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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.


5-aminolevulinic acid


acetyl tributyl citrate


basal cell carcinomas


capillary electrophoresis






3,6-dihydropyrazine 2,5-dipropionic acid


dimethyl sulfoxide


ethylendiaminetetraacetic acid


low-density lipoprotein


nuclear magnetic resonance


porphobilinogen deaminase




photodynamic therapy


photosensitizer, protoporphyrin IX


pressure-sensitive adhesive


pyrazine 2,5-dipropionic acid


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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).


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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


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.


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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).


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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.


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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).


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.


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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).


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.


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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.


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).


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).


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.


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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.


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