Polysaccharides for Drug Delivery and Pharmaceutical Applications

Chapter 7

Downloaded by UNIV OF GUELPH LIBRARY on July 27, 2012 | http://pubs.acs.org Publication Date: June 22, 2006 | doi: 10.1021/bk-2006-0934.ch007

Hyaluronan: Investigations into the Mode of Action of Hyaluronan in Topical Drug Delivery Marc B. Brown, Stuart A. Jones, Weijiang He, and Gary P. Martin Pharmaceutical Sciences Research Division, King's College London, 150 Stamford Street, London SE1 9NH, United Kingdom

Hyaluronan (HA) is a polyanionic, polysaccharide ubiquitous in mammals where it has a protective, structure stabilising and shock-absorbing role. The unique viscoelastic nature of H A along with its biocompatibility and non-immunogenicity has led to its use in a number of clinical applications. The ability of H A to localise therapeutic agents in the superficial layers of the skin has led to the commericalisation of Solaraze®, a 3% diclofenac in 2.5% H A gel, for the topical treatment of actinic keratoses (AKs), the third most common skin complaint in the US. However, the means by which H A enhances topical drug delivery remains unclear. The data described in this study demonstrate that H A offers promotes the delivery and localisation of drugs in the skin. In addition, it would appear that the molecular weight and concentration of H A may be critical for such drug delivery properties.

© 2006 American Chemical Society

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In Polysaccharides for Drug Delivery and Pharmaceutical Applications; Marchessault, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

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Introduction Hyaluronic acid was discovered in bovine vitreous humour by Meyer and Palmer in 1934 \ It is most frequently referred to as hyaluronan (HA) due to the fact that it exists in vivo as a polyanion and not in the protonated acid form. H A is ubiquitous in that it is distributed widely in vertebrates and is present as a 2 component of the cell coat of many strains of bacteria (e.g. A. Streptococcus) . Commercially produced H A is isolated either from animal sources, within the synovial fluid, umbilical cord, skin, and rooster comb, or from bacteria through a process of fermentation or direct isolation. The molecular weight of H A is heavily dependent on its source however, refinement of these isolation processes has resulted in the commercial availability of numerous molecular weight grades extending up to a maximum of 5,000,000 daltons . Extensive studies on the chemical and physicochemical properties of H A and its physiological role in humans, together with its versatile properties, such as its biocompatibility, nonimmunogenicity, biodegradability and viscoelasticity, have proved that it is an ideal biomaterial for cosmetic, medical and pharmaceutical applications. Several of H A ' s important physicochemical properties are molecular weight dependent and therefore discrete differences in function over the wide range of commercially available molecular weights enables H A to be used in a diverse set of applications. In addition, chemical modification of H A can produce a more mechanically and chemically robust material which still retains its biocompatibility and biodegradability. Methods detailing the synthetic transformation of H A and the resulting applications in cosmetic, medical and pharmaceutical applications have been extensively reviewed previously and are not discussed in further detail here. 3

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Physical, Chemical and Biological Properties of H A The primary structure of H A comprises an unbranched linear chain with repeating units of of D-glucuronic acid and N-acetyl-D-glucosamine, linked together through alternating β and βι glycosidic bonds. Hydrophobic faces exist within the secondary structure of H A , formed by the axial hydrogen atoms of about 8 C H groups on the alternating sides of the molecule. Such hydrophobic patches, energetically favour the formation of a meshwork-like β-sheet tertiary structure as a result of molecular aggregation . The tertiary structure is stabilised by the presence of inter-molecular hydrogen bonding . The hydrophobic and hydrogen bonding interactions allow large numbers of molecules to aggregate leading to the formation of molecular networks (matrices) of H A . H A in aqueous solution has been reported to undergo a transition from Newtonian to nonNewtonian flow with increasing molecular weight, concentration or shear rate. 1 3

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143 In addition, the higher the molecular weight and concentration of H A , the higher the viscoelasticity the solutions possess . The viscoelasticity of H A in aqueous solution is pH-dependent and effected by the ionic strength of the environment in which it is placed . l l 1 2


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H A in combination with other glycosaminoglycans (GAGs) such as dermatan sulphate, chondroitin sulphate and keratin sulphate are prominent in tissues such as the skin. H A is found to exist together with protein cores (aggrecans) to which the other G A G s are also attached. The most important property of these molecules is their ability to bind to water and this induces the proteoglycans to become hydrated such that a gel-like system is formed. H A is found in almost all vertebrate organs, but most abundantly in the extracellular matrix of soft connective tissues. H A is particularly abundant in mammalian skin where it constitutes a high fraction of the extracellular matrix of the dermis . The estimated total amount of H A in human skin has been reported to be 5 g, about a third of the total amount of H A believed to be present within the entire human body. 1 4

Cosmetic, Medical and drug delivery applications of HA H A has been extensively utilised in cosmetic products because of its viscoelastic properties and excellent biocompatibility. Application of H A containing cosmetic products to the skin is reported to moisturise and restore elasticity to the skin thereby achieving an anti-wrinkle effect, albeit no rigorous scientific proof exists to substansiate this claim. HA-based cosmetic formulations or sunscreens may also be capable of protecting the skin against U V irradiation due to the free radical scavenging properties of H A . In addition, H A , either in a stabilised form or in combination with other polymers, is used as a component of commercial dermal fillers (e.g. Hylaform®, Restylane® and Dermalive®) in cosmetic surgery. It is reported that the long-term injection of such products into the dermis, can reduce facial lines and wrinkles with fewer side effects and better tolerability compared to the use of collagen . The main side effect may be an allergic reaction, possibly due to impurities present in H A . Lin et al (1994) have investigated the feasibility of using H A as an alternative implant filler material to silicone gel in plastic surgery and H A is commonly used as growth scaffold in surgery, wound healing and embryology. In addition, administration of purified high molecular weight H A into orthopaedic joints has been reported to restore the desirable rheological properties and alleviate some of the I 5

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symptoms of osteoarthritis . The success of the medical applications of H A has led to the production of several successful commercial products which have been extensively reviewed previously . 8


144 H A has been extensively studied in ophthalmic, nasal and parenteral drug delivery. In addition, more novel applications including, pulmonary, implantation and gene delivery have also been suggested . In the majority of drug delivery applications H A acts either as a mucoadhesive, to retain the drug at its site of action/absorption, or as an in vivo release/absorption modifier, to improve drug bioavailability. 8


Administration of therapeutics to the skin using HA Topical delivery for the treatment of skin disorders offers numerous potential advantages over systemic therapies, such as those involving the use of oral or parenteral products. These include avoidance of hepatic first-pass metabolism, improved patient compliance and ease of access to the absorbing membrane, i.e. the skin. In addition, by directly administering the drug to the pathological site, any adverse effects associated with systemic toxicity can be minimised. However, the targeted delivery of drugs for the treatment of topical disorders is not trivial since percutaneous drug absorption is a partition-diffusion process. First the drug partitions from the formulation into the stratum corneum and then, after diffusing across the stratum corneum, it partitions into and diffuses across the epidermis. This partition diffusion process is repeated as the drug moves to deeper skin layers. The physiological function of the stratum corneum, the outermost and nonviable layer of the skin, is to act as a protective barrier for the body and as such, it is particularly effective at preventing the permeation of hydrophilic molecules, including some drugs, into deeper skin layers where viable cells are located. The excellent barrier properties of the stratum corneum means that in order to achieve therapeutic levels of many drugs additional methods of chemical or physical penetration enhancement are often required. However, the use of such methods can promote the transport of drug all the way through the skin into the systemic circulation. Entry into the blood stream can be desirable if systemic effects are required (as with transdermal patches) but, if the target receptor for the drug lies within the epidermis, it increases the chances of undesirable side effects. Consequently, in topically applied formulations there is a need for a system which aids drug permeation through the stratum corneum whilst avoiding further penetration all the way across the deeper layers of the skin (the dermis) and into the blood stream. Obviously, such a delivery system using traditional formulation approaches is difficult to achieve. Relatively few investigations have been conducted into the development of drug delivery technologies that facilitate targeting of drugs to the superficial layers of the skin, although one such system which is receiving increasing attention is H A " . Solaraze®, a 3% diclofenac in 2.5% H A gel for the topical treatment of actinic keratoses (AKs), has recently 20

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received regulatory approval in the US, Canada and Europe . A K is the third most common skin complaint in the US and results from excessive sun exposure. A K lesions are most common on the scalp, face, and/or dorsum of the hands in light skinned persons aged >50 years, particularly those with a history of occupational exposure to the sun. A K are now considered to be synonymous with squamous cell carcinoma (SCC) in situ with growing evidence that A K and S C C lie on a clinical, histological, cytological and molecular continuum . However, a mechanism of action to explain the topical delivery properties of H A remains to be elucidated. Thus, the aim of this study was to compare the effect of molecular weight and concentration of H A , in comparison to other glycosaminoglycans and pharmaceutically relevant polysaccharides, on the percutaneous penetration of diclofenac and ibuprofen.


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Materials and Methods Materials Sodium hyaluronate (viscosity average molecular weight (Mv) 600,000) was kindly donated by SkyePharma (London, U K ) . Sodium hylauronate (viscosity average M v 137,000) was prepared by autoclaving a solution of high M v H A at 121°C for 110 min. A l l other materials were obtained from Sigma (Poole, U K ) . All solvents used for chromatography were analytical grade. Partition cells were made on site at King's College London and were constructed from polypropylene in two parts. The lower part provided a base to support the skin sample during the partition studies, whilst the upper part, comprising a hollow threaded cylinder, was employed to constrain the skin sample in situ. The Franz cells comprised a sampling port, a donor compartment (internal diameter 0.85 cm) and receptor compartment (volume 1.80 ml) which were fixed together using a clip. The Franz cells were also made on site from glass.

Methods

Skin preparation Human skin was obtained from patients undergoing elective abdominoplastic surgery. This procedure recieved full approval from King's College London Research Ethics Committee and informed consent was obtained



146 from the donors who were females, aged between 25-40 y. Excised skin was frozen at -20°C within 2 h of surgery and stored until use. Full thickness skin sections (dermis and epidermis layers) were prepared by carefully removing subcutaneous fat and other debris using forceps and scalpel. For the epidermal sheet experiments individual portions of skin were immersed in water at 6 0 ° C for 45 s. The skin was then placed flat, dermis side down, on a corkboard and the epidermis (comprising SC and viable epidermis) was gently removed from the underlying dermis using forceps and floated onto the surface of cold water. A l l skin samples were then cut into small circular pieces using a template of similar dimensions to the partition cell or Franz cell. Skin from the same section was used for each drug study enabling comparison of the formulation effect for the same drug but not between drugs.

Skin-partitioning studies Donor solutions containing drug (10 μg/ml for diclofenac, 20 μg/ml for ibuprofen) and 1% w/w polysaccharide were prepared in deionised water (controls were drug in deionised water alone, DW) and allowed to hydrate for 24 h. The skin was mounted in the partition cells with the epidermis side up, and the unit was then placed into an air tight 50 ml glass jar containing 20 ml of the donor solution such that only the epidermal surface was exposed to the solution. The bottle was then transferred to a water bath ( 3 2 ° C ) and shaken for 48 h. Preliminary experiments showed that this time was required for equilibration to be established. Samples were then removed and assayed for drug content by H P L C . The amount of drug which partitioned into the skin was measured indirectly by the loss of compound from the donor solution. The percentage of drug partitioning into the skin was calculated from the concentration of compound in the donor solution before and after equilibrium. Control experiments were performed with no skin in the partition cell to determine any loss due to adsorption to the partition cell or glass. Results are expressed as mean + s.d for η = 4 experiments.

Franz cell studies Small sections of full thickness skin for the dermal deposition studies or epidermal sheet for the diffusion studies were mounted, stratum corneum side up, in the Franz cells. The receptor compartment was filled with pH 7.4 phosphate buffer after which the diffusion cell was placed on a stirring plate in a water bath maintained at 32°C. The receptor fluid was continuously stirred using a small teflon-coated magnetic bar to ensure complete mixing of the receptor


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fluid. The Franz cells were allowed to stand for 12 h in order to equilibrate the skin and also to remove any visceral debris that may have remained on the dermal side of the skin. After equilibration, the receptor fluid was changed by replacing with fresh buffer and any air bubbles which had accumulated inside the receptor compartment or at the skin/receptor interface, were removed by tilting the cell. In the dermal deposition experiments 50 μg of each formulation containing 1.75% w/w diclofenac or 20% w/w ibuprofen in 1% w/w polysacharides (or deionised water) unless otherwise stated, was applied to the surface of the skin (n = 4 for each formulation) and rubbed in with a circular motion using a preweighed glass rod. Throughout the 48 h of the experiment, the receiver fluid was stirred to ensure homogeneity, and was also maintained at the same level as the skin. After 48 h, the experiment was terminated and the skin removed from the diffusion cell to enable the mass balance study to be performed in order to quantify the amount of drug on the skin, in the skin and in the receiver fluid. The extraction techniques, which involved homogenisation, collagenase digestion, ultracentrifugation and solvent extraction, were all validated and proved to be 100% efficient in recovering diclofenac and ibuprofen from all of the sample matrices. Drug concentration was measured using H P L C analysis. The diffusion experiments were performed using similar methodology to the dermal deposition studies apart from 10 μg of a saturated solution of each drug in the specified H A concentration being used. In addition, during the 48 hours of the experiment, at appropriate time intervals, 0.2 ml of the receptor fluid was withdrawn and replaced by the the same volume of fresh buffer solution. Drug concentration of each of the removed samples was measured using H P L C analysis.

HPLC analysis of drug concentration The H P L C analysis of diclofenac and ibuprofen was performed using a 15 cm χ 4.6 mm I.D. Spherisorb RP-C18 (5 μπι) column (Hichrom Ltd., Reading, Berkshire, U K ) , with a 10 mm CI8 guard column (S5ODS2-10C5) (Hichrom Ltd., Reading, Berkshire, U K ) using a C M 4000 pump and a CI-4100 integrator connected to a SpectroMonitor 3100 U V detector (all L D C Analytical, Florida, USA). Analysis was performed isocratically with a mobile phase comprising 73% phosphate buffer (45 mM, pH 7) and 27% acetonitrile: tetrahydrofiiran (7:3 v/v). A l l samples were made up in mobile phase, containing an internal standard (20 μg/ml). For diclofenac analysis, ibuprofen was used as the internal standard, and vice versa. A n injection volume of 100 μΐ was used, the samples were eluted at a flow rate of 1.10 ml/min and monitored at 273 nm.


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Results


Partition studies The effects of 1% w/w polysaccharide concentrations on the skin partitioning of ibuprofen is shown in figure 1. Similar results were found with diclofenac (data not shown). The results demonstrate that all three glycosaminoglycans: H A , Chondroitin sulfate (CS) and heparin (HP) significantly increased the partitioning of diclofenac and ibuprofen into the skin compared to the aqueous control. The extent of the increase induced by H A however was greater than that for both CS and HP at the same w/w concentrations. In contrast, N a C M C was found not to increase the partitioning of either diclofenac or ibuprofen into skin (p>0.05).

Franz cell studies

Dermal deposition studies The amount of drug on the surface of the skin and diffused through to the receptor compartment was determined 48 h after application of 1.75% w/w diclofenac or 20% w/w ibuprofen all formulated in 1% w/w polysaccharide formulations. None of the polysaccharides had a significant effect on the amount of diclofenac remaining on the skin surface compared to the control (data not shown). However, H A significantly reduced the amount of ibuprofen remaining (pCS>NaCMC^DW. Significantly more diclofenac and ibuprofen was delivered into the skin when applied in H A or C S , when compared to the other polysaccharides and the aqueous control. In addition, it was found that H A delivered significantly more drug to the skin when compared to CS (p

Polysaccharides for Drug Delivery and Pharmaceutical Applications

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