels using pig nasal mucosa in the horizontal Ussing chambermodel. Manuscript

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AbbreviationsPAA Poly(acrylic acid)LM lipophilic modificationLM-PAA Lipophilically modified poly(acrylic acid)C1342 Carbopol 1342, cross-linked PAA with lipophilic modificationC934 Carbopol 934, cross-linked PAAC940 Carbopol 940, cross-linked PAAC981 Carbopol 981, cross-linked PAAPVP polyvinylpyrrolidoneCMC carboxymethylcelluloseGlcp glucoseGlcpA glucuronic acidRhap rhamnose

BSMG mucin from bovine submaxillary glandsPSI mucin from porcine stomach

B58 Brij 58SDS Sodium Dodecyl Sulphate

DHA dihydroalprenololTS testosteroneHC hydrocortisone

TFR Tear fluid ratioSTF Simulated tear fluidcft cumulative fraction transported

pm micellar partitionLog D logarithm of the distribution coefficientLog P logarithm of the partition coefficientPapp apparent permeability coefficientD diffusion coefficientG elastic (storage) modulusG viscous (loss) modulus� shear viscosity� angular frequency

USP United States Pharmacopeia

1. Introduction

9

1. Introduction

“The colloidal condition, the gel, is one which iseasier to recognize than to define.”

Dorothy Jordan Lloyd [1].

A gel is a soft, solid or solid-like material consisting of two or more components, one ofwhich is a liquid, present in substantial quantity. A gel should, on a time scale of seconds,not flow under the influence of its own weight. The solid-like characteristics of gels can bedefined in terms of two dynamic mechanical properties: an elastic modulus, G (�), whichexhibits a pronounced plateau extending to times at least of the order of seconds; and aviscous modulus, G (�), which is considerably smaller than G (�) [2].

The term “hydrogel” was introduced by Thomas Graham in 1864 to denote silicic acidhydrates with gelatinous properties [3]. The first biological uses of gels (polymerizedmethylmethacrylate) were presented by the Institute for Macromolecular Chemistry inPrague in 1960 and involved the manufacturing of contact lenses, arteries etc. [4].

Gelation occurs via the cross-linking of polymer chains, something that can be achieved by(i) covalent bond formation (chemical cross-linking) or (ii) non-covalent bond formation(physical cross-linking). Examples of non-covalent bonds are hydrogen bonds and thecross-linking of polyelectrolytes by inorganic ions [5].

Gels have been used for the delivery of drugs for both systemic and local action, see thereview by Peppas et al. [6]. Many different methods using gels have been reported,including subcutaneous delivery for sustained release [7, 8], buccal delivery [9], delivery tothe stomach [10-14], colon [15, 16], rectum [17-20] and vagina [21].

This thesis focuses on mucosal drug delivery, exemplified by the nasal and ocular routes.The principles for controlled release also apply, of course, to other routes.

The nasal cavity is an irregularly shaped space and its folded structure results in a surfacearea of approximately 150 cm2. It is covered with a mucous membrane, which is suppliedwith viscid mucus by the goblet cells. The epithelium is ciliated, and the rapid mucociliartransport makes the mucosa an effective barrier to drug delivery [22]. Nasal administrationby gels has been studied for roxithromycin [23], insulin [24], calcitonin [24] and nifedipine[25].

Bioavailability following ocular delivery is often very low, typically 1% or less, dependingon the physicochemical properties of the drug [26]. The low bioavailability is caused byextensive precorneal drug loss by nasolacrimal drainage, and by the low drug permeabilityof the cornea [27]. The rapid elimination of the instilled drug often results in a shortduration of the therapeutic effect and, consequently, the need for frequent doses. Gels havebeen studied for ophthalmic administration of several drugs, including pilocarpine [28, 29],timolol [30], methylprednisolone [31], �-adrenoceptor blockers [32] and oligonucleotides[33].

CHAPTER 1. INTRODUCTION

10

Gel formulations with suitable rheological properties increase the contact time with themucosa at the site of absorption [23, 34-39]. The increased contact time is caused by themucoadhesive properties of the polymer in the gel and by the rheological properties of theformulation reducing the clearance by the nasal and ocular protective mechanisms.

Although many gels exhibit shear-thinning behaviour (pseudoplasticity), they cannot beeasily delivered to the nose using a normal nasal spray device. It is also difficult to apply agel to the eye. Formulations that gel upon contact with the mucosa, known as“environmentally responsive polymers” or “in situ gel-forming polymers” have beenevaluated for both the nasal and ocular routes. The phase transition can be induced by ashift in pH, (as, for example, for cellulose acetate phtalate [40]), a shift in temperature (asfor the thermogelling Poloxamer 407 [28, 41], EHEC/ionic surfactant mixtures [30]) or bythe presence of cations (as, for example, for deacetylated gellan gum [42]).

The rheological and mucoadhesive properties of a gel may give rise to a long residencetime, but this is only advantageous if the drug remains in the formulation and is releasedthroughout the complete period.

The viscosity of a solution may increase dramatically in the presence of a polymer, but thetransport conditions for a small drug molecule are apparently the same as they are in water.This phenomenon has been demonstrated for drugs in 1% agarose [43]. Themacromolecular nature of the polymers may result in their effects on macroscopic motion(flow) differing from their effects on the microscopic motion (diffusion). Flynn et al.defined two terms that describe this: “macroviscosity” and “microviscosity” [44]. Thedifferences between these two viscosities have been evaluated in pharmaceutically relevantconditions (PVP and CMC solutions) [45].

There are several ways to sustain the release of a drug from gels in order to take fulladvantage of the contact time. The drug can be dispersed in the gel, giving a concentrationthat is higher than that corresponding to the solubility of the drug [46, 47], formulated asparticles [48, 49], distributed in liposomes [50, 51], formulated in biphasic gels in a mannerthat gives zero-order release [52], interacting with cyclodextrin [53, 54] or with an oil phasethat has been included in the gel [55]. Interactions between the drug and the polymer canalso be used as will be described in Chapter 6, or surfactants can be added to theformulation and form micelles or vesicles as will be further described in Chapters 7 and 8.

This thesis describes the evaluation of pharmaceutically appropriate materials, most ofthem approved for human use, in physiologic conditions with the aim of designing optimalgel formulations.

2. Aims

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2. Aims

� To study how the polymer concentration and salt content influence gel formulations byevaluating the viscoelastic and mucoadhesive properties of ion-sensitive polymers usingrheology. Papers I-II.

� To discover ways of controlling the release of drugs from gels by using interactionsbetween the drug and the polymer and/or added surfactants. Papers III-V.

� To evaluate gel formulations on porcine nasal mucosa in the horizontal Ussing chambermodel. Paper VI.

CHAPTER 3. MATERIALS AND METHODS

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3. Materials and Methods3.1 Materials

3.1.1 Drugs, model substances and radiolabelled markersAlprenolol hydrochloride, atenolol, diphenhydramine hydrochloride, metoprolol tartrate,ethylparaben, butylparaben, p-hydroxybenzoic acid, chlorpromazine hydrochloride,tetracaine, lidocaine, orphenadrine hydrochloride, amitriptyline hydrochloride, testosteroneand hydrocortisone were purchased from Sigma (St. Louis, MO, USA). Methylparaben andpropylparaben were purchased from Apoteket AB (Stockholm, Sweden). Fluvastatinsodium and dihydroalprenolol hydrochloride were kindly provided by AstraZeneca(Mölndal, Sweden). Betaxolol was a gift from Alcon (Stockholm, Sweden). Fig. 1 showsthe structures of drugs and model substance.3H-hydrocortisone, >97% pure, 3H-dihydroalprenolol hydrochloride, >97% pure, and 3H-testosterone, >97% pure, with 1.0 mCi/ml, were purchased from NEN Life ScienceProducts (Boston, MA, USA).

3.1.2 PolymersPoly(acrylic acid) polymers with the proprietary names Carbopol 934P (C934), Carbopol940NF (C940), Carbopol 981 (C981) and Carbopol 1342NF (C1342) were gifts from BFGoodrich (Brecksville, OH, USA). The manufacturer states that C934 gel has the lowestcross-linking density, while that of C981 is intermediate and that of C940 is the highest.C1342 has a lipophilic modification consisting of an alkyl acrylate chain of C10-C30.

Deacetylated gellan gum (Kelcogel F), also called Gelrite, was a gift from the Kelcodivision of the Monsanto Company (San Diego, CA, USA).

3.1.3 SurfactantsBrij 58 (polyoxyethylene 20 cetyl ether), Brij 700 (polyoxyethylene 100 stearyl ether),sodium dodecyl sulphate (SDS) and benzalkonium in the form of the pure homologuebenzyldimethyldodecyl-ammonium bromide (BAB) were purchased from Sigma (St. Louis,MO, USA). Pluronic F-127 was bought from BASF (Parsippany, NJ, USA).

3.1.4 Other chemicalsTwo mucins were used: type III (PSI, partially purified mucin from porcine stomach) andtype I-S (BSMG, bovine submaxillary gland mucin). These were purchased from SigmaChemical Co. (St. Louis, MO, USA). All other chemicals were from Sigma, and ofanalytical or “ultra” quality. Ultra-pure water, prepared using a MilliQ Water PurificationSystem (Millipore, Molsheim, France), was used in all preparations. DPX mounting liquidwas purchased from BDH laboratory supplies (Poole, UK). The scintillation fluid Aquasafe300 plus (Zinsser Analytic) was purchased from Scintvaruhuset, Uppsala, Sweden.

3.2 Preparation of Samples

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Fig. 1 Structure of drugs and model substances used in the thesis.

3.2 Preparation of samples

The samples in Papers I-V were prepared in simulated tear fluid and the samples inPaper VI were prepared in Krebs Ringer (KR) buffer. The composition of simulated tearfluid was adopted from a tear fluid analysis [56] using 8.3 g NaCl, 0.084 g CaCl2�2H2O,1.4 g KCl in 1 liter of ultra-pure water. This is equal to 142 mM Na+, 19 mM K+ and0.6 mM Ca2+. The KR (from Sigma) was supplemented with NaHCO3 (15 mM) and CaCl2(1.2 mM). The osmolarity was adjusted to 300 ± 5 mmol/kg by the addition of NaCl to a

NH

OOH

NH

OOH

O

H

H H

OHOH

O

OH

O

OH

H

H H

CH3

CH3

NH

O

NCH3

CH3 S

N N(CH3)2

Cl

N(CH3)2

NH2

O

NH

OOH

ON(CH3)2

NH

CH3

ON(CH3)2

O

CH3

O

N(CH3)2

N

F

OHOH

COOH

NH

OOH

O

O

O

OHNH

O

OH

OR

dihydroalprenolol alprenolol hydrocortisone

orphenadrine

amitriptylinechlorpromazinelidocaine

testosterone

tetracaine

fluvastatin betaxolol atenolol

diphenhydramine metoprolol paraben esters

paraben estersa) R=Hb) R=CH3c) R=CH2CH3d) R=CH2CH2CH3e) R=CH2CH2CH2CH3


14

final concentration of 138 mM. The buffer was oxygenated using a mixture of O2 (95%)and CO2 (5%) (AGA Gas AB, Sundbyberg, Sweden).

3.2.1 Preparation of Carbopol gelsCarbopol was delivered from BF Goodrich as dry flocculated powders consisting ofprimary particles with an average diameter of around 0.2 �m. The flocculated powders(average size 2-7 �m) cannot be broken down into the ultimate particle once produced.Each primary particle contains a network of cross-linked polymer chains and will swell upto 1000 times their volume in contact with water to form a continuous gel when exposed toa suitable pH (according to manufacturer). In the work described in Papers I-V, the poly-mer powder was dispersed in simulated tear fluid containing the dissolved model drug andin some systems also surfactants. The dispersions were then stirred using magnetic stirringbars for approximately 1 h at room temperature and 1 M or 2 M NaOH, depending on thepolymer concentration, was subsequently added to neutralize the sample to pH 6.5–7.

A heavy precipitation formed in some formulations, and in these cases it was necessary toprepare the solutions of drugs and surfactants and then mix them in the ratio 1:1 withneutralized gel.

All gels were allowed to equilibrate for at least 16 h at room temperature. The pH of thegels was then adjusted to pH 7.4, simulated tear fluid was added to achieve the final desiredvolume and the gels were left for at least 90 minutes before measurement commenced. Thepolymer content of all Carbopol gels was 1% (w/w) except where otherwise stated. Thegels used in the work described in Paper VI were prepared in a slightly different manner –stock gels with double concentration were mixed with drug solutions in KR.

3.2.2 Preparation of Gelrite gelsThe polymer powder was dispersed in ultra-pure water, which contained dissolvedsurfactant for some systems. The dispersions were then stirred for 20 minutes at 100°C in awater bath and then cooled to room temperature. Drugs and surfactants were added, whenincluded, during the cooling process. The solutions were allowed to equilibrate for at least16 h. The polymer content was 0.5% (w/w), except where otherwise stated.

3.2.3 Mucin mixturesThe mucin concentration in all mixtures was 4% (w/w). Mucin was added to Carbopol gels(0.25-1.5%) together with the polymer, and the dispersions were stirred and neutralized asdescribed above. Mixtures of mucin with Gelrite gels (0.25-0.75%) were prepared bymixing the freshly prepared Gelrite solutions at 65�C with dispersed and neutralized mucinand then equilibrating at 4�C overnight.

3.3 Rheology

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3.3 Rheology

Rheological measurements were carried out using a Bohlin VOR Rheometer (BohlinReologi, Lund, Sweden), a controlled rate instrument of couette type [57]. The measuringsystem used was a concentric cylinder (C14) or a parallel plate (PP30). Silicone oil wasadded to the surface of the samples to prevent evaporation during measurements.

Carbopol gels were centrifuged at 1,500-2,000 rpm before measurement in order to removeentrapped air, and then placed into the C14 cup or between the PP30 plates. In the lattercase, the separation of the plates was adjusted to give the desired gap width.

Gelrite gels were characterized by pouring the hot, freshly prepared sample solutions intothe measuring system, which was also at a raised temperature. The samples were then keptat 90°C for an additional 20 minutes, cooled to 5° and then heated to 35°C wheremeasurements were performed. The highest temperature used for mixtures containingmucin was 65°C.

The oscillation frequencies used were 0.001-2 Hz and the strain was within the linearviscoelastic region. Shear rates of 0.1-2,000 s-1 where used during measurements ofviscosity.

3.3.1 Temperature scansThe temperature dependence of the viscoelastic properties was studied by conductingtemperature scans over the range from 90°C to 5°C. The heating and cooling rates were0.5°C/min. The sample was kept at 90°C for 20 minutes before scanning down to 5°C. Itwas kept at this temperature for 90 minutes, and then swept back up to 90°C. The elastic(G ) and viscous (G ) moduli were measured at intervals of 5 minutes during an oscillationfrequency sweep (0.05-2 Hz). The applied strain was determined by an automatic function(“auto strain”) of the instrument that determined the smallest deformation that yielded adetectable signal without destroying the structure of the material.

Measurements were also carried out to ascertain that the applied strain was within the linearviscoelastic region of the non-melted samples. The automatic function for determiningstrain was not used in the temperature scans described in Papers III-V. In these cases, lowamplitudes (within the linear viscoelastic region) were used throughout the measurement.

3.3.2 Evaluation of mucoadhesion Values of the elastic modulus (G') measured at 35°C were used to evaluate the interactionsbetween the polymer and mucin. This interaction was described by a term called the“interaction term”, denoted by �G'. This term was calculated from

'''' GGGG mpmix �� (1)

where G'mix is the elastic modulus of the mixture and G'p and G'm are the elastic modulus ofpolymer and mucin, respectively. �G' is the elastic component arising from the interactionbetween the polymer and mucin. The elastic modulus of mucin solutions was negligibly


16

small, both in water and in simulated tear fluid. Therefore the equation was simplified and,instead, �G' was calculated from

''' GGG pmix �� (2)

This simplification has also been made by Rossi et al. [58], and by other researchers.

The elastic modulus determined at the intermediate frequency of 1 Hz was used in thecalculations, since all samples had no or only a small frequency dependence and G' wasmeasured with sufficient accuracy at this frequency.

3.4 Determination of micellar partition coefficient

The aqueous solubilities of drugs and their solubilities in the presence of micelles weredetermined by placing excess amounts of the drugs in simulated tear fluid with or without1% Brij 58 and leaving the samples to equilibrate at 35�C for at least 48 hours. The sampleswere then centrifuged and the UV-absorbance of the supernatant measured.

The fraction of solute in the micellar phase, pm, can be expressed as follows:

wm

w

wm

mm n

nnn

p��

�� 1 (3)

where nm+w is the total amount of drug, nw is the amount of drug in the aqueous phase andnm is the amount of drug in the micelles. The first two (nm+w and nw) can be estimated bymeasuring solubility in the presence of micelles and the aqueous solubility respectively andby assuming that the free fraction of drug in the solubilization determination is equal to theaqueous solubility, pm can be calculated. It is assumed that pm is not affected when theconcentration of drug is lowered to that used in the release experiments.

3.5 Drug release measurements and diffusion coefficient calculation

Drug release from the gels was measured by the USP paddle method using gel containerswith a fixed volume of 6 cm3 and a surface area of 21 cm2, covered by a coarse mesh-sizeplastic net and a stainless steel net. The containers were immersed in 250 mL simulated tearfluid at 35�C and stirred at 20 rpm in a Pharma Test PTW II USP bath (Pharma TestApparatebau GmbH, Hainburg, Germany). The stirring rate was chosen to give adequateconvection and minimize surface erosion of the gels. In the drug release measurementspresented in Paper III, 1 mL samples of the dissolution medium were manually collectedand analyzed at 5 min intervals for the first hour and then for every half hour during the lastfive hours. For each removed sample, 1 mL of fresh medium was added in order to keep thevolume of the dissolution medium constant. Plastic utensils were avoided in order to

3.5 Drug Release Measurements

17

prevent lipophilic substances from being ab-/adsorbed to the utensils. The concentration ofmodel drug in the dissolution medium was determined by spectrophotometry using aUnicam UV4 spectrophotometer (Unicam Ltd, Cambridge, UK).

Papers IV-VI describe on-line measurements of the concentration that were performed bycontinuously pumping the dissolution media through a, Shimadzu UV-1601spectrophotometer (Shimadzu, Kyoto, Japan). The absorbance was measured every 150seconds for the first 45 minutes, then at 65 minutes and finally every 30 minutes until thelast measurement was made 6 hours after the first one.

Light-scattering of polymers released from the gel was compensated for by measuring theabsorbance during a drug-free release experiment. This compensation was primarily for thevery late hours since during the first hour of measurement this effect was negligible and didnot contribute to the calculated diffusion coefficient.

Paper VI describes experiments in which the release of testosterone could not be detectedusing spectrophotometric methods. In this case, radiolabelled testosterone was used in thegel, and the levels of radioactivity of samples withdrawn from the receiver media weremeasured at the above-specified time intervals. Measurement of radioactivity is describedin section 3.10.3.

One-dimensional Fickian diffusion from a gel holder under sink conditions and during theinitial part of the release can be expressed as:

21

02 �

�

�

�

�

��

�

DtCQ (4)

where Q is the amount of drug released per unit area, C0 is the initial concentration of drugin the gel, D is the diffusion coefficient of the drug in the gel and t is the time after the startof the release experiment. The equation is valid for the first 60% of the release [59, 60]. Inthe experiments described here, the gel was placed in a confined space and was not allowedto swell during the experiment. The amount of drug released plotted against the square rootof time is a straight line during the initial period, and the diffusion coefficient can becalculated from the slope of this graph.

The diffusion of a drug substance in solution with micelles present can be described by theequation:

mmff pDpDD �� (5)

where D is the experimentally measured diffusion coefficient, Df is the diffusion coefficientfor the drug substance in the aqueous phase and Dm is the diffusion coefficient for the drugtransported by micelles. Dm is equal to the diffusion of the micelle. pf and pm are thefractions of drug in the aqueous and micellar phases, respectively. This equation has beenadopted from studies of the diffusivity of charged surfactants [61, 62].


18

The size of a diffusing particle can be estimated from the Stokes-Einstein equation:

DkTRh��6

� (6)

where Rh is the hydrodynamic radius, k is the Boltzmann constant, T is the temperature, � isthe viscosity and D the diffusion coefficient.

3.6 Cryo-TEM and surface tension measurements

Cryogenic transmission electron microscopy (Cryo-TEM) was used to visualize drug-surfactant aggregates both in polymer-free solutions and in gels. A small drop of the samplewas deposited onto a grid covered by a polymer film, the excess liquid was blotted off withfilter paper and the sample remaining on the grid was vitrified in liquid ethane. The filmswere transferred to a Zeiss EM 902 transmission electron microscope and kept below –165 °C during the viewing procedures. All observations were made in the zero-loss bright-field mode at an accelerating voltage of 80 kV. Details of the method are describedelsewhere [63].

A duNoüy 8551 tensiometer (Krüss, Hamburg, Germany) was used to measure the surfacetension of drug solutions in simulated tear fluid after 10 minutes of equilibration at roomtemperature. The critical micelle concentration (CMC) was obtained from plots of thesurface tension against the logarithm of drug concentration.

3.7 The horizontal Ussing chamber

The horizontal Ussing chamber system contains six chambers, which are placed side-by-side on a water-heated block. The buffer on the receiver side was oxygenated andmaintained at 37�C, and stirred by placing the equipment on a circular shaker set at145 rpm. Immediately before inserting the mounting ring, 1.2 ml of cold, preoxygenatedKR was added to the receiver side, and an equal amount was added to the donor side afterinsertion. The setup is described in more detail in Östh et al. 2001 [64].

3.7.1 Tissue preparationNasal respiratory mucosa from 6-month-old domestic pigs was isolated at the local abattoir(Swedish Meats, Uppsala, Sweden). Each piece of mucosa was placed in KR on ice duringtransport to the lab. Circular pieces of the mucosa were placed into the mounting rings ofthe Ussing chamber resulting in an exposed surface area of 0.55 cm2 (� 8.4 mm). Thetissue was mounted in the chamber with the mucosal side upwards.

3.7.2 Viability measurementThe viabilities of the tissue before and after transport experiments were investigated bymeasuring several electrophysiological parameters; transmucosal electrical resistance (R),

3.8 Statistical Analysis

19

potential difference (PD) and short-circuit current (Isc) [65]. Chamber tops were closedduring the electrophysiological measurements, and open during the transport studies. Thevalues of the electrophysiological parameters reached a plateaux after 60 minutes. Mucosaewith stable profiles approaching plateaux at PD � -1 mV and Isc � 30 �A/cm2 were includedin the experiments. These electrophysiological criteria were set to distinguish betweenviable and non-viable mucosae [64].

3.7.3 Drug transportDrug transport across the mucosae was studied by removing the KR on the donor side andadding 100 �L of drug solution or gel to the mucosae. Samples (100 µL) were taken fromthe receiver solution and replaced with equal amounts of KR at 37�C before the addition ofthe drug formulation and at 5, 10, 15, 30, 45, 60, 75 and 90 minutes after application. Thesamples were then transferred to scintillation vials, shaken with 10 ml scintillation fluid andstored in darkness for 30 minutes before measuring the radioactivity in a liquid scintillator(Tri-Carb® Liquid Scintillation Analyzers, Model 1900CA, Packard Instrument Company,Downers Grove, IL, USA).

The apparent permeability coefficients (Papp) were calculated from:

AVkP R

app�

�

�

60 (7)

where k is the transport rate (determined from the slope obtained by linear regression of thefraction of drug absorbed drug as a function of time), VR is the volume in the receiverchamber and A the exposed surface area of the mucosa.

3.7.4 HistologyPieces of mucosa were removed from the mounting ring, rinsed with KR and placed inembedding cassettes in Bouin’s solution overnight, then stored in 4% formaldehydesolution in PBS until required. A piece of about 8 � 5 mm was then cut out from onerandomly chosen mucosa from each group in the transport study, dehydrated in EtOH andthen embedded in Historesin (Leica Microsystems, Nussloch, Germany). Thin slices (3 �m)were cut on a Leica RM 2165 (Leica Microsystems, Nussloch, Germany) microtome andstained by the following procedure: hematoxylin (5 min), tap water (10 sec), eosine (3min), 95% ethanol (2 � 10 sec), 99.5% ethanol (2 � 1 min), xylene (10 sec) and xylene (1min). Specimens were then mounted in DPX and observed and photographed in a lightmicroscope.

3.8 Statistical analysis

Results in Paper VI are given as means � standard errors of the mean (S.E.M.). Statisticalcomparisons were made using Student´s t-test (P< 0.05). The 95 % confidence interval wascalculated for the slope of the line when the fraction released was plotted as a function oft0.5. Data presented in the drug release figures are mean ± standard deviation for n=3 exceptwhen otherwise stated.


20

4. Gelation of Gelrite GelsGellan gum is a linear, anionic heteropolysaccharide secreted by the microbeSphingomonas elodea (formerly known as Pseudomonas elodea). The polysaccharide canbe produced by aerobic fermentation and then isolated from the fermentation broth byalcohol precipitation. The polymer backbone consists of glucose, glucuronic acid, andrhamnose in the molar ratio 2:1:1 [66]. These are linked together to give a tetrasacchariderepeat unit (Fig. 2). The native polysaccharide is partially esterified with L-glycerate andacetate [67] but the commercial product Gelrite has been completely de-esterified by alkalitreatment [68].

O

CH3

OH OH

O

CH2OH

OH

OH

OO

O

O

OH

OH

COOH

O

CH2OH

OH

OH

O

n

[�3)-�-D-Glcp-(1�4)- �- D -GlcpA-(1�4)- �- D - Glcp- (1�4)- �- L-Rhap-(1�]n

Fig. 2 The structure of deacetylated gellan gum.

Gelrite has been granted regulatory approval as a pharmaceutical excipient and is marketedby Merck in a controlled-release glaucoma formulation called Blocadren® Depot(Timoptic-XE®).

Formulations with the in situ gel Gelrite can be administered to nasal or ocular mucosa as alow-viscosity solution. On contact with cations in tear fluid or nasal secretion theformulation will form a clear gel [42, 69]. This is caused by cross-linking of the negativelycharged polysaccharide helices by monovalent and divalent cations (Na+, K+, Ca2+). Severalmodels have been presented to explain gellan gum gelation [70, 71]. The model proposedby Robinson et al. will be discussed in more detail here and it will be partiallysupplemented to describe a more pharmaceutically relevant situation. In an ion-freeaqueous medium, Gelrite forms double helices at room temperature (sol in Fig. 3). Thissolution has a viscosity close to that of water and the helices are only weakly associatedwith each other (by van der Waals attraction). When gel-promoting cations are presentsome of the helices associate into cation-mediated aggregates, which cross-link the polymer(gel1). On heating the polysaccharide in an ion-free environment, the polysaccharidebecomes a disordered coil. However, on heating a sample with cations present, the non-aggregated helices melt out first, and the aggregated helices (gel2) melt out at a highertemperature in a second transition [70].

Paper I describes experiments in which the capacities of the three different ions present intear fluid (Na+, K+, Ca2+) to cross-link Gelrite and form a gel were examined. The effectson the gel strength of varying the ionic and macromolecular content were also investigated.

The ionic content in the preparations described in Paper I was varied keeping theproportions of the different ions, Na+:K+:Ca2+, constant.

4.1 Effect of ionic content

21

coil

sol

gel1

gel2

Fig. 3 Model for the formation of Gelritegels on addition of cations (�). Modifiedfrom Robinson et al., reference [70].

The tear fluid ratio, TFR, is defined as the quotient:

� �

� �fluidtearinpresentionssampleinpresentionsTFR �

Thus, for example, in a solution with TFR = 2, all ions are present at twice theconcentration that they have in tear fluid. The concentrations of cations in tear fluid are:142 mM Na+, 19 mM K+ and 0.6 mM Ca2+ [56].

4.1 Effect of ionic content

Fig. 4 shows the elastic modulus, G , and the phase angle, �, for 0.5% Gelrite as a functionof ion concentration (described by TFR). G increases substantially as the ionic contentincreases, reaching a plateau at a tear fluidratio of 0.5, i.e., when the amount of ionspresent is half that of tear fluid. Between atear fluid ratio of 1 and 2, G and � werealmost constant.

At TFR values above two, the gel strength(seen as the elastic modulus) decreases andthe gels become turbid. This agrees withprevious observations and Morris et al.proposed that insoluble aggregates ofpolysaccharide will act as heterogeneousnuclei leading to the growth of microgelswhich interconnect to form a weakened gelnetwork [72]. The polysaccharide is less solubleat high salt concentrations, even at high

Tear fluid ratio

0 1 2

G'(

Pa)

10-2

10-1

100

101

102

103

104

105�

(deg

rees

)

0

10

20

30

40

50

60

70

80

90

Fig. 4 The elastic modulus, G ( ), and phaseangle, � (�), at 1 Hz of 0.5% Gelritesamples as a function of added ions.

CHAPTER 4. GELATION OF GELRITE GELS

22

temperatures, and Morris et al. suggested that thehelices clustered into progressively larger aggregates (that scatter light) as the ionicconcentration increases leading to the profile seen in Fig. 4 [73]. We have also observedthat G of 0.5% Gelrite� in a solution containing only Na+ ions decreased at high Na+

concentrations. At 426 mM, the elastic modulus was only 20% of that of a solutioncontaining Na+ at 142 mM (corresponding to Na+ in TFR = 1). The turbidity of the 426 mMNa+ sample was high and melted samples were granular, indicating that microgelaggregates are present. The high turbidity has been attributed tothe larger refractive index of the junction zones, which are moredense and more orderly packed than the more randomlydispersed gellan coils [74]. Images of networks andaggregates of gellan gum have been obtained using atomicforce microscopy [75].

In the presence of monovalent cations such as potassium,pairs of gellan double helices are aligned in an antiparallelfashion with their helix axes 9 Å apart. The helices areconnected by strong carboxylate��K+

��water��K+��

��carboxylate interactions [76]. When divalent ions suchas calcium are present, the bridge is replaced by a singleCa2+ ion so that the helices are linked by carboxy-late��Ca2+

��carboxylate interactions (Fig. 5). This is clearlystronger than the potassium/water bridge.

Divalent cations produce stronger Gelrite gels than monovalent cations since fewer ions areneeded for cross-linking. K+ is also more effective in promoting ordered structures than Na+

[77-79]. Potassium has a larger ionic radius but a weaker hydration than sodium, as isdescribed by the relative positions of these elements in the Hofmeister series [81]. Thismeans that the hydrated potassium ion is smaller than the hydrated sodium ion, and thisexplains the higher efficiency of potassium in promoting ordered structures. It is possiblethat the packing of the chains in the junction zones requires small hydrated ions forgeometrical reasons [77]. 1.0% Gelrite gels have an elastic modulus of 100 Pa when formed

by 75 mM NaCl, 50 mM KCl or 4 mMCaCl2 in the study by Miyoshi et al. [79].

Tear fluid is composed of a relatively smallproportion of the potent gel-promotingdivalent cations and a relatively largeproportion of monovalent ions. Thecontribution of K+ and Ca2+ to the gelstrength was almost negligible at 35ºC andthe elastic modulus of the tear fluid gel wasapproximately equal to that obtained withNa+ alone, see Table 1.

Fig. 6 shows how gel strength depends onpolymer concen-tration. All gels were

Fig. 5 A computergenerated space-filling model.Carboxylate oxygenatoms (black) of theleft gellan helix (darkgrey) are stronglyconnected to those ofthe right gellan helix(light grey) bycalcium ions (white).Prof. Chandra-sekaran and Dr.Janaswamy ofPurdue University,kindly provided thefigure.

Polymer concentration (%)

0 1 2

G'(

Pa)

102

103

104

105

Fig. 6 The elastic modulus, G , at 1 Hz of Gelritesamples prepared in STF as a function ofpolymer concentration.

4.2 Temperature Scans

23

prepared in simulated tear fluid, TFR = 1. Even at the lowest concentration tested(0.1%), a gel (however easily broken) with a frequency independent G was formed (phaseangle < 10�), while increasing the concentration to 0.25% produced a very firm gel withG =1500 Pa.

4.2 Temperature scans

Rheological thermal scans have beenwidely used to characterise thegelation of Gelrite [81-83]. Thistechnique can be used, as differentialscanning calorimetry (DSC) also can,to see transitions such as melting inthe material when it is heated orcooled at different rates.

Table 1 The elastic modulus, G , and phaseangle, �, for 0.5% Gelrite gels prepared in tearfluid concentration of a single ion (Ci) and intear fluid. Mean � standard deviation, n=3.

Ionpresent

Ci(mM)

G(Pa)

�

(�)K+ 19 0.34�0.05 25�4.5Na+ 142 8800�750 2.3�1.0Ca2+ 0.6 21.00�0.02 5.5�4.0Tear fluid - 10600 1.8

Fig. 7 shows how the elastic modulus is affected by heating and cooling. The heating andcooling curves do not superimpose and a thermal hysteresis is evident. The gel was notremelted since the temperature in the rheometer was only 90�C and the second cooling andheating curves were different from the first. The third cycle, however, was almost identicalto the second (data not shown). Thermal hysteresis has been observed by others [79, 84,85]. The heating curve in Fig. 7 shows that the gel starts to melt in the same temperatureinterval as that it was formed. A further transition occurs at 60�C. The elastic modulus wasshifted one decade higher, indicating formation of a different kind of structures in the gel.This unexpected phenomenon has also been reported by others [84]. Two-step changesduring heating have been discussed Morris et al., and attributed to an initial melting ofunaggregated helices accompanied by an enhanced sol fraction of “dangling ends” that cancontribute to an increased gel strength [73].

Experiments described in Paper I showed that the cooling rate of a freshly prepared and hotGelrite sample must be kept constant in order to prepare gels with reproducible

characteristics. A slow cooling givesthe gellan double helices more timeto migrate and join existing junctionzones, thereby creating largerjunction zones than those formed

Temperature (°C)

0 20 40 60 80 100

G' (

Pa)

10-1

100

101

102

103

104

Fig. 7 Temperature scan curvesshowing the elastic modulus,G , at 1 Hz during cooling(closed symbols) and heating(open symbols) for 0.5%Gelrite gels prepared in426 mM NaCl solution(TFR = 3). First temperaturescan cycle (�, ), secondtemperature sweep cycle(�, ).

CHAPTER 4. GELATION OF GELRITE GELS

24

when the gel is rapidly cooled. It has recently been reported that both the gel strength andthe turbidity are affected by the cooling rate [74].

The magnitude of the elastic modulus that we found differed considerably from the valuereported by Carlfors et al. [37]. This may be due to the polysaccharide coming fromdifferent batches or it might be due to the use of different methods for the preparation andhandling during the measurements. In particular, we formed the gels in the rheometer cupduring a controlled rate cooling process. Since the gels are very sensitive to shearing, hotsolutions of polysaccharide should be poured directly into the measuring cup and thencooled in a controlled way. In this way no shearing of the samples takes place beforemeasurement.

The experiments described in Papers III-VI showed that the addition of drugs andsurfactants has only minor or no effects on the rheological behaviour of Gelrite gels ascharacterized by temperature scan measurements. It has previously been shown that addingup to 35% fructose to 0.6% gellan solutions has little effect on the gelling temperature [74].However, the gel structure was completely lost following the addition of some compounds.This will be further discussed in Chapter 5.

5. Mucoadhesion

25

5. MucoadhesionMucoadhesive dosage forms that can stick to the site of application/absorption haveattracted considerable interest since the idea was first introduced early in the 1980s [86-98].The advantages of mucoadhesive formulations include: (i) prolonged residence time at thesite of drug absorption, and (ii) better contact with the underlying mucosa so that thediffusional path of the drug to the epithelium is shorter. Furthermore, some mucoadhesivepolymers can modulate the permeability of epithelial cells by partially opening tightjunctions [99, 100]. Carbopol 934 increase paracellular transport which is caused by thecells being depleted of extracellular Ca2+ since Carbopol has a high binding affinity forCa2+ [99, 101]. Carbopol polymers also inhibit enzymes [102] and this is also a result of thestrong binding affinity of Carbopol for Ca2+, which depletes the enzymes of calcium ions[101].

Mucoadhesive polymers interact withglycoproteins in the mucus layer thatcovers mucosal epithelial surfaces inthe body, and popular routes in whichmucoadhesive materials are used arethe nasal, ocular, buccal, vaginal,rectal and the oral route.Mucoadhesives cannot distinguishbetween adherent or shed-off mucusand this means that applicationthrough the oral route is of limitedinterest. Furthermore, if the mucusturnover is rapid, as it is, for example,in the nose, adhesion to the mucosa might not affect the bioavailability of the drug. Therheology of the formulation might be more important in such cases.

A second generation of bioadhesives, lectin-like cytoadhesives, is now in focus [103].These bioadhesives achieve more specific mucoadhesion that is independent of mucusturnover. This class of substances will probably be most useful for the oral route, ratherthan the nasal/ocular routes.

5.1 Mechanism and theory of bioadhesion

The mechanisms by which mucoadhesive bonds form are not completely clear. It isgenerally accepted that the process involves three steps [104]: Wetting and swelling ofpolymer to permit intimate contact with biological tissue, interpenetration of bioadhesivepolymer chains with mucin molecules leading to entanglement and formation of weakchemical bonds between entangled chains.

Five theories of adhesion have been developed to explain the properties of a wide range ofmaterials including glues, adhesives and paint [105]:

Facts about respiratory mucin [143]

� Mucus contains a highly swollen glycoproteinnetwork

� Protein core with carbohydrate side chains inrandom coil configuration

� Whole structure is held together withdisulphide and secondary bonds (lost whenusing commercial mucins)

� MW 3-32�106 Da, subunits of 2�106 Daconsisting of small glucopeptides of 3�105 Da

� Negative charge at physiological pH (sialicacids)

CHAPTER 5. MUCOADHESION

26

(i) The electronic theory assumes that the different electronic structures of themucoadhesive and the biological material result in electron transfer upon contact.

(ii) The adsorption theory states that the bioadhesive bond is due to van der Waalsinteractions and hydrogen bonds. This is the most widely accepted theory of adhesion.

(iii) The wetting theory uses interfacial tension to predict the degree of spreading of, forexample, a gel formulation on the mucosa, which can then be used to predict the degree ofmucoadhesion.

(iv) The diffusion theory states that interpenetration and entanglement of polymer chainsare responsible for mucoadhesion. The more structurally similar a mucoadhesive is to themucosa, the greater the mucoadhesion will be. It is believed that an interpenetration layer of0.2-0.5 �m is required to produce an effective bond.

(v) The fracture theory analyzes the force required to separate two surfaces after adhesion.It is often used for calculating fracture strengths of adhesive bonds during detachment.

5.2 Methods of characterizing mucoadhesion

Several methods are available, both in vitro and in vivo, to screen and determine themucoadhesive properties of formulations. The most commonly used in vitro methods formucosal routes are tensile strength measurements [104, 106, 107], perfusion [108] and therheological test developed by Hassan and Gallo [109]. This test will be described in moredetail below. The in vivo methods that have been used to study mucoadhesion includegamma scintigraphy [110] and transit studies of radiolabelled formulations [111].

The method developed by Hassan and Gallo is based on the changes of rheologicalproperties that occur when a potentially bioadhesive polymer is mixed with mucin. Thismethod is based on a simulated interpenetration layer (see Fig. 8) in the mucoadhesionprocess. A polymer is interpreted as being mucoadhesive if the total rheological response ofthe system is larger than the sum of the contributions from the mucin and the polymer dueto interactions between the molecules. The increase in rheological properties, described bythe interaction term (�G' in Eq. 1), may be due to mechanical interactions (entanglements)or chemical interactions (weak intermolecular forces) between the polymer and the mucinmolecules. Originally, rheological response was measured using viscosity, but during

Gel formulation

Interpenetration layer

Basal cell

Ciliated cell

Mucus layer

Basal membrane

Fig. 8 Illustration of the interpenetration layer between a gel and nasal mucosa.

5.3 Mucin Interactions

27

recent years dynamic measurements have been used and viscoelastic properties such as theelastic modulus, G , have been studied.

5.3 Mucin interactions with Carbopol 934 and Gelrite

Paper II describes the use of the Hassan and Gallo method to investigate the interactionunder physiological conditions of C934 and Gelrite with two types of commercial mucin,PSI from porcine stomach and BSMG from bovine submaxillary glands.

-15000-10000-5000

05000

10000150002000025000

0.25 0.5 0.75

Concentration /%

G' (

Pa)

A

-15000-10000-5000

05000

10000150002000025000

0.25 0.5 0.75

Concentration /%

G' (

Pa)

B

As was discussed in Section 4.2 (and in Paper I) it is very important to control the coolingrate of Gelrite. Paper II describes a method based on mixing the samples at 65�C that gavehomogeneous Gelrite-mucin mixtures and reproducible rheological data without noticeablydegrading the mucin. Mixtures of Gelrite and PSI mucin show positive interaction terms forGelrite concentrations of 0.25% and 0.5% but not for a concentration of 0.75% (Fig. 9a).High concentrations of Gelrite give very strong gels in tear fluid and the effect of the addedPSI mucin was probably too small to be seen. In contrast, the gel structure is totally

destroyed when BSMG mucin is added(Fig. 9b). Mucus sialic acid can bindcations [88] and in this way deprive Gelriteof cross-linkers. It is interesting to note thatBSMG has such a large effect on Gelrite,when only minor effects are seen when lowmolecular weight drugs or surfactants areadded (see Section 4.2).Mixtures of Carbopol with mucin (both PSIand BSMG) in ultrapure water all shownegative interaction terms for allformulations tested (0.25-1.5% polymer).This is probably due to the small amountof ions that is added from the mucin rawmaterial and that will shield the carboxylicgroups of the polymer resulting in a less

Fig. 9 Elastic modulus of Gelrite solutions, G'p (white), mixtures with mucin, G'mix (black), calculated interactionterms, �G' (striped). Mean values � standard deviation, n=3 except for mixtures with BSMG mucin wheren=1. All samples were prepared in simulated tear fluid. Values shown were obtained at a frequency of1 Hz. a) Gelrite mixtures with PSI mucin (4%). b) Gelrite mixtures with BSMG mucin (4%).

Concentration of C934 (%)

0 1 2

G' (

Pa)

0.01

0.10

1.00

10.00

100.00

1000.00

Fig. 10 The elastic modulus, G at 1 Hz of Carbopol934 in ultrapure water (�) and in simulated tearfluid ( ) as a function of polymerconcentration. Mean � standard deviation, n=3.

CHAPTER 5. MUCOADHESION

28

expanded structure. Fig. 10 shows the lowering effect of ions on the elastic modulus ofCarbopol. The cross-linked polymer gel particles are smaller when ions are present, and ahigher concentration of polymer is needed to obtain contact between particles atequilibrium swelling [112]. Weakly cross-linked poly(acrylic acid) chelates 80% of thecalcium ions in a physiological buffer [113].

In simulated tear fluid, 0.5% C934 polymer cannot form a proper gel. G depends on thefrequency and G is higher than G . This suggests that the gel particles are not in contactwith each other. The addition of PSI mucin gives a positive interaction term (Table 2). Thismay mean that the particles are forced together by an increased swelling or it may meanthat weak interactions/entanglements between the polymer and the mucin give rise to anincreased rheological response.Table 2 Elastic modulus of Carbopol 934 (G'p), Carbopol 934–mucin mixtures (G'mix) and calculated interactionterms (�G'). All samples were prepared in simulated tear fluid. Mean values (�standard deviation), n=3, exceptfor BSMG systems where n=1. Sample % polymer G'p (Pa) G'mix (Pa) �G' (Pa)C934/PSI 0.5 0.024 (0.014) 0.45 (0.057) 0.43 (0.058)

0.75 9.20 (0.36) 3.21 (0.17) –6.0 (0.40)1.0 52.0 (1.3) 15.7 (1.7) –36.3 (2.2)1.5 97.6 (4.7) 78.5 (2.4) –19.0 (5.3)

C934/BSMG 0.5 0.024 (0.014) 6.24 6.20.75 9.20 (0.36) 32.1 22.91.0 52.0 (1.3) 94.2 42.21.5 97.6 (4.7) 180 82.4

All mixtures of Carbopol with BSMG have positive interaction terms (Table 2). Thissuggests that the interaction between the polymer and BSMG mucin is stronger than thatbetween the polymer and PSI mucin. This may be due to the fact that the two types ofmucin have different structures and different molecular weights.

As a rule of thumb, the gap width of the measuring geometry in the rheologicalmeasurements should be at least 10 times greater than the diameter of the particles [114].Only if this is true does the experiment measure the bulk properties of the sample. We didnot know the diameter of the gel particles, and so we checked that the experiment reallymeasured the bulk properties by comparing the results from the concentric cylinder (with afixed gap width of 0.7 mm) with the parallel plates for which the gap widths can be varied.The bulk properties of the preparation are measured when the gap is sufficiently wide whilethere is a risk that the measured properties are correlated to the number of gel particleswithin the gap and the properties within the particles when the gap becomes narrower.Fig. 11a shows the influence of the gap width on the size and sign of the calculatedinteraction term for the mixture of Carbopol with PSI. The interaction term became positivefor gap widths less than or equal to 0.5 mm. At these small gaps the measurement morelikely characterizes the elasticity of small clusters of gel particles rather than the bulkproperties of the gel. In section 3.2.1 the particle size of Carbopol is discussed and since thesize of dry flocculated gel particles is 2-7 �m it is possible that when the particles swell, theparallel plate gap is not large enough. Fig. 11b shows that the interaction term was positive

5.3 Mucin Interactions

29

and independent of gap width for mixtures of Carbopol with BSMG mucin. This may meanthat these mixtures have a continuous gel structure without evident particulate behaviour.

-25

0

25

50

75

100

0.3 0.5 1 2

Gap width (mm)

G' (

Pa)

A

-25

0

25

50

75

100

0.3 0.5 1 2 4

Gap width (mm)

G' (

Pa)

B

To conclude, the results presented in Paper II show that positive interactions are mostlikely to be obtained with weak gels than with strong gels. There are however several issuesassociated with the method of Hassan and Gallo that make its value for interpretingmucoadhesion questionable. One mucin gave positive interaction terms when mixed withone polymer, whereas the other mucin gave negative interaction terms when mixed withthat polymer. The choice of polymer concentration was also crucial. The interactionsbetween the polymer and the mucin were interpreted as being so strong in some mixturesthat the gel network collapsed and the G decreased. The same effects was seen inexperiments described in Paper V for the strong interaction between amphiphilic drugs andlipophilically modified polymers. However, according to the model, a decrease in theinteraction term should be interpreted as the behaviour of a non-mucoadhesive polymer.The model is in this respect not relevant for the in vivo situation where strong interactionsare desirable. Furthermore, it is important to perform measurements under simulatedphysiological conditions to be sure that any change in rheology is not due to an increasedionic strength that results from the addition of mucin when using ion-sensitive polymers.The effects of several aspects of the methodology, such as the mixing temperature andcooling rate of gellan samples, the frequency at which the oscillation is performed and thegap width of the parallel plate system when measuring particulate gels, must also beconsidered.

One very important question regarding mucoadhesion concerns which component of themucoadhesive joint is the weakest: the dosage form, the mucus layer or the interface? Therheological method cannot, unfortunately give information about the weakest region, sinceonly the interpenetration layer is simulated. However, provided that appropriate measuringparameters are employed, the “Hassan and Gallo” method will demonstrate possibleinteractions between the polymer and mucin. In Paper II some polymers were shown to bemucoadhesive and thus interesting to use for nasal and ocular drug delivery.

Fig. 11 Dependence of the elastic modulus on gap width for 0.75% Carbopol, G'p (white), mixtures withmucin, G'mix (black), and the calculated interaction terms, �G' (striped). All samples were preparedin simulated tear fluid, n=1. Values shown were obtained at a frequency of 1 Hz. a) mixtures withPSI mucin. b) mixtures with BSMG mucin.

CHAPTER 6. INTERACTIONS BETWEEN POLYMER AND DRUG

30

6. Interactions between polymer and drugIt is widely recognised that the choice of polymer can be used to control the release ofdrugs from gels. The diffusion and rate of drug release are affected by the mesh size andswelling of the polymer matrix [115]. For drugs of low molecular weight, the polymermatrix does not constitute any diffusional hindrance. It has been shown by Upadrashta et al.that the diffusion coefficients of drug molecules (paracetamol, ibuprofen and indomethacin)in an uncharged hydrogel (1% agarose) are essentially the same as in water [43].

However, the polymers in many gel formulations have been chosen for their ability tointeract with the drugs and in this way release them in a controlled manner. The nature ofthese interactions obviously depends on the properties of the drug (for example LogP, pKa,MW) and of the solvent (for example pH, ionic strength). Properties of the polymer onwhich the interactions depend include its charge density [116] and its flexibility [117].Several research groups have grafted lipophilic components onto polymers [118, 119].Mixtures of polymers containing one inert polymer and one highly interacting polymerhave been studied [120]. Block copolymers such as Poloxamer which can form gelsconsisting of large populations of micelles have been used to incorporate drugs and thusaffect the rate of release [47, 121-123]. Poly-orthoesters of low molecular weight and withbasic excipients (Mg(OH)2) that will affect the polymer hydrolysis have been used toconstruct sustained release gels [124].

6.1 Polymers used in the formulations

The Carbopol polymers used in the experiments described in this thesis were all cross-linked poly(acrylic acid) polymers. The manufacturer states that the C934 gel has thelowest cross-linking density, while that of C981 is intermediate and that of C940 is thehighest. C1342 has a lipophilicmodification consisting of an alkylacrylate chain of C10-C30.

6.2 Effects on gel strength

Rheology is often a good method forstudying interactions between polymersand other compounds such as added drugsor surfactants. Fig. 12 shows that theelastic modulus, G , of a 1% C1342 gelcontaining 18 mM alprenolol was higherthan that of the drug-free gel. This effectwas not seen when a more hydrophilic drug(atenolol) was added to the C1342 gel nor

Fig. 12 The elastic modulus, G , and phase angle, �,(dashed) of gels containing atenolol in C934 ( ),C1342 ( ) and alprenolol in C934 (�), C1342 (�).Data for pure C934 gels ( ) and C1342 gels (�).

Frequency (Hz)0.10 1.00

G' (

Pa)

100

101

102

103

� (d

egre

es)

0

10

20

30

40

50

60

70

80

90

alp+C1342

at+C1342pure C1342

alp+C934pure C934

at+C934

6.2 Effects on gel strength

31

when alprenolol or atenolol were formulated in gels that lacked the lipophilic modification(C934) (Paper V). We suggest that alprenolol interacts with the lipophilic grafts on C1342and that this causes the increase in elastic modulus. Alprenolol has amphiphilic properties,and this means that both electrostatic and lipophilic interactions between it and the gel arepossible. A number of drugs, including several �-adrenoceptor blocking agents exhibitsurface activity as a consequence of the amphiphilic character of the drug. The surfaceactivity depends on the nature of the hydrophobic and hydrophilic portions of the drugmolecule [125].

Fig. 13 shows the elastic modulus of formulations of C1342 with a number of well-recognized [125] amphiphilic drugs. The G s observed for C1342 gels with alprenolol,diphenhydramine and lidocaine were higher than those of the pure C1342 gel. However,these drugs did not strengthen gels that lack the lipophilic modification (for example C934).For the more hydrophilic atenolol, which lacks amphiphilic properties, no increase in thegel strength was observed.

The concentration of drug also affects the extent of the interaction. For example,amitriptylin did not increase G at the concentration used in the experiment shown inFig. 13 (18 mM), but did increase G when the concentration of drug was lowered to 9 mM(Fig. 14). This concentration dependence was also seen for alprenolol anddiphenhydramine. The rheology of lipophilically modified polymer gels following theaddition of amphiphilic molecules has been discussed by Piculell et al. [126]. G initiallyincreases as more surfactant is added, passes through a maximum, and then decreases to avalue lower than the G obtained in the absence of surfactant. This is due to the ability ofthe surfactant to form mixed micellar cross-links with the lipophilic modifications on thepolymer, leading to an increase of the elastic modulus of the gel matrix [126]. When

pure C1342 gel

alprenolol

amitriptylin

chlorpromazine

diphenhydraminelidocaine

orphenadrine

tetracaineatenolol

G' (

Pa)

0

50

100

150

200

250

pure C1342 gel

alprenol. 4.5mM

alprenolol 9mM

alprenolol 18mM

alprenolol 36mM

alprenolol 72mM

amitript. 9 mM

amitript. 18 mM

diphenh. 9 mM

diphenh. 18 mM

diphenh. 45 mM

G' (

Pa)

0

50

100

150

200

250

�

�

�

�

Fig. 13 The elastic modulus, G of 1% C1342 gelscontaining 18 mM of various drugs. Thereference line shows the G of a drug-free1% C1342 gel.

Fig. 14 The elastic modulus, G of 1% C1342 gelswith various concentrations of alprenolol,amitriptylin and diphenhydramine. Precipitationoccurred for some gels (�) and rheologicalcharacterization was not possible in one case(�).

CHAPTER 6. INTERACTIONS BETWEEN POLYMER AND DRUG

32

72 mM alprenolol was added to 1% C1342 the gel collapsed (Fig. 14) due to the positivelycharged drug shielding the negatively charged carboxylic acid groups on the polymer. Thecharges on the polymer are essential for the swelling and gel structure. This type of collapseor precipitation has previously been reported by Senan et al. [127] for LM-PAA, but is notseen when the polymer is uncharged, for example the system described by Piculell et al.[126] and discussed above.

The experiments described in Paper III showed that butylparaben and other uncharged andlipophilic drug model substances (as well as the charged hydrophilic drugs discussedabove) do not affect the gel strength of C1342. It seems that an appropriate balancebetween the charged head group and the hydrophobic portion of the drug is essential foreffects on the rheology.

6.3 Effects on drug release

The rate of release of alprenolol could be controlled by formulating the drug in differentpoly(acrylic acids) (PAA) with different cross-linking density, different concentrations andsome also with lipophilic modifications (Fig. 15). The rate of release from C1342 (LM-PAA) was slowest, while that from C934 (which has the lowest cross-linking density) wasfastest. The polymer concentration also affected the rate of release. High concentration

resulted in a slower releasesince there was morepolymer present that thedrug could electrostaticallyor hydrophobically interactwith. The polymerconcentration, however,can seldom be used as aneffective tool for tailoringthe release rate since theformulation must havegood rheological propertiesin order to have a longresidence time at the site ofdrug absorption.

Butylparaben did notinfluence the gel strength ofC1342, but it probablyinteracted with thelipophilic modifications ofthe polymer. This

interaction resulted in the different release rates presented in Fig. 16. The choice of polymeraffected the rate of release of butyl-paraben in the same way as that of alprenolol; releasewas fastest from the least cross-linked polymer and slowest from the lipophilic polymer

Time (minutes)

0 50 100 150 200 250 300 350 400

Frac

tion

rele

ased

0.0

0.2

0.4

0.6

0.8

1.0

Fig. 15 The release of alprenolol from gel formulations containing 0.5%polymer (open symbols), 1% (grey) and 2% (closed). C934 ( ),C940 ( ) and C1342 ( ).


33

C1342. The effect of polymer concentration was somewhat different. The release curve of0.5% C1342, had a quite different shape than those of 1% and 2% C1342 (Fig. 16).

The effects on the release rate of the size and lipophilicity of the drug were evaluated bystudying the release from C1342 of a homologous series of paraben esters (Fig. 17). Therate of release decreased with increasing lipophilicity (and with increasing size) of the ester,mainly due to stronger lipophilic interactions with the polymer.

Fig. 16 The release ofbutylparaben from gelformulations containing0.5% polymer (opensymbols), 1% (greysymbols) and 2% (closedsymbols). C934 ( ),C940 ( ) and C1342 ( ).

Time (min)

0 60 120 180 240 300 360

Frac

tion

rele

ased

0.0

0.2

0.4

0.6

0.8

1.0

Fig. 17 The fraction released(mean � standard deviation)from Carbopol 1342 gels ofp-hydroxybenzoic acid (�),methylparaben (�),ethylparaben (�),propylparaben (�) andbutylparaben (�).

Time (minutes)

0 50 100 150 200 250 300 350 400

Frac

tion

rele

ased

0.0

0.2

0.4

0.6

0.8

1.0

CHAPTER 7. INTERACTIONS BETWEEN POLYMER, DRUG AND NON-IONIC SURFACTANTS

34

7. Interactions between polymer, drug and non-ionicsurfactants

Including surfactant micelles in the formulation will give the drug more components tointeract with, leading to a slower release of the drug. This principle has previously beenused in analytical chemistry, as the basis of a separation method in which hydrophobiccompounds are extracted by immobilized micelles in gels [128].

When poly(oxyethylene) surfactants such as Brij 58 are added to poly(acrylicacid) polymers, the aggregation number is 15% smaller than they are when the surfactant ispresent as free surfactant micelles [129]. The polymer binds at the micellar head groupsurface and this intimate interaction leads to a reduction of the surfactant mobility.

7.1 Interactions between drug and surfactant

Surfactants that form micelles canincrease the solubility of sparingly solubledrugs by incorporating the drug into thelipophilic core. The degree of micellarpartition, pm, can be estimated from Eq. 3by comparing the aqueous solubility of adrug with its solubility in the presence ofmicelles. As the lipophilic part of thesubstance gets bigger, the micellarpartition increases (Table 3).

Table 3 The aqueous solubility at 35�C, partitioncoefficient, (log P) and micellar partition, (pm)of parabens in 1% surfactant solutions.

Substance Solubility log P pm(10-3 M) (SDS)

p-OH-benz. ac. high 1.58 -Methylparaben 32 1.96 0.15Ethylparaben 11 2.57 0.30Propylparaben 4 3.04 0.75Butylparaben 1.5 3.57 0.95

Drugs that are charged at the physiological pH(such as the amphiphilic alprenolol anddiphenhydramine) may interact with thesurfactants to form mixed micelles. The extent ofthis interaction cannot be determined usingsolubility measurements due to the high solubilityof the drug. This means that pm cannot becalculated in this case.


In a formulation containing drug, polymer andsurfactant, three kinds of interaction may affect therelease of the drug: (i) The drug may interact withthe polymer, (ii) the drug may interact with the

Fig. 18 The fraction released (mean � standarddeviation) from Carbopol 1342 gels with 1%Brij 58 micelles. Data for p-hydroxybenzoicacid (�), methylparaben (�), ethylparaben(�), propylparaben (�) and butylparaben (�).

Time (min)

0 60 120 180 240 300 360

Frac

tion

rele

ased

0.0

0.2

0.4

0.6

0.8

1.0


35

surfactant, and (iii) the surfactant may interact with the polymer matrix. The rate of drugrelease can be successfully controlled by controlling these interactions.

When 1% of the non-ionic micelle-forming surfactant Brij 58 was included in C1342formulations (Fig. 18), paraben esters were released more slowly than they were releasedfrom formulations without surfactants (Fig. 17, see Section 6.3). This is due to theincreased number of lipophilic sites in the formulation with which the drug can interact.Large structures such as micelles have low diffusion coefficients, and it is also possible thatthe micelles interact with the polymer and form immovable complexes with it (Paper III).

The effects of the identity of the polymer and theaddition of Brij 58 on the diffusion coefficient canbe seen in Fig. 19. When the lipophilicity wasincreased an increased interaction of the parabenwith both the LM and the micelles resulted in adecreased diffusion coefficient.

The micellar partition, pm, and the diffusioncoefficients of paraben both in the absence (Df)and in the presence (D) of surfactants in a C934gel, allow the diffusion coefficient of the micelles(Dm) to be estimated from Eq. 5. This gives anestimated micellar diffusion coefficient of 1�10-6

cm2/s. The Stokes-Einstein equation (Eq. 6) thenallows the micellar radius to be estimated to 22 Å,which is fairly close to the value reported (47 Å)from low-angle light-scattering measurements[130].

It is not expected that charged drugs will interactstrongly with non-ionic micelles. Diphenhydr-amine is amphiphilic and probably forms mixedmicelles with non-ionic surfactants. Fig. 20 showsthat the initial release of diphenhydramine issignificantly slower when Brij 58 is present. After3h the curves intersect. The presence of micellesdoes not affect the rate of drug release as much aschanging the identity of the polymer (for examplefrom C934 to C1342).

The addition of non-ionic surfactants does,however, effect the gel strength of PAA gels. Inthe presence of 1% Brij 58, the elastic modulus of

all three polymers, C934, C940 and C1342, were only 60-80% of the corresponding valuesin the absence of surfactant. We suggest that Brij 58 interacts with cross-links and lipo-philic modifications of the polymer in a way that decreases the swelling of the gel particlesso that the elasticity of the gel decreases. This can explain the cross-over in Fig. 20.

log P

0 1 2 3 4

D (1

0-6cm

2 /s)

0

2

4

6

8

10

Fig. 19 The diffusion coefficient (mean with95% c. i.) of substances with differentlog P values in C934 without micelles(�),with micelles ( ), C1342 withoutmicelles (�) with micelles ( ).

Time (minutes)

0 50 100 150 200 250 300 350 400

Frac

tion

rele

ased

0.0

0.2

0.4

0.6

0.8

1.0

Fig. 20 The release (mean � standarddeviation) of diphenhydramine fromgels of C934 (�), C1342 (�) andC1342 with 1% Brij 58 ( ).

CHAPTER 8. INTERACTIONS BETWEEN POLYMER, DRUG AND IONIC SURFACTANTS

36

8. Interactions between polymer, drug and ionicsurfactants

For an effective drug treatment it is important to prolong the drug release from aformulation as long as the formulation stays at the place of drug absorption. Charged drugsare hydrophilic and are thus only slightly absorbed over physiological membranes.Lipophilic interactions of the drug with micelles or polymers are often weak and thesecannot be used in controlled release formulations. This means that there is a pressing needfor a method of achieving controlled release of charged drugs.

8.1 Interaction between drug and surfactant

A mixture of 18 mM diphenhydramine and 1% (36 mM) SDS in simulated tear fluid wasquite turbid but homogeneous. Cryo-TEM images of the mixture showed that vesicles werepresent (Fig. 21a), and it was these that cause the light scattering. Vesicles formedspontaneously when the positively charged, surface active drug diphenhydramine and thenegatively charged surfactant SDS were mixed. The mixture was prepared using anordinary magnetic stirrer that gave vesicles with a very wide size distribution that rangedfrom 50 nm in diameter to at least 900 nm. The spherical and smooth vesicles wereunilamellar or oligolamellar and some had open membranes (Fig. 21b). The presence offlaws in the vesicle surface is, however, not a major concern since the drug is notencapsulated in the vesicle, but incorporated into the vesicle bilayer.

A B

C D

Fig. 21 Cryo-TEM images(bar = 200 nm) of:

(a) 18 mM diphenhydra-mine and 36 mM SDS(solution)

(b) 18 mM alprenolol and36 mM SDS (solution)

(c) 36 mM diphenhydra-mine and 36 mM SDS(solution)

(d) 18 mMdiphenhydramine and72 mM SDS(solution).


37

Vesicles formed over a fairly wide range of drug:surfactant ratios, but decreasing theamount of SDS to 0.5%, a level that is equimolar to the diphenhydramine concentration,resulted in an unstable mixture and a two-phase system. Vortex treatment and subsequentcryo-TEM of this system confirmed the presence of vesicles in addition to droplets of themomentarily dispersed phase (Fig. 21c).

A doubled concentration of SDS (2%) mixed with 18 mM diphenhydramine produced aclear solution with high viscosity (Paper IV). Cryo-TEM showed long thread-like andhighly branched micelles that formed a bicontinuous structure (Fig. 21d). Bicontinuousworm-like micelles have previously been reported for equimolar mixtures ofcetylpyridinium chloride and sodium salicylate [131]. The ions present in the simulated tearfluid affect the formation of the vesicles and the thread-like micelles. It is known that anincreased ionic strength can cause micellar growth and a higher viscosity due to theshielding of the charges of the head groups [132]. We also observed this effect (Fig. 22).

These solutions are all totally clear. Therheological properties of thread-like micellarsolutions formed by mixing cationic andanionic surfactants depend on the surfactantconcentration [133].

Vesicles formed also when metoprolol,betaxolol or alprenolol at a concentration of 18mM was mixed with 1% SDS (Fig. 21b). Fornegatively charged drugs, such as fluvastatin,vesicles formed when the drug was mixed withpositively charged surfactants such as BAB.

Charged drugs that have a lower lipophilicity,such as atenolol, produced clear solutions withunaffected viscosity when mixed at aconcentration of 18 mM with SDS atconcentrations between 0.5% and 2%

(Paper IV). This indicates that no vesicles or thread-like micelles formed. The amphiphilicnature of such drugs is not pronounced, which explains the lack of vesicle formation.


When the diphenhydramine vesicles were prepared in gels, they appeared similar to thosethat formed in solution, although the form was more faceted (Fig. 23a). This is due to bothhydrophobic and electrostatic interactions between the drug and the polymer [134]. Drugrelease from this formulation (1% SDS, 18 mM diphenhydramine) was much slower thanthat from a surfactant-free system (Fig. 24). When the SDS concentration was increased to2%, the drug in the thread-like micelles (shown in Fig. 21d) was released more slowly thanfrom a surfactant-free system. The release from the thread-like micelle gel was more rapidthan the release from the vesicle bilayer that formed at a SDS concentration of 1%. A two-

Shear rate (s-1)

10-1 100 101 102 103 104

Visc

osity

, � (P

a s)

10-3

10-2

10-1

100

Fig. 22 The shear viscosity, �, of mixed micellesformed by 36 mM diphenhydramine and139 mM SDS in ultra-pure water (�),physiological saline (�) and simulated tearfluid (�).

CHAPTER 8. INTERACTIONS BETWEEN POLYMER, DRUG AND IONIC SURFACTANTS

38

phase system similar to that shown in Fig. 21c formed at a SDS concentration of 0.1% anda release experiment with this composition showed that the release rate of diphenhydramineis only slightly slower than that of a surfactant-free system (Fig. 24).

Fig. 25 shows that the release of 18 mMalprenolol from C1342 gels can be accuratelycontrolled by varying the surfactant concentration.The addition of 1% SDS gave the slowest releasesince in this case the drug formed vesicle bilayerswith SDS. The release rate could be controlled byadding non-ionic surfactants and thereby formingmixed aggregates where the charge density isprobably lower (Paper IV). Results obtained froma formulation containing 0.5% Brij 58 and0.5% SDS (Fig. 25) show that the drug wasreleased more rapidly than it was released from aformulation containing only 0.5% SDS. Brij 58might also decrease the amount of vesicles andin this way increase the rate of release.

It is interesting to note that the vesicles thatformed with alprenolol and SDS whenformulated in C1342 were small. Most of theaggregates were smaller than 50 nm in diameter,see Fig. 23b. We believe that the lipophilicmodifications of C1342 interact with the vesiclesand cause a decrease of their size. Thisinteraction also caused an increase in the elasticmodulus. G was 123 Pa for 1% C1342 withoutdrug, 182 Pa for C1342 with 18 mM alprenololand 258 Pa for the formulation containing bothalprenolol and 1% SDS. The appearance ofvesicles of diphenhydramine and SDS changedlittle when the unmodified PAA polymer C934was added (Fig. 23a). Dilute solutions of

A B

Fig. 23 Cryo-TEM images(bar = 200 nm) of:

(a) 18 mM diphenhydramineand 36 mM SDS inC934 gel

(b) 18 mM alprenolol and36 mM SDS in C1342gel.

Time (minutes)

0 50 100 150 200 250 300 350 400

Frac

tion

rele

ased

0.0

0.2

0.4

0.6

0.8

1.0

Fig. 24 The release (mean � standard deviation,n=3) of diphenhydramine from C934formulations with no SDS (�), 0.1% SDS ( ),1% SDS ( ) and 2% SDS ( ).

Time (minutes)

0 50 100 150 200 250 300 350 400

Frac

tion

rele

ased

0.0

0.2

0.4

0.6

0.8

1.0

Fig. 25 The release (mean � standard deviation,n=3) of alprenolol from C1342 gels with noSDS (�), 1% Brij 58 ( ), 0.5% SDS + 0.5%Brij 58 (�), 0.5% SDS (�) and 1% SDS (�).


39

hydrophobically modified polymers induce the formation of clusters of vesicles [134] or oforganized “bead-on-strings” structures of micelles [135].

Fluvastatin was released more slowly from a C1342 gel than from a C934 gel (Fig. 26).The lipophilic modifications in C1342 and the different cross-linking densities of the twopolymers may explain the difference in the rate of release. When the oppositely chargedcationic surfactant benzalkonium bromide (BAB)was added, the release of fluvastatin was slowerthan it was in the surfactant-free formulations.This was due to the formation of vesicles (PaperIV). The concentrations of fluvastatin and BAB inthese formulations were both 1/5 of the concen-tration used in the previously discussed examples.It was necessary to use such low concentrationsbecause the interaction between the cationic BABand the anionic C1342 was so strong that precipi-tation may occur at higher concentrations of BAB.The strong interaction between non-cross-linkedPAA and the oppositely charged C16TAB atvarious stoichiometric ratios is discussed byFundin et al. [136].

Fig. 27 summarizes the diffusion coefficientsof drugs with various lipophilicities in diffe-rent formulations. The diffusion coefficientvaried only slightly in the gels without surfac-tants. As was discussed in Paper III (and seenin Fig. 19), the interaction between an un-charged drug (exemplified with the parabenesters) and Carbopol polymers does not giverise to a sustained release of drugs with log Dvalues of 2 or less. The diffusion coefficientdid depend on the lipophilicity when oppo-sitely charged surfactants were present, andthe slowest release was observed for drugswith a log D higher than –1. In this case,vesicles containing the drug formed whenoppositely charged surfactant was present.Atenolol, which has a log D below –1, was

released slightly but significantly slower when SDS was present in the PAA gels (PaperIV). We believe that this is due to the formation of mixed micelles or to an interactionbetween the drug and the oppositely charged micellar surface. Such an interaction haspreviously been reported by Gerakis et al. [137]. The interactions between drugs andoppositely charged surfactants in drug delivery using polymer matrix tablets have also beendescribed [138, 139].

Time (minutes)

0 50 100 150 200 250 300 350 400

Frac

tion

rele

ased

0.0

0.2

0.4

0.6

0.8

1.0

Fig. 26 The release (mean � standard deviation,n=3) of fluvastatin from C934 (�), C1342 (�),C934 with 0.2% BAB ( ) and C1342 with0.2% BAB ( ).

log D

-2 -1 0 1 2

Diff

usio

n co

effic

ient

(10-6

cm2 /s

)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

5.

1. 2.

3.

4.

6.

Fig. 27 The diffusion coefficient (mean with 95% CI)of six drugs with varying lipophilicities (log Dvalues) in C934 (�), C1324 (�), C934 withsurfactants ( ) and C1342 with surfactants ( ).1. atenolol, 2. metoprolol, 3. alprenolol, 4.betaxolol, 5. fluvastatin, 6. diphenhydramine.

CHAPTER 9. EVALUATION OF GELS IN THE USSING CHAMBER

40

9. Evaluation of gels in the Ussing chamber

The nasal route is attractive for systemic delivery of drugs since the relatively largeabsorption surface is well vascularized and the epithelium is relatively leaky. Further,metabolism in the gastrointestinal tract and during the first pass through the liver is avoided[98]. Administration by this route is easy, relatively inexpensive, suitable for self-application and does not require sterile equipment. The plasma concentration profilesobtained after nasal administration of small (<500 Da) lipophilic compounds are similar tothose seen after intravenous administration [140, 141].The horizontal Ussing chamber is a development of the traditional, vertical Ussingchamber, and it can be used to study tissues such as the nasal mucosa that have an air

interface in vivo [64]. The membrane ismounted in a horizontal position and asubstance or formulation is applied on themucosal side without having to add buffer.

Electrophysiological parameters aremeasured in the Ussing chamber method inorder to evaluate the viability of themembrane and the toxicity of the testsubstances. It is necessary to have buffer onboth sides of the membrane during thesemeasurements, but the buffer can beremoved during absorption studies in thehorizontal chamber leaving the upper,mucosal side dry. This makes it possible toevaluate a wide range of formulationsincluding pharmaceutical powders and gels.

9.1 The evaluated formulations

The experiments described in Paper IIIshowed that the release of uncharged drugsfrom C934 gels could be controlled byadding Brij 58. This finding was used in theexperiments described in Paper VI to sustainthe release of the uncharged steroidshydrocortisone and testosterone. These twodrugs have different log Ds, and so themicellar partition coefficients and hence therates of release differ. This can be seen inFig. 28, which shows that the rate of releaseof the more lipophilic testosterone in the

Time (minutes)

0 10 20 30 40 50 60 70 80 90 100

Frac

tion

rele

ased

0.0

0.2

0.4

0.6

0.8

1.0

Fig. 28 The release (mean � standard deviation,n=3) from C934 of testosterone ( , �) andhydrocortisone ( , �) with 1% Brij 58 (opensymbols) and without surfactant (closed s.).

Time (minutes)

0 10 20 30 40 50 60 70 80 90 100

Frac

tion

rele

ased

0.0

0.2

0.4

0.6

0.8

1.0

Fig. 29 The release (mean � standard deviation,n=3) of dihydroalprenolol from C934 formu-lations with no SDS (�) and 1% SDS ( ).

9.2 Transport over porcine nasal mucosa

41

presence of micelles was considerably slower than that of hydrocortisone.

Paper IV shows that the release of amphiphilic drugs from C934 gels can be controlled bymixing the drug with oppositely charged surfactants to produce vesicles. This principle wasevaluated in Paper VI by forming vesicles of dihydroalprenolol (DHA) and SDS and thenmeasuring the rate of release in vitro. The presence of SDS clearly reduced the rate ofrelease (Fig. 29).

9.2 Transport over porcine nasal mucosa

Figs. 30-32 show the cumulative fractions of applied drug that were transported over thenasal mucosa as a function of time. Mucosal permeability (Papp) can be calculated from thedata obtained for drugs in solution. For dihydroalprenolol Papp was 2.6�10-6

�1.4�10-6 cm/s,for testosterone 4.0�10-6

�1.9�10-6 cm/s and for hydrocortisone 2.4�10-6�1.6�10-6 cm/s.

There were no significant differencebetween cumulative fractions transported(CFT) of drug when formulated in a C934gel or in a solution.

The transport (CFT) of testosterone in theUssing chamber was significantly slower inthe presence of Brij 58 micelles (Fig. 31)than in their absence, which is consistentwith the sustaining effect seen in vitro(Fig. 28) The release from the formulationis the rate-limiting step in this case. TheS.E.M. (standard error of mean) wassmaller for experiments carried out in thepresence of Brij 58, which might indicatethat it is the properties of the formulation,and not the thickness or properties of themucosa that control the transport(Paper VI). The thickness and properties ofthe mucosa both have a very high degree ofvariability.

Since only minor effects were seen on the in vitro release rate when the more hydrophilichydrocortisone was evaluated, no further transport studies in the Ussing chamber wereperformed.

The transport of dihydroalprenolol was examined at two concentrations, 0.7 mM and18 mM shows. Fig. 30 shows that the transport rate was slower and that the S.E.M. wassmaller for formulations with SDS than surfactant-free formulations. This indicates that therelease rate from the formulation was rate-limiting.

Time (minutes)

0 10 20 30 40 50 60 70 80 90 100

Frac

tion

trans

porte

d

0.000

0.000

0.002

0.003

0.004

0.005

0.006

0.007

Fig. 30 The fraction transported (+ S.E.M.) overporcine nasal mucosa of dihydroalprenolol(DHA) in solution ( , n=6), gel (�, n=6), solution0.7 mM DHA with 1.4 mM SDS ( , n=2),solution 18 mM DHA with 36 mM SDS ( , n=4),gel 0.7mM DHA with 1.4 mM SDS (�, n=2) andgel 18 mM DHA with 36 mM SDS (�, n=6).

CHAPTER 9. EVALUATION OF GELS IN THE USSING CHAMBER

42

In the Ussing chamber, it is also possible to evaluate any changes in the viability of themucosa due to substances in the applied formulation. The viability was seriously effectedby the presence of 1% Brij 58. The viability of the mucosae was also affected inexperiments using formulations with SDS (both at concentration 0.04% and 1%), althoughin a complex manner. A 0.05% solution of SDS disrupts the ciliary beat frequency (CBF),whereas a 0.03% solution does not [142]. Since SDS is vesicle-bound in our study the freeconcentration that can have a negative effect on the mucosa is hard to estimate.

Time (minutes)

0 10 20 30 40 50 60 70 80 90 100

Frac

tion

trans

porte

d

0.0000.0000.0020.0030.0040.0050.0060.0070.0080.0090.0100.0110.0120.0130.014

Time (minutes)

0 10 20 30 40 50 60 70 80 90 100

Frac

tion

trans

porte

d

0.000

0.000

0.002

0.003

0.004

0.005

0.006

0.007

The decrease in viability and problems with toxicity that are associated with the use ofsurfactants in formulations depend strongly on the concentration of surfactant used. Theconcentrations used in the formulations described in the Paper VI and evaluated for theirtoxicity were the same as those used in the previous papers on drug release (Papers III-V).However, the drug concentrations were chosen so that they would give acceptable detectionin the spectrophotometric methods used and not primarily to be pharmaceutically relevant.The formulations used in clinical practice often have a lower drug concentration than thosewe have used, and excipients such as surfactants can then be included at correspondinglylower concentrations. The choice and concentration of surfactants also depends on theintended route of administration.

Fig. 31 The fraction transported (+ S.E.M.) overporcine nasal mucosa of 0.1 mM testosteronein solution ( , n=4), gel (�, n=4) and gel with1% Brij 58 (�, n=3).

Fig. 32 The fraction transported (+ S.E.M.) overporcine nasal mucosa of 0.1 mMhydrocortisone in solution ( , n=4) and gel(�, n=5).

10. Conclusions

43

10. ConclusionsGelrite gels are formed in tear fluid even when the concentration of Gelrite is only 0.1%.The most important gel-promoting ion in vivo is Na+. Samples containing concentrations ofGelrite of the order of 0.5% form gels even when the concentration of ions is10-25% of thatfound in tear fluid. The rheological method for evaluating mucoadhesion can give highlyvarying results depending on what experimental settings and the type of mucins used. Thepolymer concentration is particularly important. Furthermore, positive interaction terms aremost likely to be obtained with weak gels.

The work presented in this thesis shows that it is feasible to control the release ofuncharged drug substances from gels by using the lipophilic interactions between the drug,the polymer and micelles in which the drug can be distributed. Vesicles generally formwhen amphiphilic drugs and oppositely charged surfactants are mixed in certain ratios, andthis phenomenon can be used to design controlled-release gel formulations of chargeddrugs.

The mucosal transport rate of drugs can be controlled by the rate of release from theformulation, and the Ussing chamber method can be used to evaluate the toxicity of theformulations.

ACKNOWLEDGEMENTS

44

Acknowledgements

The studies in this thesis were carried out at the Department of Pharmacy, UppsalaUniversity.

The financial support of the Knut and Alice Wallenberg Foundation, the Swedish NationalBoard for Industrial and Technical Development (NUTEK), AstraZeneca, EthicalsPharmaceuticals and Pharmacia Corp. are gratefully acknowledged.

I wish to express my sincere gratitude to:

Dr Katarina Edsman, min handledare för din entusiasm, din förmåga att alltid prioritera ossdoktorander och ditt stöd i min utveckling till en självständig forskare.

Professor Christer Nyström och professor Göran Alderborn för tillhandahållande avändamålsenliga lokaler, instrument och en kreativ och stimulerande forskningsmiljö.

Helene Hägerström, medförfattare, för trevligt samarbete, roliga stunder, goda idéer samtovärderlig hjälp med korrekturläsning av manuskript.

Karin Östh, medförfattare, för trevligt och glatt samarbete. Speciellt tack för att du ställdeupp och jobbade så intensivt med vårt pek och hjälpte mig när jag skrev sammanfattningen.

Lena Strindelius, min rumskompis och goda vän, för alla seriösa och oseriösa diskussioneroch alltid lika underhållande Eskilhistorier.

Dr. Hans Evertsson, Dr. Andreas Hugerth och Dr. Maria Dahlin för givande laborativtsamarbete och trevliga diskussioner.

Stefano Bonfante, Anja Lahi och Tobias Bramer för experimentella bidrag och trevligastunder på lab.

Dr. Katarina Edwards och Göran Karlsson för givande samarbete när det gäller cryo-TEM.

Professor Chandrasekaran and Dr. Janaswamy, Purdue University, Indiana, for providingimages of gellan.

IF stiftelse, CD Carlssons stiftelse och Apotekare C.R. Leffmans stipendiestiftelse för attjag fick möjlighet att resa på konferenser och kurser.

Eva Nises-Ahlgren för all hjälp med det praktiska kring avhandlingsarbetet och för att duhåller reda på institutionen.

BMCs forskningsverkstad för kunnig och snabb problemlösning

References

45

Dr. Erik Björk, medförfattare, Professor Sven Engström och Dr Johan Carlfors förvärdefulla diskussioner.

Johan Gråsjö för mycket trevliga och lärorika diffusa diskussioner.

Dr Lucia Lazorová, för givande laborativt samarbete, för vänskap och för att du är en sådanglädjespridare.

Magnus Köping-Höggård, Dr Raouf Ghaderi, Christel Johansson, Jonas Berggren, ochKarin Östh för trevligt sällskap på konferensresor till Boston och Indianapolis. Jag villockså tacka er, liksom även Staffan Tavelin, Dr Patric Stenberg, Åsa Tunón, SitaramVelaga och alla nuvarande och före detta anställda på institutionen för att ni gjort min tidsom doktorand minnesvärt angenäm både på BMC och utanför.

Dr Mikael Hedeland för trevliga stunder utanför lab men också för att ha påverkat mig tillatt börja där.

Mamma, pappa och mormor för ert oändliga stöd under alla år.

Min Linda. Mitt allt.

Tack!

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