Li Wei Ma

On the pharmacokinetics of topically applied 5-aminolevulinic acid and two of its esters

The kinetics of protoporphyrin IX (PpIX) production in normal tissues and WiDr tumors of mice were studied after topical application of 5‐aminolevulinic acid (ALA) and its methyl ester and hexyl ester. ALA and ALA esters were applied on a spot of 1.0 cm diameter on normal skin and on skin overlaying tumors. PpIX production was studied by fluorescence measurements. ALA induced PpIX not only on the spot of application but also on remote skin areas. This was not found for the ALA esters. They produced PpIX only on the spot of application. Thus, ALA, but neither its esters nor PpIX, is passing into the circulation. The time needed for ALA to enter the circulation through normal skin was about 5 hr. Even when looking normal, the skin overlaying tumors was more permeable to ALA than normal skin. Thus, when applied on the tumor, ALA induced PpIX on remote skin areas without any lag phase. Mainly, PpIX was found in all tissues although small amounts of a porphyrin with an excitation peak at about 400 nm, supposedly uroporphyrin and/or coproporphyrin, were found, notably in remote skin areas. An altered stratum corneum of the skin overlaying tumors probably contributes to the tumorselectivity, although in the present tumor system less PpIX was found in tumors than in muscles. This is probably related to biochemical and physiological conditions in this particular tumor, since i.p. injection of ALA also leads to less PpIX formation in the tumor than in skin/muscle tissue. Nevertheless, it seems evident that ALA can diffuse more easily from the skin surface and down to the vasculature in the tumor than in the normal tissue and that this leads to a higher concentration of PpIX in the tumor than would have been found if the physiological factors relevant for drug diffusion were the same for tumors as for skin/muscles.

Phototransformations of 5-Aminolevulinic Acid–induced Protoporphyrin IX in vitro: A Spectroscopic Study¶

Abstract Human adenocarcinoma cells of the line WiDr were incubated with 5-aminolevulinic acid to induce protoporphyrin IX (PpIX) and then exposed to laser light of wavelength 635 nm. The PpIX fluorescence decreased with increasing exposure. The decay rate was slightly dependent on the initial PpIX concentration. The PpIX fluorescence was halved by a fluence of about 40 J/cm2. Several fluorescing photoproducts were formed. The main one, supposedly the chlorin-type photoprotoporphyrin (Ppp), had a fluorescence excitation spectrum stretching out to about 680 nm with a maximum at around 668 nm. The formation kinetics of this product was dependent on the initial PpIX concentration. Moreover, it was selectively bleached by exposure to light at 670 nm. A photoproduct with an emission maximum at 652 nm, different from Ppp, remained after this exposure. Traces of a photoproduct(s) with fluorescence emission slightly blueshifted compared with that of PpIX, supposedly water-soluble porphyrins, were also detected after light exposure.

Pharmacology of protoporphyrin IX in nude mice after application of ALA and ALA esters

Aminolevulinic acid (ALA), ALA methylester (ALA‐Me) and ALA hexylester (ALA‐Hex) were topically applied for 5 and 20 hr, respectively, on normal skin of mice. The distribution of protoporphyrin IX (PpIX) induced in 7 different tissues by these drugs was determined either by spectrofluorometric measurements with an optical fibre probe or by chemical extraction of PpIX from the tissues. The results from these 2 types of measurements were compared. Both methods showed that ALA and the esters induced similar amounts of PpIX at the skin spot where they were applied and that the esters produced much less PpIX at remote skin spots (i.e., spots outside the location where the drugs were applied) than ALA did, notably after 20 hr application. After 20 hr of drug application ALA produced much more PpIX in liver, intestine and lungs than the esters did. In contrast with the direct fluorescence measurements, the extraction method showed detectable amounts of PpIX in liver, intestine and lung after application of the esters, notably of ALA‐Me. The discrepancy is probably related to the fact that the pigmented tissues absorb light and, therefore, the direct fluorescence readings are misleading. Notably in the liver, which contains high concentration of light‐absorbing pigments, very weak direct fluorescence was seen. In no case there was any accumulation of PpIX in muscle tissue nor in brain. The esters seem to penetrate less into the circulation than ALA, and PpIX formed by them in the skin is faster cleared than PpIX formed from ALA. This is also true after oral and i.p. administration of the drugs.

Intracellular Localisation of Hypericin in Human Glioblastoma and Carcinoma Cell Lines

Abstract. Hypericin, a natural polycyclic quinone extracted from Hypericum perforatum, has been recently shown to be a powerful sensitiser for photodynamic therapy (PDT). However, its intracellular localisation remains unclear and contradictory. In the present work we compared the intracellular localisation of hypericin in three cultured cell lines (adenocarcinoma cells WiDr, carcinoma cells NHIK 3025 and glioblastoma cells D54Mg) with the distribution of fluorescent probes specific to lysosomes (LysoTracker Blue DND-22), mitochondria (MitoTracker Green FM) and endoplasmic reticulum (ERTracker Blue-White DPX). It was shown that the hypericin staining pattern was different compared to the intracellular distribution of mitochondria or lysosomes. Hypericin was concentrated in the perinucleolar cytoplasmic area mainly on one side of the nucleus – the region rich in endoplasmic reticulum and Golgi. Sometimes nuclear envelope was also stained. Plasma membrane was not stained but the dye was often accumulated in the intercellular space between the tightly contacting WiDr cells in colonies. Hypericin concentrations of 10 μM or less were not toxic for WiDr cells in the dark. Orange light (λmax≈600 nm; 6 mW/cm2) killed the cells stained with 1 μM hypericin with LD50∼ 1J/cm2.

Protoporphyrin IX accumulation in cells treated with 5‐aminolevulinic acid: Dependence on cell density, cell size and cell cycle

Human colon adenocarcinoma cells (WiDr) and Chinese hamster lung fibroblasts cells (V79) were incubated with different concentrations of 5‐aminolevulinic acid (ALA), and the production of protoporphyrin IX (PpIX) was studied using several techniques. The amount of PpIX produced per cell increased with increasing ALA concentration according to different kinetics for the 2 cell lines. For both cell lines a cell density dependency of the PpIX synthesis was observed. For saturating ALA concentrations, 2–3 times more PpIX was produced per cell at a density of 5 × 104 than at a density of 5 × 103 cells/cm2. The photosensitivity of cells appeared to increase even more than the PpIX content, indicating a cooperative effect in inactivation. The PpIX production rate increased with cell size and was about 1.9 times higher for cells in the G2 + M phase than for cells in the G1 phase of the cell cycle. Neither cell size nor cell cycle distribution were significantly dependent on cell density. Int. J. Cancer 75:134–139, 1998.© 1998 Wiley‐Liss, Inc.

The stability of 5-aminolevulinic acid in solution

5-Aminolevulinic acid (ALA) is being assessed for photodynamic therapy of cancer and other diseases worldwide. However, its stability properties in solution are not well understood yet. The breakdown of ALA in pH-buffered solutions was examined in this work. Solutions of ALA in PBS buffered to physiological pH were found to be unstable, leading to a breakdown product that absorbs photons around 278 nm. The ability of the solution to stimulate porphyrin production in cells is gradually lost upon breakdown, though the kinetics for this are different from those for formation of the UV absorbing product. It is likely, therefore, that several chemical pathways contribute to the breakdown of dissolved ALA at physiological pH. Temperature studies of the formation kinetics of the UV absorbing product also indicate that a complex formation process is involved.

Comparison of the photobleaching effect of three photosensitizing agents: Meso-tetra(m-hydroxyphenyl)chlorin, meso-tetra(m-hydroxyphenyl)porphyrin and photofrin during photodynamic therapy

V79 cells (Chinese hamster lung fibroblasts) were incubated with meso-tetra(m-hydroxyphenyl)chlorin (mTHPC), meso-tetra(m-hydroxyphenyl)porphyrin (mTHPP) or Photofrin and exposed to light. Light exposure resulted in the photobleaching of all three drugs and a change in the shape of the fluorescence emission spectrum of Photofrin, but no significant spectral change for mTHPC or mTHPP. The rate of photobleaching of mTHPC fluorescence was much higher than that of mTHPP and Photofrin. However, there was only a slight increase in the rate of photobleaching with increasing mTHPC concentration in the cells. It was proposed that the light-induced photobleaching of mTHPC may influence its ability in cell photoinactivation and change the shape of survival curve of the cells treated by photodynamic therapy (PDT). Photobleaching of mTHPC resulted in the need for increased light exposure for cell inactivation at low mTHPC concentrations. These results suggest that mTHPC photobleaching may play a role in protecting normal tissue from damage by PDT and enhancing the therapeutic ratio of PDT.

Photobleaching of Hypericin Bound to Human Serum Albumin, Cultured Adenocarcinoma Cells and Nude Mice Skin¶

Abstract Hypericin is a promising photosensitizer for photodynamic therapy (PDT) characterized by a high yield of singlet oxygen. Photobleaching of hypericin has been studied by means of absorption and fluorescence spectroscopy in different biological systems: in human serum albumin solution, in cultured human adenocarcinoma WiDr cells and in the skin of nude mice. Prolonged exposure to light (up to 95 min, 100 mW/cm2) of wavelength around 596 nm induced fluence-dependent photobleaching of hypericin in all studied systems. The photobleaching was not oxygen dependent, and singlet oxygen probably played no significant role. Emission bands in the spectral regions 420–560 nm and above 600 nm characterize the photoproducts formed. An emission band at 615–635 nm was observed after irradiation of cells incubated with hypericin or of mouse skin in vivo but not in albumin solution. The excitation spectrum of these products resembled that of hypericin. Hypericin appears to be more photostable than most sensitizers used in PDT, including mTHPC and Photofrin.

In vitro and in vivo photosensitization by protoporphyrins possessing different lipophilicities and vertical localization in the membrane

Abstract Photodynamic therapy (PDT) is being evaluated in clinical trials for treatment of various oncologic and ophthalmic diseases. The main cause for cell inactivation and retardation of tumor growth after photoactivation of sensitizers is very short-lived singlet oxygen molecules that are produced and have limited diffusion distances. In this paper we show that the extent of biological damage can be modulated by using protoporphyrin, which was modified to increase its lipophilicity, and which also places the tetrapyrrole core deeper within the membrane by the carboxylate groups being anchored at the lipid:water interface. The uptake of the parent molecule (PPIX) and its diheptanoic acid analogue (PPIXC6) by WiDR and CT26 cells was investigated by fluorescence microscopy and by fluorescence intensity from the cells. The uptake of PPIXC6 increased almost linearly with incubation length for over 24 h, whereas for PPIX only 1 h was needed to reach maximal intracellular concentration. Fluorescence microscopy of both cell lines indicated that both drugs were distributed diffusely in the plasma membrane and cytoplasm, but remained outside the nucleus. The efficiency of in vitro inactivation of WiDr and CT26 cells increased with the length of the alkylcarboxylic chain. Tumors in mice that were treated with PPIX-PDT grew more slowly than control tumors. However, tumors that were given PPIXC6 followed by light exposure showed a significant delay in their growth.

Enhanced antitumour effect of photodynamic therapy by microtubule inhibitors

The combination of photodynamic therapy (PDT) and the microtubule (MT) inhibitor, vincristine (VCR) or taxol, was studied in the CaD2 mammary tumour model in mice. Meso-tetra(di-adjacent-sulphonatophenyl) porphine (TPPS2a) was used as a photosensitizer. An enhanced antitumour effect was found when VCR, at an almost non-toxic dose (1 mg/kg1, was injected i.p. into the mice 6 h before PDT, while no such enhanced effect was observed when the same dose of VCR was given either 12 or 24 h before PDT or immediately before PDT. Furthermore, it was found that the number of mitotic cells increased 4-5-fold 6 h after the injection of VCR into the mice. VCR did not enhance the sensitivity of normal skin to PDT. Combination of PDT and taxol was also studied. The antitumour activity of PDT could be increased by taxol when the drug (35 mg/kg) was administered i.p. either 6 h prior to PDT or immediately after or before PDT. No significant enhancement in PDT efficiency was found when PDT with photofrin was combined with VCR.