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ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION TO TOPICAL PHOTODYNAMIC THERAPY
  4. DRUG DELIVERY AND DIFFUSION
  5. CHARACTERISTICS OF TOPICAL ALA-BASED PDT
  6. TISSUE DISPOSITION OF ALA FOLLOWING TOPICAL ADMINISTRATION
  7. TISSUE PENETRATION ENHANCEMENT STRATEGIES
  8. ENHANCEMENT OF PROTOPORPHYRIN IX FORMATION FOLLOWING TOPICAL ALA DELIVERY
  9. FORMULATION OF ALA-CONTAINING TOPICAL DRUG DELIVERY SYSTEMS
  10. CONCLUSION
  11. REFERENCES

Topical photodynamic therapy is used for a variety of malignant and pre-malignant skin disorders, including Bowen's Disease and Superficial Basal Cell Carcinoma. A haem precursor, typically 5-aminolevulinic acid (ALA), acting as a prodrug, is absorbed and converted by the haem biosynthetic pathway to photoactive protoprophyrin IX (PpIX), which accumulates preferentially in rapidly dividing cells. Cell destruction occurs when PpIX is activated by an intense light source of appropriate wavelength. Topical delivery of ALA avoids the prolonged photosensitivity reactions associated with systemic administration of photo-sensitisers but its clinical utility is influenced by the tissue penetration characteristics of the drug, its ease of application and the stability of the active agent in the applied dose. This review, therefore, focuses on drug delivery applications for topical, ALA-based PDT. Issues considered in detail include physical and chemical enhancement strategies for tissue penetration of ALA and subsequent intracellular accumulation of PpIX, together with formulation strategies and drug delivery design solutions appropriate to various clinical applications. The fundamental aspects of drug diffusion in relation to the physicochemical properties of ALA are reviewed and specific consideration is given to the degradation pathways of ALA in formulated systems that, in turn, influence the design of stable topical formulations.


Abbreviations
ALA

5-aminolevulinic acid

ATBC

acetyl tributyl citrate

BCC

basal cell carcinomas

CE

capillary electrophoresis

DDC

3,5-diethoxycarbonyl-1,4-dihyrocollidine

DEF

desferrioxamine

DHPY

3,6-dihydropyrazine 2,5-dipropionic acid

DMSO

dimethyl sulfoxide

EDTA

ethylendiaminetetraacetic acid

LDL

low-density lipoprotein

NMR

nuclear magnetic resonance

PBGD

porphobilinogen deaminase

PDD

photodiagnosis

PDT

photodynamic therapy

PpIX

photosensitizer, protoporphyrin IX

PSA

pressure-sensitive adhesive

PY

pyrazine 2,5-dipropionic acid

INTRODUCTION TO TOPICAL PHOTODYNAMIC THERAPY

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION TO TOPICAL PHOTODYNAMIC THERAPY
  4. DRUG DELIVERY AND DIFFUSION
  5. CHARACTERISTICS OF TOPICAL ALA-BASED PDT
  6. TISSUE DISPOSITION OF ALA FOLLOWING TOPICAL ADMINISTRATION
  7. TISSUE PENETRATION ENHANCEMENT STRATEGIES
  8. ENHANCEMENT OF PROTOPORPHYRIN IX FORMATION FOLLOWING TOPICAL ALA DELIVERY
  9. FORMULATION OF ALA-CONTAINING TOPICAL DRUG DELIVERY SYSTEMS
  10. CONCLUSION
  11. REFERENCES

Photodynamic therapy (PDT) is a clinical treatment that combines the effects of visible light irradiation with subsequent biochemical events that arise from the presence of a photosensitizing drug (possessing no dark toxicity) to cause destruction of selected cells (1). The photosensitizer, when introduced into the body, accumulates in rapidly dividing cells and a measured light dose of appropriate wavelength is then used to irradiate the target tissue (2,3). This activates the drug through a series of electronic excitations and elicits a series of cytotoxic reactions, which can be dependent on, or independent of, the generation of reactive oxygen species (4).

PDT has progressed considerably from the early application of sunlight and hematoporphyrin derivative, to the use of Photofrin®, and to second-generation preformed photosensitizers and topical (surface) application of the prodrug 5-aminolevulinic acid (ALA), which leads to in situ synthesis of protoporphyrin IX (5). Topical PDT is now used for a variety of malignant and premalignant skin disorders and has been reviewed comprehensively (6,7). Clinical acceptance of topical PDT, in particular, has been accredited to the pioneering work of Kennedy et al. in 1990 (8). The results of this first clinical trial of topical PDT exploited the tumor-selective accumulation of the photosensitizer, protoporphyrin IX (PpIX), following topical cutaneous application of ALA. A 90% clearance rate was achieved in 80 lesions treated with 20% w/w ALA in an o/w cream followed 3–6 h later by local illumination from a 500-W lamp equipped with a 600-nm long-wave-pass filter. The popularity of ALA, as the most commonly studied agent for PDT, is clearly evident in the number of published articles on the topic, which has increased markedly from 2 in 1991 to about 3000 in 2003.

The detailed mechanism of action of PDT has been discussed extensively elsewhere (9–11). Briefly, it results from the interaction of photons of visible light of appropriate wavelength with intracellular concentrations of photosensitizing molecules. Photosensitizers have a stable electronic configuration, which is in a singlet state in their lowest or ground energy level (10). This means that there are no unpaired electron spins (12,13). Following absorption of a photon of light of specific wavelength, a molecule is promoted to an excited state, which is also a singlet state and is short lived with a half-life between 10−6 and 10−9 s (10,11). The photosensitizer can return to the ground state by emitting a photon as light energy, or, in other words, by fluorescence, or by internal conversion with energy lost as heat. Alternatively, the molecule may convert to the triplet state. This conversion occurs via intersystem crossing, which involves a change in the spin of an electron (14). The triplet-state photosensitizer has lower energy than the singlet state but has a longer lifetime.

The singlet-state sensitizer can interact with surrounding molecules via Type I reactions, while the triplet-state sensitizer can interact with its surroundings via Type II reactions. The former type of reaction leads to the production of free radicals or radical ions via hydrogen or electron transfer. These reactive species, after interaction with oxygen, can produce highly reactive oxygen species, such as the superoxide and peroxide anions, which then attack cellular targets (9). However, Type-I reactions do not necessarily require oxygen and can cause cellular damage directly, through the action of free radicals, which may include sensitizer radicals. Type-II reactions, by contrast, require an energy transfer mechanism from the triplet-state sensitizer to molecular oxygen, which itself normally occupies the triplet ground state (3). Although possessing a short lifetime of approximately 10−6 seconds, a sufficient concentration of highly cytotoxic singlet oxygen is produced to induce irreversible cell damage (9,11). In addition, the photosensitizer is not necessarily destroyed, but can return to its ground state by phosphorescence without chemical alteration and may be able to repeat the process of energy transfer many times (14). Alternatively, the sensitizer may return to ground by transferring its energy to molecular oxygen and may even be destroyed by photobleaching due to oxidation (15). Evidently, many effects of PDT are oxygen dependent and rely on the oxygen tension within the target tissue. Type-I and Type-II reactions can occur simultaneously and the ratio between the two depends on the photosensitizer, substrate, oxygen concentration and sensitizer to substrate binding (9). Singlet oxygen is, however, widely believed to be the major damaging species in PDT (1,2,10). Due to its extreme reactivity, singlet oxygen has a short lifespan in a cellular environment and limited diffusivity in tissue, allowing it to travel only approximately 0.1 μm (16). This, combined with the fact that normal tissue may not contain photosensitizers or may not be perfused by blood vessels damaged by PDT, means that this normal tissue is normally unaffected by exposure to light (2).

DRUG DELIVERY AND DIFFUSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION TO TOPICAL PHOTODYNAMIC THERAPY
  4. DRUG DELIVERY AND DIFFUSION
  5. CHARACTERISTICS OF TOPICAL ALA-BASED PDT
  6. TISSUE DISPOSITION OF ALA FOLLOWING TOPICAL ADMINISTRATION
  7. TISSUE PENETRATION ENHANCEMENT STRATEGIES
  8. ENHANCEMENT OF PROTOPORPHYRIN IX FORMATION FOLLOWING TOPICAL ALA DELIVERY
  9. FORMULATION OF ALA-CONTAINING TOPICAL DRUG DELIVERY SYSTEMS
  10. CONCLUSION
  11. REFERENCES

The clinical success of topical ALA-based PDT is in part influenced by pharmaceutical and physicochemical considerations, such as tissue-penetration characteristics of ALA, its ease of application and stability in the applied dose. These, in turn, are critically dependent on formulation factors, notably the design of the topical drug delivery system. Before any formulation design can be considered, it is important to appreciate the permeation process within a dosage form matrix, this being a fundamental mechanism by which most semisolid formulations control the drug-delivery process.

Molecular diffusion is a passive-transport mechanism that always directs a chemical system toward a state of thermodynamic equilibrium. It is a random process and the driving force behind the mass transfer that occurs from applied topical dosage systems. If applied to skin, for example, the depth of drug penetration can be such that the drug is localized in cutaneous sites or it may extend deeper to the underlying capillary network and bring about transdermal delivery into the systemic circulation. Diffusion of drug molecules occurs from areas of high concentration to those where the concentration is lower and is described by Fick's first law of mass flow. The movement of drug through a cross-section of area, S, in unit time, t, is known as the flux, J. This is a vector that gives the direction and magnitude of the transport. Considering the one-dimensional form along the x component, the flux is shown to be proportional to the concentration gradient, as shown in Eq. 1,

  • image(1)

where M is the amount of drug diffusing through a distance, x, and D is the diffusion coefficient.

Most drug-release experiments use a receptor and donor phase separated by a membrane, as shown in Fig. 1. The membrane can be a biological barrier, such as excised skin, or a model membrane, such as silicone sheeting (17). During the initial part of the experiment, a nonsteady state exists, where Fick's second law describes how the concentration of diffusant varies at a distance, x, through the membrane with respect to time. After a period of time, a quasi-stationary state (pseudosteady state) is established, where the concentration gradient (δC/δx) across the membrane remains constant and does not vary with respect to time. During this state, it is possible to express Eq. 1 as

image

Figure 1. Schematic representation of a typical side-by-side diffusion cell apparatus. The concentrations across the cells are represented in the top part of the figure. The concentration gradient across the membrane of thickness h is shown at steady state. Cd is not expected to equal C1 unless the partition coefficient of the drug into the membrane from the donor phase is unity.

  • image(2)

where Cd and Cr are the concentrations in the donor and receptor phases, respectively, h is the thickness of the membrane and K is the partition coefficient into and out of the membrane from the phases.

Drug release from a cream- or ointment-type delivery system can be evaluated using a diffusion cell, such as that shown in Fig. 1, but with some modification. Diffusion of the drug through the semisolid matrix becomes an important consideration. If the donor phase is replaced by a sample of the formulation, the membrane can either be rate limiting in terms of drug permeating to the receiver phase or can simply act as a means to separate the phases. In the latter case, the membrane poses little resistance to drug diffusion. The membrane may be dispensed with altogether in cases where a hydrophobic formulation is poorly miscible in an aqueous receiver phase.

Incorporating a drug with limited solubility in a semisolid matrix results in a suspension-type formulation. Even moderate loadings will inevitably possess a substantial fraction of drug in the solid phase. In this case, the Higuchi equation is applicable, as shown in Eq. 3.

  • image(3)

where Q is the amount of drug depleted per unit area of matrix, Cs is the solubility of drug in the matrix and A is the total drug per unit volume. When ACs, which is the case here, Eq. 3 can be differentiated with respect to t to yield Eq. 4.

  • image(4)

It is clear from Eq. 4 that the rate of release can be increased by increasing the total amount of drug in the formulation, its solubility and its diffusion coefficient. Ordinarily, drug release that is under matrix (bulk formulation) control will display linear √t kinetics.

Under conditions where the drug release greatly exceeds diffusional resistance at any interfaces between the donor and receiver phases and the drug has significant solubility in the vehicle, then Eq. 3 is expected to hold. It must be remembered that the solubility of ALA is high in aqueous formulations so that ACs is no longer considered appropriate. However, if the stratum corneum exerts a significant resistance to diffusion, which is known to be the case, then barrier resistance must be considered similarly. In these cases, when the stratum corneum is believed to be the rate-controlling factor, Eq. 2 can be used, providing the stratum corneum thickness, h, is known and remains constant.

ALA is a particularly water-soluble molecule. Even when loadings as high as 20% w/w are used, no solid drug exists and all is present in the dissolved state. In most topical systems used during ALA-based PDT, it is clear that A < Cs and the following solution to Fick's law can be used when a pseudosteady state is assumed:

  • image(5)

and differentiating with respect to t gives the rate of release;

  • image(6)

where Mt is the amount of solute released in time, t, per unit area and providing the skin acts as a perfect sink and is not a barrier to permeation. This is unlikely to be true when ALA is applied to intact skin. However, the disordered structure associated with many superficial lesions (18) and the lack of a keratinized barrier in certain mucous membranes, such as the oral cavity (19) and vulval epithelium (20), may present situations where Eq. 5 is valid.

It is clear in Eq. 5, as with Eq. 3, that Mt, vst is linear. This demonstrates that the mass of ALA released from a semisolid matrix, such as a cream vehicle, does not remain constant with respect to time. Thus, the mass of ALA released during the initial application phase is more than that after a period of time has elapsed. It is also clear that drug release can be enhanced by increasing A and D. Indeed, these strategies have been manifest in the high loadings characteristic of cream-based preparations (21–23) and the use of alkyl esters of ALA (24), the latter prodrug approach increasing the diffusivity of the parent compound through lipid-rich regions of the stratum corneum.

Equation 6 defines the relationship between the diffusivity of the drug and the rate of release. The diffusivity of ALA through a matrix with less hydrophilic character, such as an emulsion system with a high proportion of lipid, is expected to be low. This will not be so when using a more hydrophilic vehicle, such as a structured aqueous hydrogel, where upon the water solubility of ALA will permit a more rapid flux through the matrix. Permeation through the stratum corneum arising from a crystalline ALA deposition on the skin surface is most unlikely, unless dissolution occurs beforehand.

CHARACTERISTICS OF TOPICAL ALA-BASED PDT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION TO TOPICAL PHOTODYNAMIC THERAPY
  4. DRUG DELIVERY AND DIFFUSION
  5. CHARACTERISTICS OF TOPICAL ALA-BASED PDT
  6. TISSUE DISPOSITION OF ALA FOLLOWING TOPICAL ADMINISTRATION
  7. TISSUE PENETRATION ENHANCEMENT STRATEGIES
  8. ENHANCEMENT OF PROTOPORPHYRIN IX FORMATION FOLLOWING TOPICAL ALA DELIVERY
  9. FORMULATION OF ALA-CONTAINING TOPICAL DRUG DELIVERY SYSTEMS
  10. CONCLUSION
  11. REFERENCES

The ideal photosensitizer is one that shows a high tumor-to-normal-tissue ratio, exhibits rapid accumulation in tumor tissue and is cleared efficiently from the body (25,26). Localization of preformed photosensitizers in neoplastic tissue has been shown, though its mechanism is not completely understood. Preformed, lipophilic sensitizers, such as the porphyrins and phthalocyanines, when administered intravenously, are believed to be transported in the bloodstream bound to lipoproteins, such as low-density lipoproteins (LDL) (14,27). Tumor-cell membranes are known to possess disproportionately high numbers of LDL receptors (28), leading to active accumulation of photosensitizer molecules at close proximity to tumor cells. Photosensitizers may also accumulate in tumors due to abnormalities in the local microvasculature, including disordered blood supply and enhanced vascular permeability (27,29).

ALA is a small, water-soluble, prodrug that is a naturally occurring precursor in the biosynthetic pathway of heme. Administration of excess exogenous ALA avoids the negative feedback control that heme exerts over its biosynthetic pathway. Due to the limited capacity of ferrochelatase to convert PpIX into heme, the presence of excess exogenous ALA in cells induces accumulation of PpIX (8,22,30). This effect is pronounced in sebaceous glands and also in neoplastic cells. It has been reported that certain types of neoplastic cells have not only reduced ferrochelatase activity but also enhanced porphobilinogen deaminase (PBGD) activity (3,9,31). Low ferrochelatase activity may be of lesser import to deficiencies in mitochondrial energy generation because tumor cells typically have low activities of mitochondrial cytochrome oxidase and utilize glycolysis rather than oxidative phosphorylation (32). In addition, certain malignant cells have low iron stores, a characteristic of proliferating cells, leading both to increased expression of transferrin receptors and, importantly, to decreased conversion of PpIX into heme (33,34). Porphobilinogen deaminase is considered to have the lowest activity in the heme biosynthetic pathway (35,36) and the reason for its upregulation in certain tumor cells has not yet been elucidated (9). It is generally considered to be rate limiting in the ALA-induced synthesis of PpIX in neoplastic cells (37). Notwithstanding this, other reports have failed to find a clear relationship connection between high PBGD and/or low ferrochelatase activity and PpIX accumulation (38–40).

To date, clinical applications of PDT have been limited to areas of the body easily amenable to irradiation from laser or incoherent light sources. Consequently, PDT has been primarily investigated as a treatment for tumors and neoplasias of the skin, bladder, mouth and female reproductive tract. Compounds of high molecular weight (>500 Daltons) have inherently low permeabilities of the stratum corneum barrier of the skin (17). Therefore, notwithstanding a few isolated studies (41,42), preformed photosensitizers, which are generally large, highly conjugated molecules, are not commonly used in topical PDT. This, coupled with their inherent lack of selectivity, means that ALA, a photosensitizer prodrug with a relatively low molecular weight of 167.8 Daltons, is the most frequently employed agent in modern topical PDT.

PDT, based on topical application of ALA, has been successfully used in the treatment of basal cell carcinoma (8,18,21,43), actinic keratosis (22,23,44), Bowen's disease (45–47), vulval intraepithelial neoplasia (20,48,49), vulval Paget's disease (50) and cervical intraepithelial neoplasia (51). Due to the highly selective accumulation of PpIX in neoplastic cells resulting from topical application of ALA, the technique has also found use in the photodiagnosis (PDD) of neoplastic lesions of the mouth (19), bladder (52,53) endometrium (54) and cervix (55). Illumination of the treated area with UV light causes reddish-pink PpIX fluorescence in neoplastic tissue, while the surrounding healthy tissue appears blue. The technique often allows detection of subclinical lesions, which may be missed by conventional means of examination.

PDT using topically applied ALA, in addition to producing successful therapeutic outcomes with excellent tissue preservation and no scarring, does not give rise to cutaneous phototoxicity (18,56). Thus, ALA-PDT can be repeated often without causing accumulation of PpIX in normal skin (11). This is particularly important when the aim of treatment is primarily palliative. This is in contrast to conventional PDT using the older preformed sensitizers, such as hematoporphyrin derivative. Repeated administration of such agents leads to high photosensitizer levels in normal skin and severe phototoxic reactions after sun exposure (8,10).

TISSUE DISPOSITION OF ALA FOLLOWING TOPICAL ADMINISTRATION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION TO TOPICAL PHOTODYNAMIC THERAPY
  4. DRUG DELIVERY AND DIFFUSION
  5. CHARACTERISTICS OF TOPICAL ALA-BASED PDT
  6. TISSUE DISPOSITION OF ALA FOLLOWING TOPICAL ADMINISTRATION
  7. TISSUE PENETRATION ENHANCEMENT STRATEGIES
  8. ENHANCEMENT OF PROTOPORPHYRIN IX FORMATION FOLLOWING TOPICAL ALA DELIVERY
  9. FORMULATION OF ALA-CONTAINING TOPICAL DRUG DELIVERY SYSTEMS
  10. CONCLUSION
  11. REFERENCES

ALA is a small molecule (167.6 Daltons), favoring its diffusion into cutaneous tissue from a topical delivery system. However, the hydrophilic nature of ALA, as evident from its low octanol: water partition coefficient of 0.03 (57), does impair permeation markedly because skin presents an essentially hydrophobic barrier to the permeation of exogenously applied agents. As a result, topically applied ALA penetrates intact stratum corneum poorly, making it the principal barrier to effective absorption (58,59). Fortuitously, the disordered stratum corneum and disrupted epithelial barriers offered by many neoplastic lesions allow enhanced ALA penetration due to poor continuity in intercellular lipid structures. This further improves the selectivity of PpIX accumulation and explains why ALA can be successfully employed for diagnostic purposes. However, its low lipophilicity (60) does prevent effective penetration into hyperkeratotic lesions (18,23,61) and may even facilitate efflux via the local microcirculation from deep nodular lesions (62).

The clinical success of PDT relies on achieving a threshold concentration of ALA after topical application that induces therapeutic levels of protoporphyrin IX (PpIX) in abnormal cells. Failure to achieve this threshold leads to insufficient amounts of PpIX and irradiation of the target cells will then not generate enough singlet oxygen to eradicate the lesion successfully. Clearly, it is important that this threshold is evaluated. Cell culture experiments have demonstrated that concentrations of interstitial ALA must reach levels between 0.01 mg·ml−1 and 0.17 mg·ml−1 before sufficient PpIX is produced to cause a significant (>90% kill) level of neoplastic cell death upon illumination with an optimized dose of red light (15,24,63,64).

Numerous literature reports describe both in vitro and in vivo penetration of topically applied ALA into tissue. However, while a range of techniques have been used to assess the final penetration depth, few report on the specific ALA concentrations found at various depths from the plane of surface absorption. Most studies to date have used fluorescence microscopy to investigate the formation of PpIX in tissue after topical application of ALA. In the majority of cases, an ALA-containing vehicle is applied topically to normal or diseased skin of animal or human volunteers. The formulation is generally left in place for 4–6 h before a biopsy is taken from the application site. Sectioning of the biopsy allows the fluorescence intensity of PpIX to be evaluated using suitable microscopy with excitation wavelengths around 400 nm and emission wavelengths from 600 to 700 nm (3,65). Alternatively, PpIX is extracted from dissolved tissue samples and determined using fluorescence spectrophotometry with similar excitation and emission wavelengths as above (33,66,67). It is clear that most studies tend to be qualitative in nature, comparing PpIX fluorescence with background autofluorescence. They serve, simply, to give an indication of depth of PpIX formation and, by inference, ALA penetration. Konig et al. (68) reported PpIX fluorescence at a depth of 0.6 mm, 6 h after topical ALA application in patients with skin tumors. Szeimies et al. (69) reported PpIX fluorescence at a depth of 0.3 mm in basal cell carcinomas (BCC). In contrast, Pahernik et al. (70) reported PpIX fluorescence at depths as low as 3 mm in hamster skin tumor models.

Wennberg et al. (71) used microdialysis to quantify ALA in normal skin and BCC after topical application of ALA (20% w/w) in an aqueous gel. A microdialysis tube was inserted intracutaneously at a depth of 0.5 mm and samples were taken at regular time intervals for analysis by high-performance liquid chromatography. The concentration of ALA in BCC at a depth of 0.5 mm was found to range between 0 mg·mL−1 and 0.52 mg·mL−1. No ALA could be detected in normal skin at this depth. While this study was able to quantify ALA concentrations at a depth of 0.5 mm, the drug concentrations above and below this depth could not be assessed nor could the depth of ALA penetration.

Casas et al. (58), using liquid scintillation spectrometry, showed that ALA could penetrate model mouse tumors down to depths of 5 mm, although the majority of drug was found in the upper 2 mm of tissue. ALA concentrations at the different depths were not reported, in contrast with the studies carried out by Ahmadi et al. (72) and McLoone et al. (73). In these studies, the authors used liquid scintillation spectrometry to determine the concentrations of ALA at varying depths from the surface of nodular BCC. Concentrations of ALA as high as 20 mg·mL−1 were detected at depths as low as 2 mm in these nodular lesions. However, once the integrity of the stratum corneum is more intact, as in normal skin, then permeation is shown to slow. Johnson et al. (74) used autoradiography to demonstrate that the majority of a topically applied ALA dose did not penetrate porcine skin much deeper than the lower reaches of the stratum corneum. The same authors, using scintillation spectrometry, reported that ALA pasting through the entire stratum corneum barrier only achieved depths of 100–150 μm in underlying tissue. The importance of the stratum corneum as a barrier to ALA penetration was illustrated by Donnelly et al. (75). The authors showed, using autoradiography, that topically applied ALA could penetrate vaginal tissue, which possesses no stratum corneum barrier, down to depths of at least 6 mm.

TISSUE PENETRATION ENHANCEMENT STRATEGIES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION TO TOPICAL PHOTODYNAMIC THERAPY
  4. DRUG DELIVERY AND DIFFUSION
  5. CHARACTERISTICS OF TOPICAL ALA-BASED PDT
  6. TISSUE DISPOSITION OF ALA FOLLOWING TOPICAL ADMINISTRATION
  7. TISSUE PENETRATION ENHANCEMENT STRATEGIES
  8. ENHANCEMENT OF PROTOPORPHYRIN IX FORMATION FOLLOWING TOPICAL ALA DELIVERY
  9. FORMULATION OF ALA-CONTAINING TOPICAL DRUG DELIVERY SYSTEMS
  10. CONCLUSION
  11. REFERENCES

The more common means of drug administration have been used for ALA delivery, such as the oral (76) and parenteral (77) routes. Although PpIX accumulation and prolonged patient photosensitization are not problematic, side effects, such as nausea (78) and abnormalities of liver function, are of concern (79,80). In addition, it has been reported that ALA, when given systemically, can permeate across the blood-brain barrier, but the clinical implications of this observation are still unclear (81). These factors ensure that the topical route remains a viable alternative, especially when the neoplastic lesion is superficial in nature. In such cases, the diffusion of ALA and its ability to reach deep sites becomes an important consideration, especially through the stratum corneum, and one that will influence the expected clearance rate, In a small number of studies, this barrier to ALA diffusion has been bypassed completely and the drug has been administered via intracutaneous injection directly into skin tumors (82,83). Generally, though, more conventional strategies to enhance ALA penetration through intact stratum corneum have been devised. These include chemical methods, such as the use of chemical derivatives of ALA or penetration enhancers and physical methods, such as tape stripping, curettage and iontophoresis.

Chemical methods for enhanced tissue diffusion

The lipophilicity and molecular weight of a drug substance are considered to be the primary determinants of diffusivity through stratum corneum (84). This layer of the epidermis comprises mostly anucleate cells and is associated with providing the principal barrier function in respect of transdermal delivery of drugs. The stratum corneum has been portrayed as the often cited ‘brick and mortar’ model in which keratinized cells are embedded in a mortar of lipid bilayers. The intercellular route of drug diffusion will not, therefore, be an accessible phase for either very polar or charged species.

Enhanced permeability through the lipid networks in the stratum corneum can be undertaken by chemical derivatization of ALA (85,86). This prodrug strategy imparts a greater lipophilicity to the parent ALA compound, usually accomplished by formation of a labile bond at the amino (87,88) or, most commonly, the carboxylic acid group of the parent ALA molecule. Table 1 shows the octanol/water partition coefficients and the stratum corneum/ water partition coefficients of ALA and several commonly employed ALA esters. It can be seen clearly that increasing the alkyl chain length of ALA esters significantly enhances lipophilicity and the ability to partition into the stratum corneum. However, within a homologous series, drug permeability usually increases with log Poct up to a maximum, at which point transport becomes limited due to sequestration of the drug within the lipophilic barrier. Consequently, following topical application to skin, PpIX fluorescence induced by ALA esters is localized to the site of application, while fluorescence arising from ALA-induced PpIX is typically observed at distant locations. This effect may be directly attributable to prodrug sequestration within the stratum corneum, whereas the more hydrophilic ALA is able to penetrate to vascular networks more effectively. However, because PpIX and ALA are completely cleared from the body within 24 h of administration (89), prolonged and undesirable widespread cutaneous photosensitivity is not problematic.

Table 1.  Log Poct and log PSC/W for ALA and its ester derivatives
Compound*Mol. wt. (Da)log Poctlog PSC/W
  1. *Hydrochloride salt (HCl.H2N-CH2-CO-CH2-COOR1= general structure).

  2. Poct is the partition coefficient between octanol and aqueous buffer solution (pH 7.4, 21°C), as calculated by Uehlinger et al. (110). PSC/W is the partition coefficient between stratum corneum and water, as calculated by De Rosa et al. (91).

ALA167.6−1.5−1.4
Methyl-ALA181.6−0.90.2
Butyl-ALA223.81.40.3
Hexyl-ALA251.81.80.9
Octyl-ALA279.62.61.0

Numerous in vitro and in vivo studies have been carried out to assess the ability of ALA esters to enhance penetration and PpIX production. The majority of in vitro investigations reveal that increased amounts of ALA esters, relative to the parent compound, only penetrate stratum corneum after prolonged application times, sometimes approaching 30 h. Working within a framework of clinically relevant application times, such as 4 or 6 h, no significant difference is observed in amounts of ALA or ALA-esters penetrating stratum corneum, regardless of ester alkyl chain length (90–92). The in vivo studies have typically investigated PpIX production in the skin of human volunteers (93–95) or nude mice (65,86,96,97) following topical application of ALA or one of its esters. Again, significant lag times are generally observed before PpIX fluorescence induced by ALA esters becomes greater than that induced by ALA. These observations are in contrast with those found in PDD of dysplasia and early bladder cancer, which showed that the ALA hexyl ester could not only reduce instillation times but induce a twofold increase in fluorescence signals with a 20 times lower concentration of ALA hexyl ester compared with ALA (98). These findings were attributed to the different properties of the urothelial and stratum corneum permeability barriers.

Chemical methods for enhanced cellular penetration

ALA-induced formation of PpIX depends on the penetration of ALA through the cell membrane. Being a zwitterion with pKa values of 4 (carboxylic acid group) and 8.9 (amino group) (58) the lipophilicity of ALA is unlikely to change significantly in the physiological pH range. It is expected, therefore, that ALA is unlikely to enter cells by passive diffusion alone. In Salmonella typhimurium (99) and Escherichia coli (100), the dipeptide permease is probably responsible for ALA transport across the cell membrane. In eukaryotic cells, the uptake mechanisms are not clear. ALA uptake may rely on an active transport mechanism, as exemplified by that in Saccharomyces cerevisieae, which shows an apparent Km of 0.1 mM at an optimum extracellular pH of 5.0 (101). Mammalian cells may possess additional cell-type dependent mechanisms (9) because ALA uptake in rat cerebellum particles, for example, was found to be nonsaturable up to 4 mM of ALA (24). In addition, Rud et al. (102) showed that ALA is transported into human adenocarcinoma cells by β-amino acid and γ-aminobutyric acid carriers and is Na+ and partly Cl dependent. The PEPT1 and PEPT2 transporters have also been identified as potential transporter systems for ALA uptake (103,104).

The methyl ester of ALA has been shown to be taken up actively by WiDr cells using transporters of nonpolar amino acids (105). However, longer chain aliphatic ALA esters are not transported by these carriers and it has been postulated that they may enter cells by either passive diffusion or endocytosis (106,107). Once in the cell, the esters may be converted to ALA by nonspecific esterases. Cell culture studies have demonstrated that aliphatic straight chain ALA esters, up as far as the hexyl ester in the homologous series, induce higher levels of PpIX in neoplastic cells more rapidly than the parent compound (24,64,97,108–111), presumably due to their nonrequirement for a saturable active transport mechanism. The optimum PpIX fluorescence in intact murine mammary cancer cell spheroids (275–350 μm) was shown using 0.05 mM hexyl ALA, almost 200 times lower than the optimum concentration of ALA (10 mM). This indicated that not only did the interior cells maintain esterase activity and porphyrin synthesis but that hexyl ALA diffused efficiently to the spheroid interior (112).

It has been suggested that lower concentrations of ALA esters, with shorter application times, would increase the efficacy of PDT and PDD (106). However, with the exception of the case of bladder instillation, topical application of ALA esters does not seem to provide such an advantage. The results obtained from in vitro cell culture studies demonstrate that the time required for cleavage of the ester group to yield free ALA appears to be insignificant and does not seem to limit the usefulness of ALA esters. Retention in, and gradual release from, the stratum corneum, as discussed above, in combination with poor release from the topically applied vehicle may, therefore, be mostly responsible for the observed lag times before significant in vivo PpIX production. In spite of this, ALA methyl ester has been shown to be effective for PDT of nodular basal cell carcinoma (60,113,114), where ALA PDT has historically produced poor results (115,116). However, it should be pointed out that these clinical studies used curettage/debulking to remove the stratum corneum and some of the carcinoma before treatment, and also routinely used a 1–2 treatment cycles that each involved two treatments a week apart. Nevertheless, a topical cream containing 16% w/w methyl ALA (Metvix®, Photocure, Norway) has received market authorization in the United States and Europe.

Formulation-based penetration enhancers

Penetration enhancers can be incorporated into topical dosage forms to increase diffusivity of drug compounds through stratum corneum. Their use, particularly that of dimethyl sulfoxide (DMSO) (60,58,117), has, arguably, been the most commonly employed strategy to enhance tissue penetration of ALA to date (118). Importantly, they generally reduce the barrier resistance in a reversible and innocuous manner (119). The lipid-protein-partitioning theory (120–122) attributes the potential modes of action of penetration enhancers to one or more of three main mechanisms,

  • 1
    disruption of the highly ordered structure of stratum corneum lipids,
  • 2
    interaction with intracellular protein,
  • 3
    improved partitioning of a drug, coenhancer or cosolvent into the stratum corneum.

DMSO is an aprotic solvent, extensively studied in transdermal drug-delivery research. It may act by association with, and solubilization of, stratum corneum lipids and proteins, thereby altering the conformation and barrier properties of the skin (123,124). It is known to be a malignant cell differentiator and has been shown to induce increased activity of cellular ALA synthase, ALA dehydratase and porphobilinogen deaminase in culture (125,126). Clearly, these effects occurring as a result of inclusion of DMSO in topical ALA-containing formulations may act synergistically to any enhancement in ALA penetration. Its effectiveness is known to be highly concentration-dependent, meaning that relatively high concentrations of 60% w/w and above were normally used (127). At such high concentrations, DMSO produces erythema and wheals (128), irreversible skin damage (129), delamination of the stratum corneum and denaturation of its proteins (130). Production of dimethyl sulfide, a DMSO metabolite, produces halitosis. However, some evidence has shown that lesser amounts may be effective also. The addition of 20% w/w DMSO to an o/w emulsion containing 10% w/w ALA has been shown to produce a significant increase in the flux of ALA across skin in vitro (131). Moreover, a 2.5-fold increase in PpIX production was observed in vivo relative to the same formulation without the penetration enhancer. Similarly, glycerol monoleate, an ester of oleic acid, has been shown to increase the in vitro perrnation and retention of ALA at concentrations up to 20% w/w (132).

The formation of a neutral ion pair is a strategy used to overcome the poor permeability of charged drug molecules through stratum corneum. A neutral complex is formed by combining the charged permeant with an oppositely charged, lipophilic species. Enhanced partitioning into the stratum corneum is followed by dissociation to yield the charged species once again (133). Auner et al. (118) used lipophilic counter ions in combination with the penetration enhancers phloretin and 6-ketocholestanol to increase permeation of ALA through porcine skin in vitro. Cationic and anionic counter ions enhanced ALA permeation at pH 7.0 and 4.0, respectively, and the combination of 6-ketocholestanol and cetylpyridinium chloride at pH 7.0 was considered a potential strategy for increasing ALA penetration.

Physical methods

Skin penetration of drugs can be enhanced by the application of physical procedures. One example is Wolf's skin-stripping technique (134), which removes successive layers of the stratum corneum. Loveday (135) used adhesive tape to show that the penetration of salicylic acid is markedly increased with increased numbers of strippings. Curettage, tape stripping and dermabrasion and delipidation of the stratum corneum with acetone scrubs are techniques that have all been used, prior to ALA or ALA-ester application. Their purpose is to remove the stratum corneum and keratinized debris overlying lesions and, thereby, enhance drug penetration (43,60,62,136,137). In a semiquantitative study, Van den Akker et al. (65) demonstrated reduced PpIX production due to the reservoir capacity of the stratum comeum after topical application of hexyl ALA. When the stratum comeum was removed by tape stripping, PpIX production equalized between topically applied hexyl ALA and ALA. De Rosa et al. (91) quantified the amounts of topically applied ALA or ALA esters retained in the stratum comeum in vitro. The authors showed that, if the barrier could be removed by tape stripping prior to drug application, penetration could be increased significantly.

An increase of between 5- to 20-fold in the transdermal permeation of drugs, such as inulin and mannitol, is observed following application of ultrasound energy (138,139). This effect arises mostly by the generation of heat or cavitation (140,141). Photomechanical waves of this type appear to interact directly with cells and tissue by mechanical forces (142). Although tissue damage is a potential outcome, ultrasound has been shown to induce or enhance the delivery of molecules across the plasma membranes of cells in vitro without loss of viability (143–145). Ma et al. (146) proposed that ultrasound could increase the low intrinsic blood circulation in tumors (147), resulting in a relatively selective uptake of drugs into these cellular masses. The authors showed an increase of 45% in the amount of PpIX produced by transplanted human adenocarcinoma tumors in mice after 1–2 h following ultrasound treatment. Lee et al. (148) showed that a single photomechanical wave (110 × 10−9 s) generated by a 23 × 10−9 s Q-switched ruby laser produced a remarkable 680% increase in ALA penetration through human skin in vivo.

The skin is maintained at a constant temperature of approximately 32°C. The use of an occlusive formulation can cause local skin temperature to rise to 37°C. It is generally accepted that an increase in skin temperature will result in an increase in drug penetration (84). Van den Akker et al. (149) have shown recently that the in vitro penetration of ALA through mouse skin was significantly enhanced when the skin was maintained at 37°C rather than 32°C.

Iontophoresis of topical ALA solutions has been used to increase penetration depths, increase tissue concentrations and reduce topical application times (74,150,151). The technique uses an electrical potential difference to facilitate drug delivery into the skin. The drug, which is normally charged, is dissolved in a suitable vehicle and placed in contact with an electrode of similar polarity. The second electrode is placed in close proximity to complete the circuit. When the current flows, the drug is repelled from the electrode of similar polarity and is attracted toward the oppositely charged electrode, thus driving the drug into the skin by electro-repulsion. To a lesser extent, a process of electroosmosis establishes a convective solvent flow simultaneously in the direction of anode to cathode. This enhances the transport of cations and of neutral, polar compounds (133). Lopez et al. (152) showed that, at pH 7.4, ALA delivery increased linearly with concentration and the drug traversed skin by electroosmosis. When the pH of the topically applied vehicle was reduced to 4.0, electrorepulsion became the principal mechanism due to neutralization of the net negative charge of the skin. The increased electrorepulsive delivery was balanced by a decrease in electroosmotic delivery and, hence, the net ALA flux was similar at both pH values. Interestingly, iontophoresis delivered the same mass of ALA in 10 min as that delivered by several hours of passive diffusion alone.

ALA esters have a net positive charge at physiological pH. Because of this, electrorepulsion can be used to enhance the delivery of methyl ALA over and above that of ALA by approximately 50 times (153,154). The magnitude of this effect gradually decreased with increasing chain length within a homologous series of ALA esters. Conversely, Gerscher et al. (155) could detect no significant difference between the levels of PpIX induced in vivo after iontophoresis of solutions of ALA, butyl ALA or hexyl ALA. This may have resulted from the acidic solutions used in vivo, which converted a greater fraction of ALA to the cationic form and elevated hydronium concentration. The latter carries charge more efficiently than ALA esters and reversed the direction of electroosmotic flow (107). Nevertheless, it must be borne in mind that conversion of ALA and its esters into clinically useful concentrations of PpIX may take several hours in vivo, regardless of how quickly the drug is delivered in the first instance. Moreover, in skin, iontophoresis preferentially carries the drug into the hair follicles, so it may not provide uniform dosage (133). Therefore, the real clinical benefits of iontophoretic delivery for topical PDT are yet to be proven. Further in vitro studies demonstrating both increased depths of penetration of ALA or its esters and increased concentrations in skin are clearly required before iontophoresis becomes a viable method in clinical PDT.

ENHANCEMENT OF PROTOPORPHYRIN IX FORMATION FOLLOWING TOPICAL ALA DELIVERY

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION TO TOPICAL PHOTODYNAMIC THERAPY
  4. DRUG DELIVERY AND DIFFUSION
  5. CHARACTERISTICS OF TOPICAL ALA-BASED PDT
  6. TISSUE DISPOSITION OF ALA FOLLOWING TOPICAL ADMINISTRATION
  7. TISSUE PENETRATION ENHANCEMENT STRATEGIES
  8. ENHANCEMENT OF PROTOPORPHYRIN IX FORMATION FOLLOWING TOPICAL ALA DELIVERY
  9. FORMULATION OF ALA-CONTAINING TOPICAL DRUG DELIVERY SYSTEMS
  10. CONCLUSION
  11. REFERENCES

Iron chelation

Numerous attempts have been made to enhance the accumulation of cellular PpIX, by either inducing enzymatic activity in the heme cycle or by attempting to stop the insertion of elemental iron and subsequent formation of heme. Iron chelation remains the most popular approach, endeavoring to minimize the amount of elemental iron available as a substrate for ferrochelatase. Examples include ethylenediaminetetraacetic acid (EDTA) and desferrioxamine (DEF), the structures of which are shown in Fig. 2. Both show a high affinity for Fe3+, but different degrees of membrane permeability, with DEF considerably higher than that of EDTA. The hydroxypyridones are a relatively new series of iron-chelating agents, which have low molecular weights and greater lipophilicity than DEF. They can be administered orally and may bind iron in a ratio of 3:1 as compared with 1:1 for DEF (156). One of note is 3,5-diethoxycarbonyl-1,4-dihyrocollidine (DDC), a methylated dihydropyridine, which not only sequesters Fe3+ but N-alkylates porphyrins and has a direct bearing on ferrochelatase activity (33).

image

Figure 2. Chemical structures of (A) desferrioxamine (DEF), (B) ethylenediamine-tetraacetic acid (EDTA) and (C) dimethyl sulfoxide (DMSO).

Hanania and Malik (157) incubated K562 human leukemic cells with 50 mM ALA and 10 μM EDTA for 4–5 h and found that the cell viability after irradiation (380 nm, 0.5–2 J·cm−2) was reduced by three orders of magnitude. The cell viability of cells incubated with ALA alone was reduced by two orders of magnitude. Berg et al. (33) investigated the effects of EDTA, DEF and DDC on the formation of PpIX in ALA-treated cells. V79 and WiDr cells were incubated with varying concentrations of ALA alone (0.025–1 mM) or with the same concentrations of ALA plus varying concentrations (1–1000 μM) of the other three agents individually. Both EDTA and DEF were most effective at enhancing PpIX formation at their highest concentrations. Their effects were most pronounced at the lowest ALA concentrations. This was attributed to the demonstrable tendency of ferrochelatase to become saturated when PpIX levels are high due to the presence of excessive amounts of ALA. DEF was found to be marginally more effective at enhancing PpIX formation than EDTA and increased PpIX formation by 3 and 1.4 times in V79 and WiDr cells, respectively, relative to ALA alone. At the lowest ALA concentration, DEF increased PpIX formation 44- and 3.5-fold in V79 and WiDr cells, respectively, relative to ALA alone. The increased effectiveness of DEF compared with EDTA was attributed to the ability of DEF to enter cells and the affinity of EDTA for cations other than iron. DDC, despite increasing PpIX formation in rat and mouse hepatocytes, decreased PpIX formation in the cell lines studied. It was suggested that DDC may not be effective in nonhepatic cells.

There is evidence to suggest that exercising an iron-chelation strategy based on compounds, such as EDTA or DEF, may actually reduce the selectivity of ALA-based PDT by causing PpIX accumulation in normal cells. This is illustrated by the enhanced PpIX production in V79 cells when compared with WiDr cells, as discussed previously. The latter are derived from a human adenocarcinoma, while V79 cells are derived from normal mouse fibroblasts. This finding may be explained by the reduced activity induced by iron chelation in normal cells (V79) becoming equivalent to the inherently low ferrochelatase activity in malignant cells (WiDr). To confirm this, Curnow et al. (156) reported the doubling of PpIX formation in normal rat colon cells after intravenous administration of ALA and hydroxpyridone iron chelators when compared with ALA alone. Conceivably, normal cells adjacent to the target malignant cells may be damaged by irradiation and, if iron chelators and ALA were given systemically, widespread cutaneous photosensitivity could occur.

Enzymatic induction

In addition to its penetration-enhancing properties, DMSO (Fig. 2C) is believed to be a potentiator of malignant cell differentiation (59). At certain concentrations, it can induce various enzymes of the heme biosynthetic pathway. This is at least partially due to transcriptional stimulation (2). DMSO has been shown to induce increased activity of ALA synthase, ALA dehydratase and porphobilinogen deaminase in cultured cells (125,126). However, there is little published data regarding any subsequent increase in PpIX in cells treated with a combination of ALA and DMSO compared with those treated with ALA alone. DMSO is frequently included in ALA-containing vehicles intended for topical application and it has been reported that it causes an increase in PpIX accumulation in treated tissue (58,131). Whether this is due to its role as a penetration enhancer or by way of inducing enzymes in the heme pathway is not clear. What is clear is that DMSO allows ALA to penetrate deeper into both normal skin and skin tumors (131) and, hence, it may have a role in improving the success of PDT treatment of deep or highly keratinized lesions (43). Formulations containing ALA, DMSO and EDTA have been described (117). Inclusion of EDTA in the topically applied vehicle leads to more PpIX formation than with ALA and DMSO alone. However, the effect is limited by the inability of EDTA to penetrate into the skin significantly, even in the presence of DMSO (59).

Levamisole and lonidamine, both inhibitors of respiration and glycolysis, were shown to have a synergistic effect with ALA-based PDT in killing V79 cells (158). Lonidamine decreased PpIX synthesis significantly, while levamisole significantly enhanced it. The authors concluded that the observed synergism between lonidamine and PDT was due to the effect of the former on energy metabolism. Conversely, it was unclear whether levamisole disrupted energy metabolism in this study. Its synergistic effect was attributed to the increased rate of PpIX production.

pH, oxygen tension and temperature effects

Normal tissue is expected to have an extracellular pH of 7.0–7.4, whereas that seen in the cellular matrix of an archetypal tumor can vary between pH 6.5 and pH 6.8 (159). This difference has been shown to influence the outcome of ALA-induced PDT (160). Experiments on cells in culture demonstrated that PpIX formation is maximal at an extracellular pH of around 7.4 and is reduced at pH values above or below this (63,111,161). This is despite the cellular uptake of ALA being maximal around pH 5 (102) and was attributed to the pH dependency of the activity of porphobilinogen deaminase (PBGD), which is maximal at pH 7.4 (161). Although PpIX production is optimal around pH 7.4, the rate of cell kill by PDT at lower extracellular pH values remains mostly unchanged (160,162). This may be attributed to increased susceptibility to free-radical toxicity or alteration of cellular repair enzymes under acidotic conditions (162). In addition, the retention of formed PpIX in cells is enhanced at lower pH values (pH 5.8–6.8), such as those common in tumor masses (63). It has been suggested that PpIX becomes protonated, acquires increased lipophilicity and becomes strongly bound to lipid structures (37). Extracellular pH values below pH 6, however, may influence sensitivity to PDT by inducing cellular quiescence (163). Quiescent cells are less sensitive to PDT than proliferating cells (164). Uehlinger et al. (110) have suggested that ALA formulations for topical or intravesical administration be adjusted to pH 7.4, as topically applied products can influence local extracellular pH (165) and, hence, PpIX formation. This is difficult to do, given the instability of ALA at neutral and alkaline pH (52). In practice, formulating topical ALA-containing vehicles at pH 7.4 is a debatable requirement because, although less PpIX is formed at slightly lower pH, the rate of cell kills remains similar (162).

It is generally accepted that the oxygen tension in normal tissue varies between 5–10%. In tumor tissue, a range of 0–5% is more common. These low oxygen tensions are believed to reduce the formation of PpIX, as demonstrated in bladder cancer cells in vitro (160). In this work, cells incubated at reduced oxygen tensions (0, 2.5 and 5%) demonstrated less PpIX formation. This finding was attributed to the oxygen dependence of the enzymes of the heme biosynthetic pathway. It was also shown that no PDT-induced cell damage resulted when the oxygen tension was 0% during irradiation. These findings have helped to advocate fractionation of the light dose, allowing tumor reoxygenation and enhanced efficacy of PDT (83,166,167).

Moan et al. (36) showed that PpIX formation in human skin was strongly temperature dependent, with an activation energy of around 17 kcal·M−1. Juzenas et al. (168) reported that, for skin temperatures of 12 and 42°C, PpIX formation was significantly increased at the higher temperature. Indeed, Moan et al. (23) reported that elevating the temperature of human skin from 31 to 36°C during a 15-min period of ALA application resulted in a 50% increase in the amount of PpIX formed. The increased production of PpIX at slightly elevated skin temperatures was attributed to increased activity of porphobilinogen deaminase (PBGD) because it has an activation energy similar to that of PpIX formation. Moreover, PBGD is believed to be rate limiting with respect to PpIX formation in neoplastic cells (36,37,169). Therefore, induction of local hyperthermia may enhance PBGD activity, PpIX production and the success rate of PDT. In addition, hyperthermia causes hypoxic cells to become more susceptible to PDT (117) so the success rate of PDT may be further enhanced. In contrast, Von Beckerath et al. (170) showed that, if ALA was applied topically to mouse skin 24–48 h after exposure to erythemogenic doses of UV radiation, significantly less PpIX was produced than in unexposed skin. Temperature elevation arising from arteriolar dilation did not enhance ALA-induced PpIX production. Instead, a decrease was noted that probably arose from altered ALA penetration through the stratum corneum, altered metabolizing ability of UV-exposed skin or a combination of both.

FORMULATION OF ALA-CONTAINING TOPICAL DRUG DELIVERY SYSTEMS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION TO TOPICAL PHOTODYNAMIC THERAPY
  4. DRUG DELIVERY AND DIFFUSION
  5. CHARACTERISTICS OF TOPICAL ALA-BASED PDT
  6. TISSUE DISPOSITION OF ALA FOLLOWING TOPICAL ADMINISTRATION
  7. TISSUE PENETRATION ENHANCEMENT STRATEGIES
  8. ENHANCEMENT OF PROTOPORPHYRIN IX FORMATION FOLLOWING TOPICAL ALA DELIVERY
  9. FORMULATION OF ALA-CONTAINING TOPICAL DRUG DELIVERY SYSTEMS
  10. CONCLUSION
  11. REFERENCES

Stability of ALA

ALA undergoes an enzymatically induced dimerization to give porphobilinogen in the biosynthetic pathway of heme (30). Two molecules of ALA participate in a Knorr condensation reaction to give porphobilinogen, with the elimination of two molecules of water, as shown in Fig. 3. The enzyme responsible for this reaction is ALA dehydratase, known to exist in almost all living organisms (171). ALA belongs to the class of α-aminoketones, which dimerize readily under alkaline conditions (172). The formed dihydropyrazines can further oxidize to pyrazine-type derivatives.

image

Figure 3. Condensation of two molecules of 5-aminolevulinic acid to give porphobilinogen and water, catalyzed by 5-aminolevulinic acid dehydratase.

Formulation of ALA into drug-delivery systems for photodynamic therapy has necessitated a clear understanding of its degradation pathways. Under alkaline conditions, the formation of 3,6-dihydropyrazine 2,5-dipropionic acid (DHPY), porphobilinogen and pseudoporphobilinogen, via an open-chain dimeric ketimine, from ALA, has been postulated previously (173). Furthermore, the oxidation of DHPY to pyrazine 2,5-dipropionic acid (PY) has also been reported (174). The possible condensation products of ALA, formed under alkaline conditions and in the absence of enzymes, are shown in Fig. 4 (175–177). In addition to the cyclic degradation products presented, it has also been suggested that ALA may undergo a polymerization reaction in solution (178).

image

Figure 4. Possible condensation reactions involving 5-aminolevulinic acid. Adapted from Bunke et al. (179).

Early reports detailing the degradation mechanism of ALA were conflicting, a situation not helped by the diversity of stability test conditions and analytical methods used for investigation. However, Bunke et al. (179), using capillary electrophoresis (CE) and nuclear magnetic resonance spectroscopy (NMR), showed that only two condensation products for ALA existed in alkaline media in the absence of enzymes. DHPY was formed initially and then oxidized to PY, which is the major degradation product in aerated solutions. Neither porphobilinogen nor pseudoporphobilinogen was formed under such conditions. Novo et al. (52), De Blois et al. (180) and Gadmar et al. (178), using UV spectroscopic methods, have come to similar conclusions.

The stability of ALA in aqueous solution has been shown to be dependent on pH, concentration, temperature and degree of oxygenation of the solution (52,178,180,181). Two molecules of ALA can only react to form DHPY when the amino group of the ALA is deprotonated, before being oxidized to PY in aerated media (52). The pH dependence of the reaction of ALA can be explained on the basis of the acid-base equilibria of this amino acid (182), which is shown diagrammatically in Fig. 5. The values of the acid dissociation constants (pKa) of ALA have been shown to be around pK1= 4.0 and pK2= 8.3 (52,183). These values indicate that the zwitterion is the major species present in the pH range between 5 and 7.5. The anion, a species with a deprotonated amino group, is the only one able to react with the ketone group of a neighboring molecule to yield the cyclic dihydropyrazine, DHPY. This explains the strong pH dependence of the reaction because the concentration of the anion increases with pH. Consequently, ALA solutions demonstrate stability at low pH, where the anion is not expected. De Blois et al. (180) showed that ALA solutions, of an initial concentration of 0.1% w/w and pH 4.0, were still within the pharmaceutically acceptable range of 90–100% w/w ALA (184,185) after 128 days storage at 21°C. At pH 8.0, the ALA content declined below the 90% w/w limit within a few days. Elfsson et al. (183) found that solutions of ALA buffered to pH 2.35 were stable over a period of 37 days, even when stored at 50°C. The half-lives for the decomposition of ALA at pH 4.81 and pH 7.00 at 50°C were 257 and 3 h, respectively. Novo et al. (52) showed that a 0.3 M solution of ALA in distilled water had a pH of 2 and was completely stable under various conditions of storage. A decreasing pH in degrading solutions of ALA has been a common observation (52,180). This may be explained by a shift in the zwitterion-anion balance as the equilibration is reestablished (Fig. 5).

image

Figure 5. pH-dependent equilibria occurring in aqeous solutions of 5-aminolevulinic acid (ALA). Adapted from Novo et al. (52).

The degradation of ALA has been shown experimentally to follow second-order kinetics (180,181). Doubling the concentration of ALA in a solution of a given temperature and pH quadruples its rate of decomposition. De Blois et al. (180) showed that a solution at pH 5.0, with an initial ALA concentration of 0.5% w/w, had ALA concentrations that were still higher than 90% w/w after 178 days storage. Solutions with initial ALA concentrations of 2, 5 and 10 % w/w had ALA contents that dropped below the 90% limit after 150, 94 and 29 days, respectively. Elfsson et al. (183) used Arrhenius plots to interpret the results from accelerated storage testing of ALA solutions. The authors showed that a 1% w/w solution of ALA, stored at pH 7.53, would have a shelf life, or t90, the time it takes for a substance to lose 10% of its initial mass (184,185), of 1.9 h at 20°C. The shelf life (t90) of a 10% w/w solution of ALA at pH 7.53 would be as short as 10 min at 20°C. Elfsson et al. (183) went on to show that, for every 10°C rise in temperature, the rate of ALA degradation increased by a factor of around 1.5. The effects of both elevated temperature and concentration on ALA degradation and stability are consistent with that found for most drug substances (186,187).

The initial second-order degradation of ALA to give DHPY has been shown to be reversible by acidification, on condition that no oxygen is present in the reaction mixture (179). If oxygen is present, then DHPY is irreversibly converted to PY and the ALA lost is irretrievable. During degradation, aqueous solutions of ALA are reported to undergo color changes over time, changing from colorless to yellow and then to red/orange (179,180). The yellow coloration is attributed to DHPY, based on UV studies of deaereated solutions (52), while the red color is attributed to PY (179).

Drug-delivery strategies

A knowledge of the combined effects of pH, oxygen concentration and ALA loading has led to the rational development of various formulation strategies for maintaining the stability of ALA in solution or semisolid matrices. Addition of neither EDTA (178,183) nor antioxidants (179) to ALA solutions was able to prevent degradation. Most workers have advised dissolving ALA in solutions buffered to around pH 2.0 to maintain long-term stability. Due to the potential for cutaneous irritancy at such pH values, ALA solutions, buffered to physiological pH values, such as pH 5.5 or pH 7.4, are normally prepared immediately prior to use. To date, the stability of chemical derivatives of ALA has not been reported. Presumably, the aliphatic alkyl esters would possess similar stability profiles as the parent drug. However, derivatization at the amino group, such as in the compounds described by Berger et al. (87), may provide a simple step in preventing unwanted dimerization.

The majority of studies conducted thus far on ALA stability have been primarily concerned with ALA dissolved in aqueous solutions. As the applicability of ALA-based PDT embraces new clinical challenges, such as oral cancer (188) and gynecological neoplasias (189), drug delivery using low-viscosity aqueous solutions is not an appropriate course of action. Clearly, retention at these sites becomes problematic due to difficulties in keeping solutions in place. Novel dosage forms designed to circumvent these problems have been described recently in the literature and initial results are promising. However, if such systems are to gain marketing approval from regulatory bodies, such as the FDA, then the stability of ALA or the ester derivative contained in the dosage form will have to be demonstrated over prolonged storage times. Moreover, the biological safety of each of the individual degradation products formed in a particular formulation must also be established.

Drug-delivery vehicles

The process of rational dosage form design takes into account the physiochemical properties of the drug and dosage form, the nature of the biological barriers to drug delivery, the anatomy of the body site to which the drug must be delivered and the desired drugrelease kinetics. The rapid development of topical PDT based on ALA and its ester derivatives has seen numerous reports focused primarily on the drug type and the treatment outcome. Due to the success of these initial investigations, topical PDT has become an established treatment option for a variety of superficial lesions. These developments have not been matched by a corresponding optimization in the design of suitable dosage forms. This point has been highlighted as the important next step in the development of topical PDT (107,190,191).

Most reported work describing ALA-based PDT for the treatment of surface lesions details application of ALA topically, followed by irradiation 2–6 h later (32,61). The drug is dissolved or dispersed in a solution, cream, ointment or gel formulation containing drug concentrations that vary from 10 to 30% w/w (3). While the most commonly employed formulation is 20% w/w ALA in an o/w cream (192), Jeffes et al. (44) carried out a doseresponse study and found no significant difference between clinical outcomes for actinic keratosis lesions treated for 3 h with 10, 20 or 30% w/w ALA in a cream base. Various other semisolid vehicles for delivery of ALA and its esters have also been described. Table 2 provides an overview of the various types of liquid and semisolid dosage forms investigated to date for topical PDT.

Table 2.  Summary of the various types of liquid and semisolid dosage forms containing ALA or its derivatives investigated to date for topical PDT
Formulation typeCommentReferences
Creams, ointments and gelsMost commonly used clinicallyKennedy et al. (30),
   Peng et al. (32),
   Morton et al. (192)
Creams, ointments and gels containing adjuvantsAddition of penetration enhancers, iron chelators to enhance penetration and increase PpIX production. Addition of local anesthetics to reduce pain of treatmentDijkstra et al. (23)
  Soler et al. (43), Harth et al. (118),
  De Rosa et al. (131)
Cubic-phase gelEnhanced ALA penetration and stabilityTurchiello et al. (210)
Bioadhesive gelsDelivery of ALA to oral and esophageal lesionsBourre et al. (211), Tsai et al. (212)
Liposomes and nanoparticlesApplied in suspension form.Hurlimann et al. (56),
 Enhanced ALA penetration and stabilityCasas et al. (58),
  Pierre et al. (213),
  Casas et al. (214).
PowdersMixed with solution prior to application. EnhancedHillemanns et al. (189), DUSA
 ALA stability. May be difficult to keep in position at certain body sitesPharmaceuticals (215)

With the exception of the Levulan® Kerastick, topical application of ALA to superficial lesions generally conforms to an archetypical arrangement where an ALA-containing semisolid is applied to the lesion and covered with an occlusive dressing (136). However, no specific details are normally given on the amount of product applied to the area. This arbitrary approach means that comparing the results from separate reports and deciding on an optimum dose are extremely difficult because widely varying amounts of topical product and, hence, active drug may be applied in each case. Where applied quantities are reported, extreme variation from study to study is observed. These reports have indicated that between 10 and 200 mg of the ALA-containing vehicle is applied per square centimeter of lesion (4,23,18,46,62,136,193). Maintaining ALA-containing vehicles under occlusion on lesions of the trunk, head or limbs from application until irradiation does not present significant difficulties. However, the topical delivery of ALA to gynecological or oral lesions is problematic. Currently, photosensitizers are delivered to cervical lesions using a drug solution in a cervical cap. Treatment failure has been found to occur if the cap becomes dislodged (194,195). ALA is delivered to the vulva using topically applied creams and solutions covered with occlusive dressings (1,196). In practice, occlusive dressings are poor at staying in place, particularly around the female lower reproductive-tract area, where shear forces are high in mobile patients. They interfere with movement, micturition and bowel opening, thereby causing additional distress for the patient. Moreover, determination of an exact and appropriate dose of drug for successful treatment is very difficult, given that there is no control over cream thickness under occlusion (197). Oral lesions are generally treated using parenterally administered preformed sensitizers and patients are, therefore, subjected to the typical side effects associated with such agents (198).

Although numerous reports have centered on clinical efficacy of topical semisolids containing ALA or its esters, the release kinetics of their payloads from such vehicles have been poorly investigated. Donnelly et al. (199) assessed release of ALA from Porphin® cream (20% w/w ALA in Unguentum Merck®) across a model membrane in vitro. The authors found that only approximately 40% of the drug was released over 6 h, a typical in vivo application time. The poor release was attributed to the inability of the hydrophilic ALA to diffuse through the vehicle, which has a high lipid content. Similar poor release was observed from Psoralon® cream (10% w/w ALA in a lipophilic base) across excised stratum corneum and epidermis (57). Moreover, Winkler and Muller-Goymann (90) reported an almost 4-h lag phase before significant amounts of ALA were released across excised stratum corneum from Excipal®-Fettcreme (a w/o cream base) containing 10% w/w ALA. De Rosa et al. (91) showed a more efficient release of ALA than its more lipophilic esters across hairless mouse skin in vitro from an o/w cream over 6 h. The authors attributed this to the fact that ALA was dissolved in the aqueous external phase of the cream and, hence, was immediately available for release. The more lipophilic esters were dissolved in the dispersed phase and, therefore, had to partition into the external phase before diffusing to the skin surface for release. The authors recommended that taking the physiochemical properties of the drug and dosage form into account would lead to design of topical vehicles allowing enhanced delivery of lipophilic ALA esters relative to the hydrophilic parent compound. Indeed, Winkler and Muller-Goymann (90) have reported a 10–fold greater flux of butyl ALA across excised stratum corneum compared with ALA when released from a w/o cream.

The issue of drug stability within a cream vehicle and rapid dimerization will always arise when formulation is buffered at neutral or basic environments. Due to the risk of severe cutaneous irritation. ALA-containing dosage forms are not commonly formulated at low pH. Consequently, shelf lives are short. Porphin® cream must be discarded 6 months after purchase (200). The standard storage method is at −20°C, with the product applied immediately upon removal. This causes discomfort for patients, particularly those with gynecological lesions. Novel strategies circumventing this stability issue have included mixing ALA powder with its vehicle immediately prior to application. DUSA Pharmaceuticals (Tarrytown, NY, USA) have patented a roller-ball applicator (Levulan® Kerastick) consisting of two sealed compartments, one containing ALA, the other containing ethanol, isopropyl alcohol, water, a surfactant and propylene glycol. The contents of the two compartments are mixed before application. It is applied multiple times to the lesions, and commonly, to the whole face and scalp, or other body sites. Other approaches have included encapsulating ALA within solid polymeric nanoparticles (56) or multilamellar liposomes (58), which are then suspended in a lotion for topical application. Formulating vehicles that achieve a compromise between reduced cutaneous irritancy and a sufficiently low pH to allow short-term ALA stability have been reported (52,201).

Bretschko et al. (202) described the formulation of an adhesive patch, loaded with a defined amount of ALA, as a potential dosage form for topical PDT. Crystalline ALA was dispersed in a pressure-sensitive adhesive (PSA) matrix. In vitro drug-release studies demonstrated a 20-h lag phase before significant amounts of ALA were released from the patch across excised stratum corneum. The reason given for the delay before significant amounts of ALA were released was that the dispersed drug had to dissolve before diffusion could occur. Such a system, therefore, requires moisture to activate release. Moisture, however, compromises the adhesion of PSA devices (203) and the patch may not stay in place long enough to be clinically effective, especially in wet environments, such as the mouth or the lower female reproductive tract. A similar ALA-containing PSA patch was described by Lieb et al. (57). In this system, based on Eudragit® NE, the release of the dispersed crystalline ALA across excised stratum corneum was enhanced by the incorporation of the plasticizer acetyl tributyl citrate (ATBC) into the formulation. ATBC increased the flexibility of the formed patch, increasing the influx of water. No lag phase was observed in the drug-release profile. However, only 2.5% of the ALA loading was released from the patch across excised stratum corneum and epidermis after 5 h. There was no mention of drug release from a similar system described by Pons et al. (204). However, pressure-sensitive patches containing ALA were used in the successful PDT of actinic keratosis, basal and squamous cell carcinomas. Such systems may be capable of maintaining stability of ALA or its esters on prolonged storage due to the fact that the drug is in the solid state. However, the poor release and inability to adhere in wet environments may limit their commercial success.

An alternative approach to patch production was described by McCarron et al. (205). The authors described a novel bioadhesive patch cast from an aqueous gel containing poly(methyl vinyl ether/ maleic anhydride). In contrast with the pressure-sensitive systems described above, ALA was dissolved in the matrix of the patch and no lag time was observed in the ALA release profile. The patch released approximately 60% of its drug loading across a model membrane over 6 h. The system was subsequently shown to be capable of delivering high levels of ALA down to depths of approximately 2.5 mm in vaginal tissue (206). The formulation was highly flexible and capable of adhering even in wet environments and was used in the successful PDT of extramammary Paget's disease and lichen sclerosus of the vulva (50,207). Similar systems were described by Manivasager et al. (208,209) for the delivery of ALA and its methyl ester for the purposes of PDD, although drug release was not evaluated.

CONCLUSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION TO TOPICAL PHOTODYNAMIC THERAPY
  4. DRUG DELIVERY AND DIFFUSION
  5. CHARACTERISTICS OF TOPICAL ALA-BASED PDT
  6. TISSUE DISPOSITION OF ALA FOLLOWING TOPICAL ADMINISTRATION
  7. TISSUE PENETRATION ENHANCEMENT STRATEGIES
  8. ENHANCEMENT OF PROTOPORPHYRIN IX FORMATION FOLLOWING TOPICAL ALA DELIVERY
  9. FORMULATION OF ALA-CONTAINING TOPICAL DRUG DELIVERY SYSTEMS
  10. CONCLUSION
  11. REFERENCES

PDT is enjoying a period of intense investigation. Although still widely regarded as an experimental technique, its status and value within modern clinical practice continues to grow. This is due in no small part to a unique set of characteristics, which combine localized cytotoxicity with lesion-specific destruction and a low side-effect profile. Undoubtedly, its benefit to dermatological treatment of various skin conditions, both neoplastic and nonneo-plastic, is remarkable, arising from excellent response rates and impressive cosmetic outcomes.

Although clinical application of ALA and its biomolecular effects within target cells remain as primary research themes, the design and evaluation of delivery systems required for effective photosensitizer administration have been less well addressed. ALA compound combines poor stability in aqueous media with physicochemical properties that preclude efficient drug permeation through intact skin. Similarly, bespoke preparations, such as cream- and ointment-type formulations, have obvious limitations and restrict PDT to areas, such as flat skin, where the preparation will stay in place for the extended durations typically needed. The diffusivity of ALA in these formulations will depend on the resistance exerted on ALA migration. Lipid-rich formulations, such as semisolid emulsions, retard ALA release in comparison with hydrophilic formulations, such as those based on bioadhesive gels and patches (199). These more innovative formulations have opened up previously inaccessible regions to PDT, such as the vulva. Further development of these types of systems will permit applications, such as those pertinent to the oral cavity and upper female reproductive tract, for example, which have not been readily amenable. This will undoubtedly widen the appeal of ALA-based PDT to other difficult therapeutic challenges.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION TO TOPICAL PHOTODYNAMIC THERAPY
  4. DRUG DELIVERY AND DIFFUSION
  5. CHARACTERISTICS OF TOPICAL ALA-BASED PDT
  6. TISSUE DISPOSITION OF ALA FOLLOWING TOPICAL ADMINISTRATION
  7. TISSUE PENETRATION ENHANCEMENT STRATEGIES
  8. ENHANCEMENT OF PROTOPORPHYRIN IX FORMATION FOLLOWING TOPICAL ALA DELIVERY
  9. FORMULATION OF ALA-CONTAINING TOPICAL DRUG DELIVERY SYSTEMS
  10. CONCLUSION
  11. REFERENCES
  • 1
    Gannon, M. J. and S. B. Brown (1999) Photodynamic therapy and its applications in gynaecology. Br. J. Obs. Gynae. 106, 12461254.
  • 2
    Peng, Q., K. Berg, J. Moan, M. Kongshaug and J. M. Nesland (1997) 5–Aminolevulinic acid-based photodynamic therapy: principles and experimental research. Photochem. Photobiol. 65, 235251.
  • 3
    de Rosa, F. S. and M. V. L. B. Bentley (2000) Photodynamic therapy of skin cancers: sensitizers, clinical studies and future directives. Pharm. Res. 17, 14471455.
  • 4
    Fritsch, C., K. Lang, W. Neuse, T. Ruzicka and P. Lehmann (1998) Photodynamic diagnosis and therapy in dermatology. Skin Pharmacol. Appl. Skin Phys. 11, 358373.
  • 5
    Daniell, M. D. and J. S. Hill (1991) A history of photodynamic therapy. Austral. New Zeal. J. Surg. 61, 340348.
  • 6
    Moan, J. and Q. Peng (2003) An outline of the hundred-year history of PDT. Anticancer Res. 23, 35913600.
  • 7
    Henderson, B. W. and T. J. Dougherty (1992) How does photodynamic therapy work Photochem. Photobiol. 55, 145157.
  • 8
    Kennedy, J. C., R. H. Pottier and D. C. Pross (1990) Photodynamic therapy with endogenous protoporphyrin IX: basic principals and present clinical experience. J. Photochem. Photobiol. B: Biol. 6, 143148.
  • 9
    Kalka, K., H. Merk and H. Mukhtar (2000) Photodynamic therapy in dermatology. J. Am. Acad. Dermatol. 42, 389413.
  • 10
    Konan, Y. N., R. Gurny and E. Allemann (2002) State of the art in the delivery of photosensitizers for photodynamic therapy. J. Photochem. Photobiol. B: Biol. 66, 89106.
  • 11
    Dougherty, T. J., C. J. Gomer, B. W. Henderson, G. Jori, D. Kessel, M. Korbelik, J. Moan and Q. Peng (1998) Photodynamic therapy. J. Nat. Cancer Inst. 90, 889905.
  • 12
    Kalyanasundaram, K. (1992) Photochemistry of Polypyridine and Porphyrin Complexes. Academic Press, London .
  • 13
    Isaacs, N. S. (1992) Physical Organic Chemistry. Longman Scientific and Technical, Essex , UK .
  • 14
    Oschner, M. (1997) Photophysical and photobiological processes in the photodynamic therapy of tumours. J. Photochem. Photobiol. B: Biol. 39, 118.
  • 15
    Moan, J., G. Streckyte, S. Bagdonas, O. Bech and K. Berg (1997) Photobleaching of protoporphyrin IX in cells incubated with 5-aminolevulinic acid. Int. J. Cancer 70, 9097.
  • 16
    Moan, J. (1990) On the diffusion length of singlet oxygen in cells and tissues. J. Photochem. Photobiol. B: Biol. 6, 343347.
  • 17
    Govil, S. K. (1988) Transdermal drug delivery systems. In Drug Delivery Devices (Edited byP.Tyle), Chapter 3. Marcel Dekker, New York .
  • 18
    Caimduff, F., M. R. Stringer, E. J. Hudson, D. V. Ash and S. B. Brown (1994) Superficial photodynamic therapy with topical 5-aminolevulinic acid for superficial primary and secondary cancer. Br. J. Cancer 69, 605608.
  • 19
    Leunig, A., M. Mehlmann, C. Betz, H. Stepp, S. Arbogast, G. Grevers and R. Baumgartner (2001) Fluorescence staining of oral cancer using a topical application of 5-aminolevulinic acid: fluorescence micro scopic studies. J. Photochem. Photobiol. B: Biol. 60, 4449.
  • 20
    Fehr, M. K., R. Hornung, V. A. Schwarz, R. Simeon, U. Haller and P. Wyss (2001) Photodynamic therapy of vulvar intraepithelial neo plasia iii using topically applied 5-aminolevulinic acid. Gyn. Oncol. 80, 6266.
  • 21
    Calzavara-Pinton, P. G. (1995) Repetitive photodynamic therapy with topical 5-aminolevulinic acid as an appropriate approach to the routine treatment of superficial non-melanoma skin tumours. J. Photochem. Photobiol. B: Biol. 29, 5357.
  • 22
    Meijnders, P. J. N., W. M. Star, R. S. De Bruijn, A. D. Treurniet-Donker, M. J. M. Van Mierlo, S. J. M. Wijthoff, B. Naafs, H. Beerman and P. C. Levendag, (1996) Clinical results of photodynamic therapy for superficial skin malignancies or actinic keratosis using topical 5-aminolevulinic acid. Lasers Med. Sci. 11, 123131.
  • 23
    Dijkstra, A. T., I. M. L. Majoie, J. W. F. van Dongen, H. van Weelden and W. A. van Vloten (2001) Photodynamic therapy with violet light and topical 5-aminolevulinic acid in the treatment of actinic keratosis, Bowen's disease and basal cell carcinoma. Eur. Acad. Dermatol. Venereol. 15, 550554.
  • 24
    Eleouet, S., N. Rousset, J. Carre, L. Bourre, V. Vonarx, Y. Lajat, G. M. J. Beijersbergen Van Henegouwen and T. Patrice (2000) In vitro fluorescence, toxicity and photoxicity induced by 5-amino levulinic acid (ala) or esters. Photochem. Photobiol. 71, 447454.
  • 25
    Gantchev, T. G., N. Brasseur and J. E. Van Lier (1996) Combination toxicity of etoposide (VP-16) and photosensitisation with water-soluble aluminium phthalocyanine in k562 human leukemic cells. Br. J. Cancer 74, 15701577.
  • 26
    Kessel, D. (1999) Transport and localisation of m-THPC in vitro. Int. J. Clin. Pract. 53, 263267.
  • 27
    Pottier, R. and J. C. Kennedy (1990) New trends in photobiology. The possible role of ionic species in selective biodistribution of photochemotherapeutic agents toward neoplastic tissue. J. Photochem. Photobiol. B: Biol. 8, 116.
  • 28
    Jori, G., M. Beltramini, E. Reddi, B. Salvato, A. Pagnan, L. Ziron, L. Tomio and T. Tsanov (1984) Evidence for a major role of plasma lipoproteins as haematoporphyrin carriers in vivo. Cancer Lett. 24, 291297.
  • 29
    Vaupel, P., F. Kallinowski and P. Okunieff (1989) Blood flow, oxygen and nutrient and metabolic microenvironment and human tumour. Cancer Res. 49, 64496465.
  • 30
    Kennedy, J. C., S. L. Marcus and R. H. Pottier (1996) Photodynamic therapy (PDT) and photodiagnosis (PD) using endogenous photosensitization induced by 5-aminolevulinic acid (ALA): mechanisms and clinical results. J. Clin. Laser Med. Surg. 14, 289304.
  • 31
    Kennedy, J. C. and R. H. Pottier (1992) Endogenous protoporphyrin IX, a clinically useful photosensitizer for photodynamic therapy. J. Photochem. Photobiol. B: Biol 14, 275292.
  • 32
    Peng, Q., T. Warloe, K. Berg, J. Moan, M. Kongshaug K. E. Giercksky and J. M. Nesland (1997) 5-Aminolevulinic acid-based photodynamic therapy: clinical research and future challenges. Cancer 79, 22822308.
  • 33
    Berg, K., H. Anholt, O. Bech and J. Moan (1996) The influence of iron chelators on the accumulation of protoporphyrin IX in 5-aminolevulinic acid-treated cells. Br. J. Cancer 74, 688697.
  • 34
    Rittenhouse-Diakun, K., H. Van Leengoed and J. Morgan (1995) The Role of Transferrin receptor (CD71) in photodynamic therapy of activated and malignant lymphocytes using the heme precursor delta-aminolevulinic acid (ALA) Photochem. Photobiol. 61, 523528.
  • 35
    Bottomley, S. and U. Muller-Eberhard (1988) Pathophysiology of heme synthesis. Sem. Heme Synth. 25, 282302.
  • 36
    Moan, J., K. Berg, O. Gadmar, V. Lani, L. W. Ma and P. Juzenas 1999) The temperature dependence of protoporphyrin IX production in cells and tissues. Photochem. Photobiol. 70, 669673.
  • 37
    Moan, J., J. T. H. M. Vanden Akker, P. Juzenas, L. W. Ma, E. Angell-Peterson, O. B. Gadmar and V. Iani (2001) On the basis for tumor selectivity in the 5-aminolevulinic acid-induced synthesis of protoporphyrin IX. J. Porph. Phthalocyan. 5, 170176.
  • 38
    Krieg, R. C., H. Messmann, J. Rauch, S. Seeger and R. Knuechel (2002) Metabolic characterization of tumor cell-specific protoporphyrin IX accumulation after exposure to 5-aminolevulinic acid in human colonic cells. Photochem. Photobiol. 76, 518525.
  • 39
    Gibson, S. L., J. J. Havens, L. Metz and R. Hilf (2001) Is delta-aminolevulinic acid dehydratase rate limiting in heme biosynthesis following exposure of cells to delta-aminolevulinic acid Photochem. Photobiol. 73, 312317.
  • 40
    Krieg, R. C., S. Fickweiler, S. Appel, O. S. Wolfbeis and R. Knuechel (2000) Cell-type specific protoporphyrin IX metabolism in human bladder cancer in vitro. Photochem. Photobiol. 72, 22633.
  • 41
    Jin, Z. H., N. Miyoshi, K. Ishiguro, K. Takaoka, T. Udagawa, H. Tajiri, K. Ueda, M. Fukuda and M. Kumakiri (2000) Photodynamic therapy based on combined use of 5-aminolevulinic acid with a pheophoride-a derivative for murine tumors. In Vivo 14, 529534.
  • 42
    Wong, T. W., K. Aizawa, I. Sheyhedin, C. Wushur and H. Kato (2003) Pilot study of topical delivery of mono-I-aspartyl chlorin e6 (NPe6): implication of topical npe6-photodynamic therapy. J. Pharmacol. Sci. 93, 136142.
  • 43
    Soler, A. M., T. Warloe, J. Tausjo and A. Berner (1999) Photodynamic therapy by topical aminolevulinic acid, dimethyl sulfoxide and curettage in nodular basal cell carcinoma: a one-year follow-up study. Acta Dermatol. Venereol. 79, 204206.
  • 44
    Jeffes, E. W., J. L. McCullough, G. P. Weinstein, P. E. Fergin, J. S. Nelson, T. F. Shull, K. R. Simpson, L. M. Bukaty, W. L. Hoffman and N. L. Fong (1997) Photodynamic therapy of actinic keratosis with topical 5-aminolevulinic acid: a pilot dose-ranging study. Arch. Dermatol. 133, 727732.
  • 45
    Svanberg, K., T. Andersson, D. Killander, D. Wang, U. Stenram, S. Andersson-Engels, R. Berg, J. Johansson and S. Svanberg (1994) Photodynamic therapy of non-melanoma malignant tumours of the skin using topical 5-aminolevulinic acid sensitization and laser irradiation. Br. J. Dermatol. 130, 743751.
  • 46
    Morton, C. A., C. Whitehurst, H. Moseley, J. H. McColl, J. V. Moore and R. M. Mackie (1996) Comparison of photodynamic therapy with cryotherapy in the treatment of Bowen's disease. Br. J. Dermatol. 135, 766771.
  • 47
    Morton, C. A., C. Whitehurst, J. V. Moore and R. M. MacKie (2000) Comparison of red and green light in the treatment of Bowen's disease by photodynamic therapy. Br. J. Dermatol. 143, 767772.
  • 48
    Martin-Hirsch, P. L., C. Whitehurst, C. H. Buckley, J. V. Moore and H. C. Kitchener (1998) Photodynamic treatment for lower genital tract intraepithelial neoplasia. Arch. Dermatol. 134, 247249.
  • 49
    Abdel-Hady, E. S., P. Martin-Hirsch, M. Duggan-Keen, P. L. Stern, J. V. Moore, G. Corbitt, H. C. Kitchner and I. N. Hampson (2001) Immunological and viral factors associated with the response of vulval intraepithelial neoplasia to photodynamic therapy. Cancer Res. 61, 192196.
  • 50
    Zawislak, A., P. A. McCarron, W. G. McCluggage, J. H. Price, R. F. Donnelly, R. H. McClelland, S. P. Dobbs and A. D. Woolfson (2004) Successful photodynamic therapy of vulval Paget's disease using a novel patch-based delivery system containing 5-aminolevulinic acid. Br. J. Obs. Gynae. 111, 13.
  • 51
    Wierrani, F., A. Kubin, R. Jindra, M. Henry, K. Gharehbaghi, W. Grin, J. Soltz-Szotz, G. Alth and W. Grunberger (1999) 5-Aminolevulinic acid-mediated photodynamic therapy of intraepithelial neoplasia and human papillomavirus of the uterine cervix-a new experimental approach. Cancer Detec. Prev. 23, 351355.
  • 52
    Novo, M., G. Huttmann and H. Diddens (1996) Chemical instability of 5-aminolevulinic acid used in the fluorescence diagnosis of bladder tumours. J. Photochem. Photobiol. B: Biol. 34, 143148.
  • 53
    Lange, N., P. Jichlinski, M. Zellweger, M. Forrer, A. Marti, L. Guillou, P. Kucera, G. Wagnieres and H. Van den Bergh (1999) Photodetection of early human bladder cancer based on the fluorescence of 5-aminolevulinic acid hexylester-induced protopor-phyrin IX. Br. J. Cancer 80, 185193.
  • 54
    Wyss-Desserich, M. T., C. H. Sun and P. Wyss (1996) Accumulation of 5-aminolevulinic acid-induced protoporphyrin ix in normal and neoplastic human endometrial epithelial cells. Biochem. Biophys. Res. Comm. 224, 819824.
  • 55
    Keefe, K. K., E. B. Chahine, P. J. DiSaia, T. B. Krasieva, F. Lin, M. W. Berns and Y. Tadir (2001) Fluorescence detection of cervical intraepithelial neoplasia for photodynamic therapy with the topical agents 5-aminolevulinic acid and benzoporphyrin-derivative mono-acid ring. Am. J. Obs. Gyn. 184, 11641169.
  • 56
    Hurlimann, A. F., G. Hanggi and R. G. Panizzon (1998) Photodynamic therapy of superficial basal cell carcinomas using topical 5-aminolevulinic acid in a nanocolloid lotion. Dermatology 197, 248254.
  • 57
    Lieb, S., R. M. Szeimies and G. Lee (2002) Self-adhesive thin films for topical delivery of 5-aminolevulinic acid. Eur. J. Pharm. Biopharm. 53, 99106.
  • 58
    Casas, A., H. Fukuda, G. Di Venosa, A. M. Del and C. Batlle (2000) The influence of the vehicle on the synthesis of porphyrins after topical application of 5-aminolevulinic acid. implications in cutaneous photodynamic sensitisation. Br. J. Dermatol. 143, 564572.
  • 59
    Malik, Z., G. Kostenich, L. Roitman, B. Ehrenberg and A. Orenstein (1995) Topical application of 5-aminolevulinic acid, DMSO and EDTA: protoporphyrin ix accumulation in skin and tumours of mice. J. Photochem. Photobiol. B: Biol. 28, 213218.
  • 60
    Soler, A. M., T. Warloe, A. Berner and K. E. Giercksky (2001) A follow-up study of recurrence and cosmesis in completely responding superficial and nodular basal cell carcinomas treated with methyl 5-aminolevulinate-based photodynamic therapy alone with prior curettage. Br. J. Dermatol. 145, 467471.
  • 61
    Morton, C. A., R. M. MacKie, C. Whitehurst, J. V. Moore and J. H. McColl (1998) Photodynamic therapy for basal cell carcinoma: effect of tumour thickness and duration of photosensitizer application on response. Arch. Dermatol. 134, 248249.
  • 62
    Moan, J., L. W. Ma and V. Iani (2001) On the pharmacokinetics of topically applied 5-aminolevulinic acid and two of its esters. Int. J. Cancer 92, 139143.
  • 63
    Fuchs, C., R. Riesenberg, J. Siegert and R. Baumgartner (1997) pH-dependent formation of 5-aminolevulinic acid-induced protoporphyrin IX in fibrosarcoma cells. J. Photochem. Photobiol. B: Biol. 40, 4954.
  • 64
    Xiang, W., H. Weingandt, F. Liebmann, S. Klein, H. Stepp, R. Baumgartner and P. Hillemanns (2001) Photodynamic effects induced by aminolevulinic acid esters on human cervical carcinoma cells in culture. Photochem. Photobiol. 74, 617623.
  • 65
    Van den Akker, J. T. H. M., V. Iani, W. M. Star, H. J. C. M. Sterenborg and J. Moan (2000) Topical application of 5-aminolevulinic acid hexyl ester and 5-aminolevulinic acid to normal nude mouse skin: differences in protoporphyrin ix fluorescence kinetics and the role of the stratum corneum. Photochem. Photobiol. 72, 681689.
  • 66
    Casas, A., H. Fukuda, A. M. Del and C. Batlle (1999) Tissue distribution and kinetics of endogenous porphyrins synthesized after topical application of ala in different vehicles. Br. J. Cancer 81, 1318.
  • 67
    Tsai, J. C., H. Chen, W. Wong and Y. L. Lo (2002) In vitro/in vivo correlations between transdermal delivery of 5-aminolevulinic acid and cutaneous protoporphyrin ix accumulation and effect of formulation. Br. J. Dermatol. 146, 853862.
  • 68
    Konig, K., A. Kienle, W. H. Boehncke, R. Kaufmann, A. Ruck T. Meier and R. Steiner (1994) Photodynamic tumour therapy and on-line fluorescence spectroscopy after ala administration using 633 nm light as therapeutic and fluorescence excitation radiation. Opt. Eng. 33, 29452953.
  • 69
    Szeimeis, R. M., T. Sassy and M. Landthaler (1994) Penetration potency of topical applied 5-aminolevulinic acid for photodynamic therapy of basal cell carcinoma. Photochem. Photobiol. 59, 7376.
  • 70
    Pahernik, S., S. Langer, A. Botzlar, M. Dellian and A. E. Goetz (2001) Tissue distribution and penetration of 5-ALA induced fluorescence in an amelanotic melanoma after topical application. Anticancer Res. 21, 5964.
  • 71
    Wennberg, A. M., O. Larko, P. Lonnroth, G. Larson and A. L. Krogstad (2000) 5-Aminolevulinic acid in superficial basal cell carcinomas and normal skin: a microdialysis and perfusion study. Clin. Exp. Dermatol. 25, 317322.
  • 72
    Ahmadi, S., P. A. McCarron, R. F. Donnelly, A. D. Woolfson and K. McKenna (2004) Evaluation of the penetration of 5-aminolevulinic acid through basal cell carcinoma: a pilot study. Exp. Dermatol. 13, 17.
  • 73
    McLoone, N., R. F. Donnelly, P. A. McCarron, M. Walsh and K. McKenna (2004) Aminolevulinic acid penetration into nodular basal cell carcinomas: influence of different concentrations and application times on amounts of ala delivered via a bioadhesive patch. Clin. Photodyn. 1, 56.
  • 74
    Johnson, P. G., S. W. Hui and A. R. Oseroff (2002) Electrically enhanced percutaneous delivery of 5-aminolevulinic acid using electric pulses and a dc potential. Photochem. Photobiol. 75, 534540.
  • 75
    Donnelly, R. F., P. A. McCarron, A. D. Woolfson, A. Zawislak and J. H. Price (2004) Autoradiographic imaging of 5-aminolevulinic acid penetration through vaginal tissue. Clin. Photodyn. 1, 67.
  • 76
    Duska, L. R., J. Wimberly, T. F. Deutsch, B. Ortel, J. Haas, K. Houck and T. Hasan (2002) Detection of female lower genital tract dysplasia using orally administered 5-aminolevulinic acid induced protoporphyrin IX: a preliminary study. Gyn. Oncol. 85, 125128.
  • 77
    Loh, C. S., A. J. MacRobert, A. Bedwell, J. Regula, N. Krasner and S. G. Bown (1993) Oral versus intravenous administration of 5-aminolevulinic acid for photodynamic therapy. Br. J. Cancer 68, 4151.
  • 78
    Webber, J., D. Kessel and D. Fromm (1997) Side effects and photosensitization of human tissues after aminolevulinic acid. J. Surg. Res. 68, 3137.
  • 79
    Herman, M. A., J. Webber, D. Fromm and D. Kessel (1998) Hemodynamic effects of 5-aminolevulinic acid. J. Photochem. Photobiol. B: Biol. 43, 6165.
  • 80
    Ackroyd, R., N. Brown, D. Vernon, D. Roberts, T. Stephenson, S. Marcus, C. Stoddard and M. Reed (1999) 5-Aminolevulinic acid photosensitization of dysplastic Barrett's oesophagus: a pharmacokinetic study. Photochem. Photobiol. 70, 656662.
  • 81
    McGillian, F. B., G. G. Thompson, M. R. Moore and A. Goldberg (1974) The passage of 5-aminolevulinic acid across the blood-brain barrier of the rat. Biochem. Pharmacol. 23, 472.
  • 82
    de Blois, A. W., M. R. T. M. Thissen, H. S. De Bruijn, R. J. E. Grouls, R. P. Dutrieux, D. J. Robinson and H. A. M. Neumann (2001) In vivo pharmacokinetics of protoporphyrin IX accumulation following intracutaneous injection of 5-aminolevulinic acid. J. Photochem. Photobiol. B: Biol. 61, 2129.
  • 83
    Thissen, M., M. W. De Blois, D. J. Robinson, H. S. De Bruijn, R. P. Dutrieux, W. M. Star and H. A. M. Neumann (2002) PpIX fluorescence kinetics and increased skin damage after intracutaneous injection of 5-aminolevulinic acid and repeated illumination. J. Inv. Dermatol. 118, 239245.
  • 84
    Woolfson, A. D. and D. F. McCafferty (1993) Percutaneous Local Anaesthesia. Ellis Horwood, London .
  • 85
    Peng, Q., T. Warloe, J. Moan, H. Heyerdahl, H. B. Steen, J. M. Nesland and K. E. Giercksky (1995) Distribution of 5-aminolevulinic acid-induced porphyrins in noduloulcerative basal-cell carcinoma. Photochem. Photobiol. 62, 906913.
  • 86
    Peng, Q., J. Moan, T. Warloe, V. Iani, H. B. Steen, A. Bjorseth and J. M. Nesland (1996) Build-up of esterified aminolevulinic acid derivative-induced porphyrin fluorescence in normal mouse skin. J. Photochem. Photobiol. B: Biol. 34, 9596.
  • 87
    Berger, Y., A. Greppi, O. Siri, R. Neier and L. Juillerat-Jeanneret (2000) Ethylene glycol and amino acid derivatives of 5-aminolevulinic acid as new photosensitising precursors of protoporphyrin ix in cells. J. Med. Chem. 43, 47384746.
  • 88
    Berger, Y., L. Ingrassia, R. Neier and L. Juillerat-Jeanneret (2003) Evaluation of dipeptide-derivatives of 5-aminolevulinic acid as precursors for photosensitisers in photodynamic therapy. Bioorg. Med. Chem. 11, 13411351.
  • 89
    Rick, K., R. Sroka, H. Stepp, M. Kreigmair, R. M. Huber, K. Jacob and R. Baumgartner (1997) Pharmacokinetics of 5-aminolevulinic acid-induced protoporphyrin ix in skin and blood. J. Photochem. Photobiol. B: Biol. 40, 313319.
  • 90
    Winkler, A. and C. C. Muller-Goymann (2002) Comparative permeation studies for 5-aminolevulinic acid and its n-butyl ester through stratum corneum and artificial skin constructs. Eur. J. Pharm. Biopharm. 53, 281287.
  • 91
    de Rosa, F. S., A. C. Tedesco, R. F. V. Lopez, M. B. R. Pierre, N. Lange, J. M. Marchetti, J. C. G. Rotta and M. V. L. B. Bentley (2003) In vitro permeation and retention of 5-aminolevulinic acid ester derivatives for photodynamic therapy. J. Cont. Rel. 89, 261269.
  • 92
    Van den Akker, J. T. H. M., J. A. Holroyd, D. I. Vernon, H. J. C. M. Sterenborg and S. B. Brown (2003) Comparative in vitro percutaneous penetration of 5-aminolevulinic acid and two of its esters through excised hairless mouse skin. Lasers Surg. Med. 33, 173181.
  • 93
    Gerscher, S., J. P. Connelly, J. Griffiths, S. B. Brown, A. J. MacRobert, G. Wong and L. E. Rhodes (2000) Comparison of the pharmacokinetics and phototoxicity of protoporphyrin ix metabolized from 5-aminolevulinic acid and two derivatives in human skin in vivo. Photochem. Photobiol. 72, 569574.
  • 94
    Gerscher, S., J. P. Connelly, G. M. J. Beijersbergen Van Henegouwen, A. J. MacRobert, P. Watt and L. E. Rhodes (2001) A quantitative assessment of protoporphyrin IX metabolism and phototoxicity in human skin following dose-controlled delivery of the prodrugs 5-aminolaevulinic acid and 5-aminolaevelunic acid-n-pentlyester. Br. J. Derm. 144, 983990.
  • 95
    Peng, Q., A. M. Soler, T. Warloe, J. M. Nesland and K. E. Giercksky (2001) Selective distribution of porphyrins in skin thick basal cell carcinoma after topical application of methyl 5-aminolevulinate. J. Photochem. Photobiol. B: Biol. 62, 140145.
  • 96
    Moan, J., L. W. Ma, A. Juzeniene, V. Iani, P. Juzenas, F. Apricena and Q. Peng (2003) Pharmacology of protoporphyrin IX in nude mice after application of ALA and its esters. Int. J. Cancer 103, 132135.
  • 97
    Juzeniene, A., P. Juzenas, V. Iani and J. Moan (2002) Topical application of 5-aminolevulinic acid and its methylester, hexylester and octylester derivatives: considerations for dosimetry in mouse skin model. Photochem. Photobiol. 76, 329334.
  • 98
    Lange, N., P. Jichlinski, M. Zellweger, M. Forrer, A. Marti, L. Guillou, P. Kucera, G. Wagnieres and H. Van den Bergh (1999) Photodetection of early human bladder cancer based on the fluorescence of 5-aminolevulinic acid hexylester-induced protoporphyrin IX. Br. J. Cancer 80, 185193.
  • 99
    Elliott, T. (1993) Transport of 5-aminolevulinic acid by the dipeptide permeasein Salmonella typhimurium. J. Bact. 175, 325331.
  • 100
    Verkamp, E., V. M. Backman, J. M. Bjornsson, D. Soil and G. Eggertsson (1993) The periplasmic dipeptide permease system transports 5-aminolevulinic acidin Escherichia coli. J. Bact. 175, 14521456.
  • 101
    Moretti, B. M., S. C. Garci, C. Stella, E. Ramos, A. M. Del and C. Batlle (1993) 5-Aminolevulinic acid transportin Saccharomyces cerevisiae. Int. J. Biochem. 25, 19171924.
  • 102
    Rud, E., O. Gederaas, A. Hogset and K. Berg (2000) 5-Aminolevulinic acid, but not 5-aminolevulinic acid esters, is transported into adenocarcinoma cells by system BETA transporters. Photochem. Photobiol. 71, 640647.
  • 103
    Whitaker, C. J., S. H. Battah, M. J. Forsyth, C. Edwards, R. W. Boyle and E. K. Matthews (2000) Photosensitisation of pancreatic tumour cells by delta-aminolaevulinic acid esters. Anticancer Drug Res. 15, 161170.
  • 104
    Doring, F., J. Walter, J. Will, M. Focking, M. Boll, S. Amasheh, W. Clauss and H. Daniel (1998) Delta-aminolevulinic acid transport by intestinal and renal peptide transporters and its physiological and clinical implications. J. Clin. Invest. 101, 27612767.
  • 105
    Gederaas, O. A., A. Holroyd, S. B. Brown, D. Vernon, J. Moan and K. Berg (2001) 5-Aminolaevulinic acid methyl ester transport on amino acid carriers in a human colon adenocarcinoma cell line. Photochem. Photobiol. 73, 164169.
  • 106
    Lopez, R. F., N. Lange, R. H. Guy and M. V. L. B. Bentley (2004) Photodynamic therapy of skin cancer: controlled drug delivery of 5-ALA and its esters. Adv. Drug Del. Rev. 56, 7794.
  • 107
    Lange, N., L. Vaucher, A. Marti, A. L. Etter, P. Gerber, H. Van den Berg, P. Jichlinski and P. Kucera (2001) Routine experimental system for defining conditions used in photodynamic therapy and fluorescence photodetection of (non-) neoplastic epithelia. J. Biomed. Opt. 6, 151159.
  • 108
    Kloek, J. and M. J. Beijersbergen Van Henegouwen (1996) Prodrugs of 5-aminolevulinic acid for photodynamic therapy. Photochem. Photobiol. 64, 9941000.
  • 109
    Gaullier, J. M., K. Berg, Q. Peng, H. Anholt, P. K. Selbo, L. W. Ma and J. Moan (1997) Use of 5-aminolevulinic acid esters to improve photodynamic therapy on cells in culture. Cancer Res. 57, 14811486.
  • 110
    Uehlinger, P., M. Zellweger, G. Wagnieres, L. Juillerat-Jeanneret, H. Van den Bergh and N. Lange (2000) 5-Aminolevulinic acid and it's derivatives: physical chemical properties and protoporphyrin IX formation in cultured cells. J. Photochem. Photobiol. B: Biol. 54, 7280.
  • 111
    Hausmann, F., R. C. Krieg, E. Endlicher, I. Resenberg, K. Ruth and H. Brunner (2001) The effects of 5-aminolevulinic acid (ALA)-esters on ALA-induced protoporphyrin (PpIX) production and localization in human adenocarcinoma cell lines. Gastroenterology 120, A618.
  • 112
    Bigelow, C. E., S. Mitra, R. Knuechel and T. H. Foster (2001) ALA and ALA-hexylester-induced protoporphyrin IX fluorescence and distribution in multicell tumor spheroids. Br. J. Cancer 85, 727734.
  • 113
    Szeimies, R. M., S. Karrer, S. Radakovic-Fijan, A. Tanew, P. G. Calzavara-Pinton, C. Zane, A. Sidoroff, M. Hempel, J. Ulrich, T. Proebstle, H. Meffert, M. Mulder, D. Salomon, H. C. Dittmar, J. W. Bauer, K. Kernland and L. Braathen (2002) Photodynamic therapy using topical methyl 5-aminolevulinate compared with cryotherapy for actinic keratosis: a prospective, randomized study. J. Am. Acad. Dermatol. 47, 258262.
  • 114
    Yamauchi, P. S., N. J. Lowe, G. P. Lask, R. Patnaik, D. Moore and P. Foley (2002) Methyl aminolevulinate and photodynamic therapy in the treatment of actinic keratosis. J. Invest. Dermatol. 119, 804.
  • 115
    Pearse, A. D. (2002) Applications for topical PDT: basal cell carcinomas. Photodyn. News 5, 15.
  • 116
    Zeitouni, N. C., A. R. Oseroff and S. Shieh (2003) Photodynamic therapy of nonmelanoma skin cancers. Current review and update. Mol. Immunol. 39, 11331136.
  • 117
    Harth, Y., B. Hirshowitz and B. Kaplan (1998) Modified topical photodynamic therapy of superficial skin tumours, utilizing aminolevulinic acid, penetration enhancers, red light and hyperthermia. J. Am. Soc. Dermatol. Surg. 24, 723726.
  • 118
    Auner, B. G., C. Valenta and J. Hadgraft (2003) Influence of lipophilic counter-ions in combination with phloretin and 6-Ketocholestanol on the skin permeation of 5-aminolevulinic acid. Int. J. Pharm. 255, 109116.
  • 119
    Barry, B. W. (1987) Mode of action of penetration enhancers in human skin. J. Cont. Rel. 6, 8597.
  • 120
    Barry, B. W. (1991) The LPP theory of skin penetration. In In vitro Percutaneous Absorption: Principles, Fundamentals and Applications (Edited byR. L.Bronaugh and H. I.Maibach), p. 165. CRC Press, Boca Raton , FL .
  • 121
    Barry, B. W. (1991) Lipid-protein-partitioning theory of skin penetration enhancement. J. Cont. Rel. 15, 237.
  • 122
    Goodman, M. and B. W. Barry (1989) Lipid-protein partitioning theory of skin enhancer activity: finite dose technique. Int. J. Pharm. 57, 29.
  • 123
    Allenby, A. C., N. H. Creasy, J. A. G. Edington, J. A. Fletcher and C. Schock (1969) Mechanism of action of accelerants on skin penetration. Br. J. Dermatol. 81, 4755.
  • 124
    Henderson, T. R., R. F. Henderson and J. L. York (1975) Biological Actions of Dimethyl Sulfoxide. Academic Science, New York .
  • 125
    Galbraith, R. A., S. Sassa and A. Kappas (1986) Induction of haem synthesis in Hep G2 human hepatoma cells by dimethyl sulfoxide. Biochem. J. 237, 597600.
  • 126
    Fujita, H., M. Yamamoto, T. Yamagami, N. Hayashi, T. R. Bishop, H. De Verneuil, T. Yoshinaga, S. Shibahara, R. Morimoto and S. Sassa (1991) Sequential activation of hemes for heme pathway enzymes during erythroid differentiation of mouse friend virus-transformed erythroleukemia cells. Biochim. Biophys. Acta 1090, 311316.
  • 127
    Kurihara-Bergstrom, T., G. L. Flynn and W. I. Higuchi (1987) Physiochemical study of percutaneous absorption enhancement by dimethyl sulfoxide: dimethyl sulfoxide mediation of vidarabine (ara-A) permeation of hairless mouse skin. J. Invest. Dermatol. 89, 274.
  • 128
    Kligman, A. M. (1965) Topical pharmacology and toxicology of dimethyl sulfoxide. JAMA. 193, 796.
  • 129
    Al-Saidan, S. M. H., A. B. Selkirk and A. J. Winfield (1987) Effect of dimethyl sulfoxide concentration on the permeability of neonatal rat stratum corneum to alkanols. J. Invest. Dermatol. 89, 426.
  • 130
    Kurihara-Bergstrom, T., G. L. Flynn and W. I. Higuchi (1986) Physiochemical study of percutaneous absorption enhancement by dimethyl sulfoxide: Kinetic and thermodynamic determinants of dimethyl sulfoxide-mediated mass transfer of alkanols. J. Pharm. Sci. 75, 479.
  • 131
    de Rosa, F. S., J. M. Marchetti, J. A. Thomazini, A. C. Tedesco, M. V. Lopes and B. Bentley (2000) A vehicle for photodynamic therapy of skin cancer: influence of dimethyl sulfoxide on 5-aminolevulinic acid in vitro cutaneous permeation and in vivo protoporphyrin ix accumulation determined by confocal microscopy. J. Cont. Rel. 65, 359366.
  • 132
    Steluti, R., F. S. De Rosa, J. H. Collett and M. V. L. B. Bentley (2001) In vitro skin retention and permeation studies for 5-aminolevulinic acid: effect of a lipid penetration enhancer. Eur. J. Pharm. Sci. 13, S132.
  • 133
    Williams, A. C. (2003) Transdermal and Topical Drug Delivery. Pharmaceutical Press, London .
  • 134
    Wolf, J. (1939) Die Innere Strucktur der Zellen des Stratum Desquamans der Menschlichen Epidermis. Anat. Forsch. 46, 170202.
  • 135
    Loveday, D. E. (1961) An in vitro model for studying percutaneous absorption. J. Soc. Cosm. Chem. 12, 224239.
  • 136
    Morton, C. A., C. Whitehurst, J. H. McColl, J. V. Moore and R. M. MacKie (2001) Photodynamic therapy for large or multiple patches of Bowen disease and basal cell carcinoma. Arch. Dermatol. 137, 319324.
  • 137
    Centre for Cosmetic Dermatology (2004) Available at: http://www.drweksberg.com. Accessed 17 May 2004.
  • 138
    Levy, D., J. Kost, Y. Meshulam and R. Langer (1989) Effect of ultrasound on transdermal drug delivery to rats and guinea pigs. J. Clin. Invest. 83, 20742078.
  • 139
    Bomannan, D., G. K. Menon, H. Okuyama, P. M. Elias and R. H. Guy (1992) Sonophoresis II. Examination of the mechanism(s) of ultrasound-enhanced transdermal drug delivery. Pharm. Res. 9, 10431047.
  • 140
    Doukas, A. G. and T. J. Flotte (1996) Physical characteristics and biological effects of laser-induced stress waves. Ultrasound Med. Biol. 22, 151164.
  • 141
    Ter Haar, G. R. (1988) Biological effects of ultrasound in clinical applications. In Ultrasound: Its Chemical Physical Biological Effects (Edited byK. S.Suslick), pp. 305320. VCH, New York .
  • 142
    Doukas, A. G., D. J. McAuliffe, S. Lee, V. Venugopalan and T. J. Flotte (1995) Physical factors involved in stress-wave-induced cell injury: the effect of stress gradient. Ultrasound Med. Biol. 21, 961967.
  • 143
    Lee, S., T. Anderson, H. Zhang, T. J. Flotte and A. G. Doukas (1996) Alteration of cell membrane by stress waves in vitro. Ultrasound Med. Biol. 22, 12851293.
  • 144
    McAuliffe, D. J., S. Lee, T. J. Flotte and A. G. Doukas (1997) Stress-wave-assisted transport through the plasma membrane in vitro. Lasers Surg. Med. 20, 216222.
  • 145
    Lee, S., D. J. McAuliffe, H. Zhang, Z. Xu, J. Taitelbaum, T. J. Flotte and A. G. Doukas (1997) Stress-wave-induced membrane permeation of red blood cells is facilitated by water channels. Ultrasound Med. Biol. 23, 10891094.
  • 146
    Ma, L., J. Moan, Q. Peng and V. Iani (1998) Production of protoporphyrin IX induced by 5-aminolevulinic acid in transplanted human colon adenocarcinoma of nude mice can be increased by ultrasound. Int. J. Cancer 78, 464469.
  • 147
    Nyborg, W. L. (1982) Ultrasonic microstreaming and related phenomena. Brit. J. Cancer 45(Suppl. V), 156154.
  • 148
    Lee, S., A. Kollias, D. J. McAuliffe, T. J. Flotte and A. G. Doukas (1999) Topical drug delivery in humans with a single photomechanical wave. Pharm. Res. 16, 17171721.
  • 149
    Van den Akker, J. T. H. M., K. Boot, D. I. Vernon, S. B. Brown, L. Groenendijk, G. C. Van Rhoon and H. J. C. M. Sterenborg (2004) Effect of elevating the skin temperature during topical ALA application on in vitro ALA penetration through mouse skin and in vivo PpIX production in human skin. Photochem. Photobiol. Sci. 3, 263267.
  • 150
    Rhodes, L. E., M. M. Tsoukas, R. R. Anderson and N. Kollias (1997) Iontophoretic delivery of ALA provides a quantitative model for ALA pharmacokinetics and PpIX phototoxicity in human skin. J. Invest. Dermatol. 108, 8791.
  • 151
    Star, W. M., M. C. G. Aalders, A. Sac and H. J. C. M. Sterenborg (2002) Quantitative model calculation of the time-dependent protoporphyrin IX concentration in normal human epidermis after delivery of ALA by passive topical application or iontophoresis. Photochem. Photobiol. 75, 429432.
  • 152
    Lopez, R. F., M. V. L. B. Bentley, M. B. Delgado-Charro and R. H. Guy (2001) Iontophoretic delivery of 5-aminolevulinic acid (ALA): effect of pH. Pharm. Res. 18, 311315.
  • 153
    Gerscher, S., J. P. Connelly, G. M. J. Van Henegouwen, A. J. MacRobert, P. Watt and L. E. Rhodes (2001) A quantitative assessment of protoporphyrin IX metabolism and phototoxicity in human skin following dose-controlled delivery of the prodrugs 5-aminolaevulinic acid and 5-aminolaevelunic acid-n-pentlyester. Br. J. Dermatol. 144, 983990.
  • 154
    Lopez, R. F., M. V. L. B. Bentley, M. B. Delgado-Charro, H. Van den Bergh, N. Lange and R. H. Guy (2003) Enhanced delivery of 5-aminolevulinic acid esters by iontophoresis in vitro. Photochem. Photobiol. 77, 304308.
  • 155
    Gerscher, S., J. P. Connelly, J. Griffiths, S. B. Brown, A. J. MacRobert, G. Wong and L. E. Rhodes (2000) Comparison of the pharmacokinetics and phototoxicity of protoporphyrin IX metabolized from 5-aminolevulinic acid and two derivatives in human skin in vivo. Photochem. Photobiol. 72, 569574.
  • 156
    Curnow, A., B. W. McIlroy, M. J. Postle-Hacon, J. B. Porter, A. J. MacRobert and S. G. Bown (1998) Enhancement of 5-aminolevulinic acid-induced photodynamic therapy in normal rat colon using hydroxypyridinone iron-chelating agents. Br. J. Cancer 78, 12781282.
  • 157
    Hanania, J. and Z. Malik (1992) The effect of EDTA and serum on endogenous porphyrin accumulation and photodynamic sensitization of human K562 leukemic cells. Cancer Lett. 65, 127131.
  • 158
    Shevchuk, I., V. Chekulayev, J. Moan and K. Berg (1996) Effects of the inhibitors of energy metabolism, Ionidamine and levamisole, on 5-aminolevulinic acid-induced photochemotherapy. Int. J. Cancer 67, 791799.
  • 159
    Wike-Hooley, J. L., J. Haveman and H. S. Reinhold (1984) The relevance of tumour pH to the treatment of malignant disease. Radiother. Oncol. 2, 343366.
  • 160
    Wyld, L., M. W. R. Reed and N. J. Brown (1998) The influence of hypoxia and pH on aminolevulinic acid-induced photodynamic therapy in bladder cancer cells in vitro. Br. J. Cancer 77, 16211627.
  • 161
    Bech, O., K. Berg and J. Moan (1997) The pH dependency of protoporphyrin IX formation in cells incubated with 5-aminolevulinic acid. Cancer Lett. 113, 2529.
  • 162
    Piot, B., N. Rousset, P. Lenz, S. Eleout, J. Carre, V. Vonarx, L. Bourre and T. Patrice (2001) Enhancement of 5-aminolevulinic acid-photodynamic therapy in vivo by decreasing tumour pH with glucose and amiloride. Laryngoscope 111, 22052213.
  • 163
    Musgrove, E., M. Seaman and D. Hedley (1987) Relationship between cytoplasmic pH and proliferation during exponential growth and cellular quiescence. Exp. Cell Res. 172, 6575.
  • 164
    Schick, E., R. Kaufmann, A. Ruck, A. Hainzl and W.-H. Boehncke (1995) On the effectiveness of photodynamic therapy. Acta Dermatol. Venereol. 75, 276279.
  • 165
    Gfatter, R., P. Hackl and F. Braun (1997) Effects of soap and detergents on skin surface pH, stratum corneum hydration and fat content in infants. Dermatol. 195, 258262.
  • 166
    Van der Veen, N., H. L. L. M. Van Leengoed and W. M. Star (1994) In vivo fluorescence kinetics and photodynamic therapy using 5-aminolevulinic acid-induced porphyrin: increased damage after multiple irradiations. Br. J. Cancer 70, 867872.
  • 167
    de Bruijn, H. S., N. Van der Veen, D. J. Robinson and W. M. Star (1999) Improvement of systemic 5-aminolevulinic acid-based photodynamic therapy in vivo using light fractionation with a 75-minute interval. Cancer Res. 59, 901904.
  • 168
    Juzenas, P., R. Sorensen, V. Iani and J. Moan (1999) Uptake of topically applied 5-aminolevulinic acid and production of protoporphyrin IX in normal mouse skin: dependence on skin temperature. Photochem. Photobiol. 69, 478481.
  • 169
    Gibson, S. L., D. J. Cupriks, and J. J. Havens (1998) A regulatory role for porphobilinogen deaminase (PBGD) in delta-aminolaevulinic acid (delta-ALA)-induced photosensitization Br. J. Cancer 77, 235243.
  • 170
    Von Beckerath, M., P. Juzenas, L. W. Ma, V. Iani, L. Lofgren and J. Moan (2001) The influence of UV exposure on 5-aminolevulinic acid-induced protoporphyrin IX production in skin. Photochem. Photobiol. 74, 825828.
  • 171
    Bottomley, S. and U. Muller-Eberhard (1988) Pathophysiology of heme synthesis. Sem. Heme Synth. 25, 282302.
  • 172
    Krems, I. and P. E. Spoerri (1947) The pyrazines. Chem. Rev. 40, 290358.
  • 173
    Franck, B. and H. Stratmann (1981) Condensation products of the porphyrin precursor 5-aminolevulinic acid. Heterocycles 15, 919923.
  • 174
    Butler, A. R. and S. George (1992) The nonenzymatic cyclic dimerisation of 5-aminolevulinic acid. Tetrahedron 48, 78797886.
  • 175
    Granick, S. and D. Mauzerall (1957) Porphyrin biosynthesis in erythrocytes ii: enzymes converting 5-aminolevulinic acid to coproporphyrinogen. J. Biol. Chem. 232, 11191140.
  • 176
    Jaffe, E. K. and J. S. Rajagopalan (1990) Nuclear magnetic resonance studies of 5- aminolevulinate demonstrate multiple forms in aqueous solution. Bioorg. Chem. 18, 381394.
  • 177
    Dalton, J. T., D. Zhou, A. Mukherjee, D. Young, E. A. Tolley, A. L. Golub and M. C. Meyer (1999) Pharmacokinetics of aminolevulinic acid after intravesical administration to dogs. Pharm. Res. 16, 288295.
  • 178
    Gadmar, O. B., J. Moan, E. Scheie, L. W. Ma and Q. Peng (2002) The stability of 5-aminolevulinic acid in solution. J. Photochem. Photobiol. B: Biol. 67, 187193.
  • 179
    Bunke, A., O. Zerbe, H. Schmid, G. Burmeister, H. P. Merkle and B. Gander (2000) Degradation mechanism and stability of 5-aminolevulinic acid. J. Pharm. Sci. 89, 13351341.
  • 180
    de Blois, A. W., R. J. E. Grouls, E. W. Ackerman and W. J. A. Wijdeven (2002) Development of a stable solution of 5-aminolevulinic acid for intracutaneous injection in photodynamic therapy. Lasers Med. Sci. 17, 208215.
  • 181
    Bunke, A., H. Schmid, G. Burmeister, H. P. Merkle and B. Gander (2000) Validation of a capillary electrophoresis method for determination of 5-aminolevulinic acid and degradation products. J. Chromatogr. A 883, 285290.
  • 182
    Novo Rodriguez, M., G. Huttmann and H. Diddens (1995) Chemical instability of 5-aminolevulinic acid (ala) in aqueous solution. SPIE 2371, 204209.
  • 183
    Elfsson, B., I. Wallin, S. Eksborg, K. Rudaeus, A. M. Ros and H. Ehrsson (1998) Stability of 5-aminolevulinic acid in aqueous solution. Eur. J. Pharm. Sci. 7, 8791.
  • 184
    International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (2003) ICH-Topic Q6A: Test Procedures and Acceptance Criteria for New Drug Products: Chemical Substances. Geneva, Switzerland .
  • 185
    International Conference on Harmonisation of Technical Require ments for Registration of Pharmaceuticals for Human Use (2003) ICH-Topic Q6B: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. Geneva, Switzerland .
  • 186
    Cairns, D. (2000) Essentials of Pharmaceutical Chemistry. Pharma ceutical Press, London .
  • 187
    Martin, A., J. Swarbrick and A. Cammarata (1983) Physical Pharmacy. Lea & Febiger, Philadelphia .
  • 188
    Hopper, C. (1996) The role of photodynamic therapy in the management of oral cancer and precancer. Eur. J. Cancer 32B, 7172.
  • 189
    Kurwa, H. A., R. J. Barlow and S. Neill (2000) Single-episode photodynamic therapy and vulval intraepithelial neoplasia Type III resistant to conventional therapy. Br. J. Dermatol. 143, 10401042.
  • 190
    Dougherty, T. J. (2002) An update on photodynamic therapy applications. J. Clin. Laser Med. Surg. 20, 37.
  • 191
    Gudgin Dickson, E. F., R. L. Goyan and R. H. Pottier (2002) New directions in photodynamic therapy. Cell. Mol. Biol. 48, 939954.
  • 192
    Morton, C. A., S. B. Brown, S. Collins, S. Ibbotson, H. Jenkinson, H. Kurwa, K. Langmack, K. McKenna, H. Moseley, A. D. Pearse, M. Stringer, D. K. Taylor, G. Wong and L. E. Rhodes (2002) Guidelines for topical photodynamic therapy: a report of a workshop of the British Photodermatology Group. Br. J. Dermatol. 146, 552567.
  • 193
    Markham, T. and P. Collins (2001) Topical 5-aminolevulinic acid photodynamic therapy for extensive scalp actinic keratoses. Br. J. Dermatol. 145, 502504.
  • 194
    Monk, B. J., C. Brewer, K. Van Nostrand, M. W. Berns, J. L. McCullough, Y. Tadir and A. Manetta (1997) Photodynamic therapy using topically applied dihematoporphyrin ether in the treatment of cervical intraepithelial neoplasia. Gyn. Oncol. 64, 7075.
  • 195
    Hillemanns, P., M. Korell, M. Schmitt-Sody, R. Baumgartner, W. Beyer, R. Kimming, M. Untch and H. Hepp (1999) Photodynamic therapy in women with cervical intraepithelial neoplasia using topically applied 5-aminolevulinic acid. Int. J. Cancer 81, 3438.
  • 196
    Hillemanns, P., M. Untch, C. Dannecker, R. Baumgartner, H. Stepp, J. Diebold, H. Weingandt, F. Prove and M. Korell (2000) Photodynamic therapy of vulvar intraepithelial neoplasia using 5- aminolevulinic acid. Int. J. Cancer 85, 649653.
  • 197
    Zawislak, A., J. H. Price, P. A. McCarron, R. F. Donnelly and A. D. Woolfson (2002) Lighting up tumours—topical treatment of superficial lesions using 5-aminolevulinic acid based photodynamic therapy. N. Irl. Med. Rev. 4, 2628.
  • 198
    Hebeda, K. M., M. T. Huizing, P. A. Brouwer, F. W. Van der Meulen, H. J. Hulsebosch, J. H. Reiss, P. Oosting, C. H. N. Veenhof and P. J. M. Bakker (1995) Photodynamic therapy in AIDS-related cutaneous Kaposi's sarcoma. J. Acq. Imm. Def. Synd. Hum. Retrovirol. 10, 6170.
  • 199
    Donnelly, R. F., P. A. McCarron, A. D. Woolfson and A. Zawislak (2002) 5-Aminolevulinic acid for photodynamic therapy of vulval intraepithelial neoplasia. assay development and release from a proprietary formulation. J. Pharm. Pharmacol. 54, S16.
  • 200
    Porphin® Product Information Sheet. (1996) Crawford Pharmaceuticals, Milton Keynes, UK .
  • 201
    Chang, S. C., A. J. MacRobert and S. G. Bown (1996) Photodynamic therapy of rat urinary bladder with intravesical instillation of 5-aminolevulinic acid: light diffusion and histological changes. J. Urol. 155, 17491753.
  • 202
    Bretschko, E., R. M. Szeimies, M. Landthaler and G. Lee (1996) Topical 5-aminolevulinic acid for photodynamic therapy of basal cell carcinoma. evaluation of stratum corneum permeability in vitro. J. Cont. Rel. 42, 203208.
  • 203
    Moon, S. H. and M. D. Foster (2002) Influence of humidity on surface behaviour of pressure sensitive adhesives studied using scanning probe microscopy. Langmuir 18, 81088115.
  • 204
    Pons, P., P. Roald, B. Nestor, G. Rafael and A. Agustin (2001) Transdermic patches for photodynamic therapy of superficial lesions. Proceedings of the 29th Annual Meeting of the American Society of Photobiology, Chicago , IL .
  • 205
    McCarron, P. A., R. F. Donnelly, A. D. Woolfson and A. Zawislak (2003) Photodynamic therapy of vulval intraepithelial neoplasia using bioadhesive patch-based delivery of aminolevulinic acid. Drug Del. Sys. Sci. 3, 5964.
  • 206
    McCarron, P. A., R. F. Donnelly, B. F. Gilmore, A. D. Woolfson, R. McClelland, A. Zawislak and J. H. Price (2004) Phototoxicity of 5-aminolevulinic acid in the HeLa cell line as an indicative measure of photodynamic effect after topical administration to gynaecological lesions of intraepithelial form. Pharm. Res. 21, 18731881.
  • 207
    McCarron, P. A., R. F. Donnelly, A. D. Woolfson, A. Zawislak, J. H. Price and P. Maxwell (2004) Photodynamic treatment of lichen sclerosus and squamous hyperplasia using sustained topical delivery of aminolevulinic acid from a novel bioadhesive. Br. J. Dermatol. 151, (Suppl. 68), 105106.
  • 208
    Manivasager, V., P. W. S. Heng, J. Hao, W. Zheng, K. C. Soom and M. Olivo (2002) Macro-microscopic fluorescence imaging of human NPC xenografts in a murine model using topical vs intravenous administration of 5-aminolevulinic acid. Int. J. Oncol. 21, 10031007.
  • 209
    Manivasager, V., P. W. S. Heng, J. Hao, W. Zheng, K. C. Soom and M. Olivo (2003) A study of 5-aminolevulinic acid and its methyl ester used in in vitro and in vivo systems of human bladder Cancer. Int. J. Oncol. 22, 313318.
  • 210
    Turchiello, R. F., F. C. B. Vena, P. Maillard, C. S. Souza, M. V. L. B. Bentley and A. C. Tedesco (2003) Cubic phase gel as a drug delivery system for topical application of 5-ALA, its ester derivatives and m-THPC in photodynamic therapy. J. Photochem. Photobiol. B: Biol. 70, 16.
  • 211
    Tsai, J. C., C. P. Chiang, H. M. Chen, S. B. Huang, C. W. Wang, M. I. Lee, Y. C. Hsu, C. T. Chen and T. Tsai (2004) Photodynamic therapy of oral dysplasia with topical 5-aminolevulinic acid and light-emitting diode array. Lasers Surg. Med. 34, 1824.
  • 212
    Bourre, L., S. Thibaut, A. Briffaud, Y. Lajat and T. Patrice (2002) Potential efficacy of a 5-aminolevulinic acid thermosetting gel formulation for use in photodynamic therapy of lesions of the gastrointestinal tract. Pharmacol. Res. 45, 159165.
  • 213
    Pierre, M. B. R., A. C. Tedesco, J. M. Marchetti and M. V. L. B. Bentley (2001) Stratum corneum lipids liposomes for the topical delivery of 5-aminolevulinic acid in photodynamic therapy of skin cancer: preparation and in vitro permeation study. BMC Dermatol. 1, 14711476.
  • 214
    Casas, A., C. Perotti, M. Saccoliti, H. Fukuda A. M. Del and C. Batlle (2002) ALA and ALA hexyl ester in free and liposomal formulations for the photosensitisation of tumour organ cultures. Br. J. Cancer 86, 837842.
  • 215
    DUSA Pharmaceuticals (2001) Available at: http://www.dusapharma.com. Accessed 25 November 2001.