Development of a stable solution of 5-aminolaevulinic acid for intracutaneous injection in photodynamic therapy.

Photodynamic therapy using 5-aminolaevulinic acid (ALA) as a photosensitiser is a new treatment modality for basal cell carcinomas. Until now ALA has been used topically as a cream. As this administration route leads sometimes to insufficient penetration in the skin, an intracutaneously injectable solution of ALA was developed. The influence of pH, concentration and temperature on the degradation of ALA in aqueous solution was investigated in order to optimise the formulation of the injection. In 0.1% ALA solutions with pH values between 4 and 8 a pH dependency of ALA degradation was shown, comprising fast decomposition at pH values higher than 7, whereas at a pH value of 6 or lower the solutions remained within the range of 90-110% of the initial concentration for at least 128 days. An increase of degradation rate with increasing concentrations became evident which is consistent with the supposed second-order degradation kinetics. After accelerated stability research at 63 degrees C and 85 degrees C a shelf life of 281 days for a 0.1% ALA solution pH 5 was calculated from an Arrhenius plot. A 2% ALA solution was proven to be isotonic. From our results a 0.1-2% ALA solution with pH 5 and an appropriate amount of sodium chloride to obtain isotonicity is recommended as an injectable solution.

In vivo pharmacokinetics of protoporphyrin IX accumulation following intracutaneous injection of 5-aminolevulinic acid.

Photodynamic therapy with 5-aminolevulinic acid (ALA) derived protoporphyrin IX (PpIX) as photosensitizer is a promising treatment for basal cell carcinomas. Until now ALA has been administered topically as an oil-in-water cream in most investigations. The disadvantage of this administration route is insufficiënt penetration in deeper, nodular tumours. Therefore we investigated intracutaneous injection of ALA as an alternative administration route. ALA was administered in 6-fold in the normal skin of three 6-week-old female Dutch pigs by intracutaneous injection of an aqueous solution of ALA (pH 5.0) in volumes of 0.1-0.5 ml and concentrations of 0.5-2% and by topical administration of a 20% ALA cream. During 8 h fluorescence of ALA derived PpIX was measured under 405 nm excitation. For the injection the measured fluorescence was shown to be dose dependent. All injected doses of 3 mg ALA or more lead to a faster initial increase rate of PpIX synthesis and significantly greater fluorescence than that measured after topical administration of ALA. Irradiation (60 Jcm(-2) for 10 min) of the spots was performed at 3.5 h after ALA administration. After 48 and 96 h visual damage scores were evaluated and biopsies were taken for histopathological examination. After injection of 2 mg ALA or more the PDT damage after illumination was shown to be significantly greater than after topical application of 20% ALA. An injected dose of 10 mg ALA (0.5 ml of a 2% solution) resulted in significantly more tissue damage after illumination than all other injected doses.

Reconstituted membranes of tumour cells (proteoliposomes) induce specific protection to murine lymphoma cells.

Antigens presented on cell membranes or on liposomes are usually more immunogenic than antigens in soluble form, this being one of the reasons for the weak immunogenicity of extracted tumour-associated transplantation antigens (TATA). The main objective of this study is to solubilize TATA from tumour cells and to present them on a membrane-like structure to the immune system. Crude tumour cell membranes of SL2 lymphosarcoma cells (a spontaneously arising, weakly immunogenic tumour) were solubilized with octylglucoside or sodium deoxycholate, and reconstituted membranes (proteoliposomes) were prepared by detergent removal. Mice immunized s.c. with reconstituted membranes were protected against an i.p. challenge with tumour cells. Although octylglucoside solubilized only 41% of the membrane proteins, the reconstituted membranes were as immunoprotective as crude membranes. (Glyco)proteins were probably the major membrane components in the reconstituted membranes that induce immunoprotection, as mice immunized with preparations constituted of (glyco)lipids from SL2 cells could not reject SL2 cells. If Freund's complete adjuvant was used with the first immunization injection, no potentiation of the elicited immune responses was observed. Besides the membrane TATA, SL2 cells contained an apparently non-membrane-bound TATA, which was found in the cytoplasm. It is concluded that detergent solubilization of membranes and subsequent preparation of reconstituted membranes can be used to obtain membrane tumour-associated antigens that retain activity for induction of protective tumour immunity. The major advantage of this method is that membrane proteins are solubilized and are subsequently presented on a membrane-like structure that resembles the tumour cell membrane. On theoretical and practical grounds it provides a promising alternative for whole-cell vaccines.