Reference #: POT-1017-082325
Submit Date: 03/25/2002 12:15:21-0500

The in vivo monitoring of ALA-induced fluorescence

ABSTRACT:
The process of 5-aminolevulinic acid induced photodynamic therapy (ALA-PDT) involves the conversion of a non-fluorescent drug into the fluorescent photosensitizer protoporphyrin IX (PpIX). The pharmacokinetic process of ALA-PDT thus includes an additional biochemical reaction process that leads to the in vivo formation of the photosensitizing agent. The first fluorescent species to appear in this biosynthetic process is PpIX. By monitoring the ALA-induced PpIX fluorescence, one can obtain data on both the rate of conversion of ALA into PpIX and the pharmacokinetic behaviour of PpIX into the non-fluorescent heme. Excitation of PpIX can be done with either blue or red light, and the fluorescence can be conveniently monitored at either 635 nm or 710 nm. Interfering fluorophores can be largely eliminated by either a dual excitation or dual emission wavelength process, where one of the wavelengths favours the excitation or emission of the interfering fluorophore. Following the application of ALA (either topically, IV or IP), there is an initial phase where the PpIX fluorescence increases. This corresponds to the process of in vivo bioconversion of ALA into PpIX. If a bolus (IV or IP) of ALA is administered, a maximum PpIX fluorescence intensity is reached after ~3-5 hrs, after which the PpIX fluorescence intensity decreases as the PpIX is eliminated via its conversion to the non fluorescent heme. Topically applied ALA fluorokinetic behaviour has an additional "middle phase" during which the fluorescence intensity fluctuates according to the competitive rates of PpIX production and elimination. The changes in pharmacokinetic behaviour, as monitored by in vivo fluorescence, can be used as an indication of the extent of abnormal tissue, as well as its rate of destruction via ALA-PDT.

Keywords: 5-aminolevulinic acid, Protoporphyrin IX, Photodynamic therapy, fluorescence

Reference #: OSE-1017-724406
Submit Date: 04/01/2002 22:07:22-0500

Dermatologic Applications of PDT

ABSTRACT:
There has been rapid expansion of the use of PDT in dermatology, including FDA approval of ALA (Levulan) for actinic keratoses, and the introduction of new photosensitizers including the systemic agent HPPH (Photochlor) and topical ALA-methylester (Metvix), which is approved for keratoses and basal cell carcinomas in Europe. Multiple groups have demonstrated that ALA-based therapy is effective for nodular and superficial disease carcinomas. However, there are diverse approaches to the choice of treatment conditions, including illumination wavelengths, skin preparation, number of sequential treatments, and light dose and fluence rate. It is unlikely that these parameters have been optimized. Because the therapeutic success rate is high, it is difficult to design appropriate clinical trials to address optimization, or even to compare ALA formulations. This problem remains a challenge to the field. Topical ALA can produce high local photosensitizer levels, and pre-clinical data suggests that high light fluence rates can cause photodynamic oxygen depletion and loss of both efficiency and selectivity. Choice of illumination conditions might be guided by real-time optical measurements of photobleaching kinetics or singlet oxygen production, though clinical application of these techniques is non-trivial. There is increasing understanding of intracellular mechanisms of PDT killing and its effects on the host inflammatory and immune system. This information may lead to new rational combinations of PDT with other agents, and to the use of PDT as an immunomodulator for non-malignant skin conditions.

Keywords: ala pdt, skin cancer, photobleaching, immune effects

Reference #: 105547
Submit Date:

BREAK

ABSTRACT:

Keywords:

Reference #: MAR-1019-675289
Submit Date: 04/24/2002 13:49:40-0500

Current Status of DUSA's Development of Photodynamic Therapy (PDT) Using Levulan® (aminolevulinic acid HCl)

ABSTRACT:
Topical Levulan PDT is now approved and is being marketed in the United States by Berlex Laboratories for the treatment of pre-cancerous skin lesions (actinic keratoses). DUSA, together with its dermatology development partner, Schering AG, is now carrying out clinical studies to determine the safety and efficacy of topical Levulan PDT for the treatment of onychomycosis of the toenails, and plantar warts. Systemic, oral dosing of Levulan provides photosensitization of internal organs for PDT or Photodetection (PD). DUSA is currently supporting independent investigator studies on Levulan PDT for the prevention of restenosis after femoral artery angioplasty, and the treatment of Barretts esophagus (BE). DUSA-sponsored trials using red laser light for the treatment of BE with or without low-grade dysplasia, and for the treatment of areas of high-grade dysplasia (HGD) within BE are being carried out in the United States, using Levulan 60 mg/kg delivered orally. Four to six hours later, the area of BE is exposed to red laser light delivered through a clear balloon catheter. Patients are randomized to receive various light doses. Patients may be retreated if necessary, and are followed for up to 24 months after the initial treatment. The goal of the study is to ablate areas of BE or HGD, resulting in the regrowth of normal esophageal lining.

Keywords: aminolevulinic acid, PDT, DUSA

Reference #: MAL-1015-833991
Submit Date: 03/11/2002 01:40:42-0500

Regulation of porphyrin synthesis and the roles of cytosolic and nuclear porphobilinogen deaminase (PBGD).

ABSTRACT:
ALA-phototherapy and photodiagnosis mediated by enhanced protoporphyrin IX (PpIX) biosynthesis in tumors has important medical applications in spite of the fact that the biochemical ground is not fully explained. We have focused on the role of the rate-limiting enzyme PBGD in a variety of tumor and leukemia cells and have established its sub-cellular distribution in correlation with differentiation. PBGD subcellular distribution was confirmed by independent techniques: (a) fluorescence immuno-staining with specific anti-PBGD antibodies and (b) cellular expression of PBGD-GFP fusion proteins. Unexpectedly, a major fraction of PBGD was detected in the nucleus of all cell lines and only a minor fraction in the cytoplasm. Both C and N terminal fusion proteins revealed a major fraction of the newly synthesized PBGD in the nucleus. Stimulation of differentiation in all the cell lines induced a marked decrease in PBGD both in the nucleus and in the cytoplasm as determined by Western blotting, fluorescence immuno-localization and enzymatic activity. Over-expression of the human housekeeping PBGD (HK-PBGD), using the pHK-PBGD plasmid, induced a G1 cell cycle attenuation accompanied by specific differentiation markers. Conversely, induced differentiation of Friend erythroleukemia (FL) cells resulted in elevated PBGD expression, activity and nuclear localization which was coordinated with increased porphyrin and globin synthesis, hemoglobin assembly and morphological changeover. Nuclear localization and expression of both HK and erythroid PBGD isoenzymes were well correlated with erythroid differentiation of FL cells. These findings suggest a possible dual role for housekeeping PBGD in fast dividing tumor cells, one related to the porphyrin synthesis pathway and another coupled to a nuclear function, which might be linked to energy consumption in tumorigenesis. In FL, the erythroid PBGD is mainly directed to tetra-pyrrole synthesis during erythroid differentiation, while the housekeeping PBGD isoform may have a regulatory nuclear function.

Keywords: aminolevulinic acid, protoporphyrin, porphobilinogen deaminase, tumor

Reference #: MOA-1016-994589
Submit Date: 03/24/2002 12:04:24-0500

In vivo generation of PpIX from ALA and ALA derivatives.

ABSTRACT:
The speed of penetration of molecules into cells as well as through the stratum corneum and the epidermis into subcutaneous tissues is expected to increase with decreasing size of the molecules and with decreasing water solubility. Aminolevulinic acid (ALA) is a small, water-soluble molecule. Lipophilic ALA derivatives were introduced with the hope that they should penetrate faster and deeper into tumors or, at least, produce PpIX more tumor selectively than ALA. It has been convincingly demonstrated that lipophilic ALA derivatives produce PpIX at far lower concentrations than ALA does in cells in vitro. After topical application, however, the situation is unclear. The following facts will be discussed: 1) When applied topically on mouse skin ALA goes systemically and produces PpIX at remote localizations (skin and internal organs) while ALA esters have a much lower capacity of producing PpIX after systemic transport. This is an advantage since ALA may be slightly hepatotoxic and neurotoxic. Is this difference in pharmokinetics related to the penetration depth of the esters or to their stability in serum? 2) Methyl 5-aminolevulinate ester (ALA-Me) appears to produce PpIX more tumor selectively than ALA does. 3) PDT with ALA-Me seems to give less pain than PDT with ALA. Is this related to the penetration depth of ALA-Me, to the fact that it produces less PpIX in normal skin, or to differences in the interaction with nerve endings? 4) A given amount of PpIX produced from ALA-Me is faster cleared from the skin than a similar amount of PpIX produced from ALA. 5) Fluorescence excitation spectroscopy can be applied to estimate the depth of PpIX localization and shows small differences between ALA and ALA esters. 6) The stratum corneum is the main barrier for transport of ALA and ALA esters into subcutaneous tissues. The stratum corneum contains proteins and lipids that can bind ALA and ALA derivatives and slow down the penetration speed into subcutaneous tissues. Is the binding different for ALA and ALA esters? 7) The skin overlaying tumors is more permeable than normal skin. 8) PpIX production from ALA in tissue increases with increasing tissue temperature. 9) The lipophilicity of the vehicle is of significance for the tissue uptake of these drugs. This will be discussed in view of partition between the vehicle and the stratum corneum.

Keywords: aminolevulinic acid (ALA), ALA ester derivatives, photodynamic therapy (PDT)