MODIFICATION OF EXTRACORPOREAL PHOTOPHERISIS TECHNOLOGY WITH PORPHYRIN PRECURSORS

Abstract

The present invention relates to the use of a photoactivatable Porphyrin-Derivative in extracorporeal photophoresis (ECP) treatment, in which a patient's blood of part of it containing said Porphyrin-derivative is/are exposed to light of a wavelength which activates said photoactivatable Porphyrin-Derivative.

Claims

1. A method of extracorporeal photophoresis treatment of a patient's blood or part of such blood which comprises the blood or part thereof with an ALA-Compound which is a 5-aminolevulinic acid or an ester thereof or a salt of said acid or ester and exposing such blood or part to light of a wavelength of between and including 315 nm and 450 nm, wherein said extracorporeal photophoresis treatment is for the treatment of cancer, lymphocyte-mediated malignant and non-malignant disorders, T-Cell-mediated diseases, autoimmune diseases or for modulating an immunological response against malignancies and immunological diseases.

2. A method of claim 1, wherein the ALA-Compound has the formula I,
R.sup.2R.sup.2NCH.sub.2COCH.sub.2CH.sub.2COOR.sup.1 (I) wherein R.sup.1 represents alkyl optionally substituted by hydroxy, alkoxy, acyloxy, alkoxycarbonyloxy, amino, sulpho, aryl, oxo or halogen groups and optionally interrupted by oxygen, nitrogen, sulphur or phosphorus atoms; and wherein R.sup.2 and R.sup.2 independently of each other represent a hydrogen atom or alkyl optionally substituted by hydroxy, alkoxy, acyloxy, aikoxycarbonyloxy, amino, sulpho, aryl, oxo or halogen groups and optionally interrupted by oxygen, nitrogen, sulphur or phosphorus atoms.

3. The method of claim 1, wherein the ALA-Compound is 5-aminolevulinic acid (ALA) or ALA-methylester, ALA-ethylester, ALA-propylester, ALA-hexylester (hexyl 5-amino-4-oxopentanoate; HAL), ALA-heptylester, ALA-octylester or salts thereof, preferably is 5-aminolevulinic acid (ALA), ALA-methyester (methyl-aminolevulinate) or ALA-hexylester (hexaminolevulinate; hexyl 5-amino-4-oxopentanoate; HAL), more preferably is 5-aminolevulinic acid (ALA) or ALA-hexylester (hexaminolevulinate; hexyl 5-amino-4-oxopentanoate; HAL).

4. The method of claim 1 wherein the time between contacting said patient's blood or part of it with said 5-aminolevulinic acid, ester or salt and the exposure to light of said wavelength is between and including 5 min and 480 min, preferably is between and including 15 min and 240 min, more preferably is between and including 30 min and 120 min, most preferably is between and including 45 min and 75 min.

5. A method of extracorporeal photophoresis treatment of a patient's blood or part of such blood which contains a photoactivatable Porphyrin-Derivative which comprises exposing the patient's blood or part thereof containing said photoactivatable Porphyrin-derivative to light of a wavelength which activates said photoactivatable Porphyrin-Derivative wherein said wavelength which activates said photoactivatable Porphyrin-Derivative is between 315 nm and 450 nm, inclusive, wherein said Porphyrin-Derivative is selected from a Porphyrin-Derivative being derived from an ALA-Compound according to claim 1, preferably being protoporphyrin IX, ALA-induced protoporphyrin IX, or hexaminolaevulinate-induced protoporphyrin IX, and wherein said extracorporeal photophoresis treatment is for the treatment of cancer, lymphocyte-mediated malignant and non-malignant disorders, T-Cell-mediated diseases, autoimmune diseases or for modulating an immunological response against malignancies and immunological diseases.

6. The method of claim 1 wherein said ALA-Compound is used in combination with a psoralen like 8-methoxypsoralen (8-MOP).

7. The method of claim 1, wherein the wavelength of the light activating the compound is selected from a wave length between 380 cm and 450 nm inclusive.

8. The method of claim 1, wherein said extracorporeal photophoresis treatment is for the treatment of cancer.

9. The method of claim 1, wherein said extracorporeal photophoresis treatment is also for the treatment of lymphoma, preferably T-cell lymphoma, most preferably cutaneous T-cell lymphoma or erythrodermic cutaneous T-cell lymphoma; and/or haematological cancer, or lymphocyte leukaemia.

10. The method of claim 1, wherein said extracorporeal photophoresis treatment is also for the treatment of graft versus host disease, like cGVHD following bone marrow transplantation, chronic graft versus host disease (cGvHD) with cutaneous/mucous membrane involvement, chronic graft versus host disease (cGvHD) with hepatic involvement, acute or chronic graft versus host disease with gastrointestinal/pulmonary involvement; and/or transplant rejection like solid organ transplant, like lung, renal, or liver transplant, cardiac/heart transplant, heart transplant rejection prophylaxis, organ allograft rejection, like cardiac, pulmonary or renal allograft rejection, Bronchiolitis Obliterans Syndrome (BOS); and/or multiple sclerosis, systemic sclerosis, progressive systemic sclerosis (PSS), systemic lupus erythematosus (SLE), rheumatoid arthritis, juvenile onset diabetes mellitus, type I diabetes mellitus.

11. The method of claim 1, wherein the said part of the patient's blood exposed is selected from isolated white blood cells, or leukocyte enriched blood, or leukocyte enriched buffy coat preferably having a hematocrit value of 0% to 10% and/or having 5% to 25% of the total peripheryl blood mononuclear cell component.

12. The method of claim 1, wherein said treatment is performed at an interval of once or on two consecutive days per week to once or on two consecutive days per four weeks, or on two consecutive days every two weeks; and/or wherein said treatment is performed over a period of between 1 and 12 months, between 2 and 6 months, or for 3 months.

13. The method of claim 1, wherein the extracorporeal photophoresis treatment comprises or consists of the steps of a) Selecting a patient to whom said photoactivatable Porphyrin-Derivative or ALA-Compound has been administered (Administering to said patient said photoactivatable Porphyrin-Derivative or ALA-Compound), optionally systemically, like orally or intravenously; and/or b) collecting said patient's blood extracorporeally, optionally mixing it with an anticoagulant; and/or c) enriching one of the mononuclear cell components in said patient's blood like enriching white blood cells to create said part of patient's blood being enriched with said one of the mononuclear cell components and a remaining part, optionally by centrifugation; and/or d) optionally reinfusing said remaining part of patient's blood; and/or e) contacting said patient's blood or said part thereof with said photoactivatable Porphyrin-Derivative or ALA-Compound; and/or f) incubating said patient's blood or said part thereof with said photoactivatable Porphyrin-Derivative or ALA-Compound; and/or g) exposing said patient's blood or said part thereof to light of a wavelength which activates said photoactivatable Porphyrin-Derivative or of a wavelength of between and including 100 nm and 1000 nm, and/or h) reinfusing said treated patient's blood or said treated part thereof preferably wherein the extracorporal photophoresis treatment is comprising or consisting of the steps b) to h), a) to d) and g) to h) or steps b) to e) and g) to h), more preferably wherein in addition, the steps b) to h), steps a) to d) and g) to h) or steps b) to e) and g) to h) are performed in consecutive order in said extracorporal photophoresis treatment.

14. The method of claim 1, wherein the compound used is 5-aminolevulinic acid (ALA) or ALA-hexylester (hexaminolevulinate; hexyl 5-amino-4-oxopentanoate; HAL), preferably at a concentration between 0.1 and 10 nM or 0.5 and 5 mM or at 1 mM in said patient's blood or part thereof or at a concentration of between 0.1 and 10 mg/kg or 0.5 mg/kg and 5 mg/kg or 0.75 and 2 mg/kg of body weight, and/or in the absence of serum or in the presence of 5 to 20% or 10% serum; and/or with the time between contacting said patient's blood or part of it with ALA or HAL or salt thereof and the exposure to light of said wavelength is between and including 30 min to 120 min, 45 min and 75 mm n or 1 hour; and/or said wavelength of the light is of between and including 315 nm and 1000 nm or between and including 315 and 850 nm, preferably between and including 315 nm and 380 nm, between and including 380 and 450 nm, or between and including 315 and 450 nm; said light having a dose between and including 0.01 and 20 J/cm.sup.2.

15. An ALA-Compound being a 5-aminolevulinic acid or an ester thereof or a salt of said acid or ester for use in an extracorporeal photophoresis treatment by exposing a patient's blood or part of it having been brought in contact with said 5-aminolevulinic acid, said ester or said salt to light of a wavelength of between and including 100 nm and 1000 nm, wherein said ALA-Compound is used in combination with a psoralen like 8-methoxypsoralen (8-MOP).

16. The method of claim 5, wherein said Porphyrin Derivative is used in combination with a psoralen like 8-methoxypsoralen (8-MOP).

17. The method of claim 5, wherein the wavelength of the light activating the compound is selected from a wave length between 380 nm and 450 nm inclusive.

18. The method of claim 5, wherein said extracorporeal photophoresis treatment is for the treatment of cancer.

19. The method of claim 5, wherein said extracorporeal photophoresis treatment is also for the treatment of lymphoma, preferably T-cell lymphoma, most preferably cutaneous T-cell lymphoma or erythrodermic cutaneous T-cell lymphoma; and/or haematological cancer, or lymphocyte leukaemia.

20. The method of claim 5, wherein said photophoresis treatment is also for the treatment of graft versus host disease, like cGVHD following bone marrow transplantation, chronic graft versus host disease (cGvHD) with cutaneous/mucous membrane involvement, chronic graft versus host disease (cGvHD) with hepatic involvement, acute or chronic graft versus host disease with gastrointestinal/pulmonary involvement; and/or transplant rejection like solid organ transplant, like lung, renal, or liver transplant, cardiac/heart transplant, heart transplant rejection prophylaxis, organ allocraft rejection, like cardiac, pulmonary or renal allograft rejection, Bronchiolitis Obliterans Syndrome (BOS); and/or multiple sclerosis, systemic sclerosis, progressive systemic sclerosis (PSS), systemic lupus erythematosus (SLE), rheumatoid arthritis, juvenile onset diabetes mellitus, type I diabetes mellitus.

Description

FIGURES

[0229] FIG. 1 (referring to Example 1): A) Spectra of HAL-induced PpIX in Jurkat cells. B) Spectra of emitted light by UV-A lamps.

[0230] FIG. 2 (referring to Example 1): Photodynamic inactivation of Jurkat cells using HAL.

[0231] FIG. 3 (referring to Example 1): Photoinactivation of Jurkat cells using 8-MOP.

[0232] FIG. 4 (referring to Example 1): Dependence of Jurkat cell photoinactivation by 8-MOP on UV-A light source.

[0233] FIG. 5 (referring to Example 1): Photoinactivation of Jurkat cells by using combination of HAL with 8-MOP and irradiation by UV-A light.

[0234] FIG. 6 (referring to Example 1): Dark toxicity (relative to control).

[0235] FIG. 7 (referring to Example 1): Cell survival assay.

[0236] FIG. 8 (referring to Example 1): Fluorescence images of Jurkat cells after different treatments.

[0237] FIG. 9 (referring to Example 2): The effects of 5-ALA on CD4.sup.+ T lymphocytes.

[0238] FIG. 10 (referring to Example 2): The effects of 5-ALA on CD8.sup.+ T lymphocytes.

[0239] FIG. 11 (referring to Example 3): Photodynamic inactivation of human T-cell lymphoma cell line using HAL.

[0240] FIG. 12 (referring to Example 3): Photodynamic inactivation of human T-cell lymphoma cell line using 8-MOP.

[0241] FIG. 13 (referring to Example 4): 5-ALA dark toxicity on human leukocytes Different analysed fractions from the same treated patient blood samples.

EXAMPLE 1

Materials and Methods

[0242] Chemicals.

[0243] Hexaminolevulinate (HAL) was provided by Photocure ASA (Oslo, Norway). A fresh stock solution of HAL was prepared in a mixture (1:9) of ethanol and serum free RPMI 1640 medium (PAA Laboratories GmbH, Fisher Scientific, Norway) to a concentration of 8 mM before each experiment. The stock solution of 8-methoxypsoralen (8-MOP) was prepared in absolute ethanol and kept frozen until use. All the chemicals used were of the highest purity commercially available.

[0244] Cell Line.

[0245] The human T-cell lymphoma cell line, Jurkat (ATCC number: TIB-152), growing in suspensions at passages 4-24 was used in the study. The cells were incubated in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, Invitrogen, Norway), L-glutamine (Gibco, Invitrogen, Norway), penicillin and streptomycin (Gibco, Invitrogen, Norway). For subcultivation, the cells were diluted to a density of 310.sup.5 cells/ml every second day. For experiments, the cells were diluted to a density of 110.sup.6 cells/ml day before the start of experiment.

[0246] Fluorescence Spectroscopy.

[0247] Fluorescence excitation spectra were recorded by means of Perkin Elmer LS50B Luminescence Spectrometer (Norwalk, Conn.) at the emission wavelength of 635 nm. A 15 nm slit width and a 1.00.4 cm.sup.2 quartz cuvette were used for the measurements.

[0248] The emission spectra of the UV and blue lamps were recorded by using fibre-coupled spectrometers (USB4000, Ocean Optics, Duiven, The Netherlands and Avantes AvaSpec-204814-USB2, The Netherlands, respectively).

[0249] Photodynamic and Psoralen/UV-A Treatments.

[0250] The cells were collected from the cultivation flasks, centrifuged at 1400 rpm for 5 min and diluted in the serum-free RPMI 1640 medium to a density of 37.510.sup.5 cells/ml. Eighty microliters of cell suspension were portioned into 96 well plates. After the addition of 10 l of HAL solution or serum-free RPMI 1640 medium (controls), the cells were incubated for 4 hours in the darkness at 37 C. Approximately 5 min before the end of incubation with HAL, 10 l of 8-MOP solution or serum-free RPMI 1640 medium (controls), were added to the wells. After 4 hours incubation with HAL and 5 min incubation with 8-MOP, the samples were irradiated either with UV-A, blue light or both at the exposure times indicated in the Results section.

[0251] For the UV-A irradiation of samples, a home-made UV-A lamp (Srensen UV-A lamp, Phillips Th 20W/09) emitting light mainly in the region 340-410 nm was used (FIG. 1). For the blue light illumination of samples, the light from a bank of four fluorescent tubes (model 3026, Applied Photophysics, London, United Kingdom) emitting light mainly in the region 410-500 nm with a maximum around 440 nm was used (FIG. 1).

[0252] In Vitro Cell Proliferation Assay.

[0253] Cell proliferation was assessed with a commercially available kit using a colorimetric method based on the cellular conversion of a tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 Htetrazolium, inner salt, MTS) into a formazan product, which can be detected by 492 nm absorbance. Twenty four hours after irradiation, 20 l of MTS (Promega Corporation, Madison, Wis.) was added to each well. Absorbance at 492 nm was measured after one hour incubation at 37 C. by means of a well plate reader (Multiskan Ex, Labsystems, Finland).

[0254] In Vitro Long Term Cell Survival Assay.

[0255] Methylcellulose-based medium human MethoCult with recombinant cytokines, (H4034, StemCell Technologies, France) was used for survival assay. Immediately after treatments, 120 l of serum-free RPMI 1640 medium containing 200 Jurkat cells were added to 1.2 ml of MethoCult. The samples were treated according to the manufacturer's instructions and suspensions were transferred into 35 mm Petri dishes. They were incubated at 37 C. in a humidified incubator with 5% 00.sub.2.

[0256] Assessment of Apoptotic Cells.

[0257] Apoptotic cells were identified by fluorescence microscopy based on nuclear morphology after staining with 4.0 g/ml Hoechst 33342 (Sigma, St. Louis, Mo.) at 37 C. for 10 min. This assay was verified in our previous experiments [7]. The filter combination consisted of a 330 to 380 nm excitation filter, a 400 nm beam splitter, and a 420 nm long-pass emission filter. 2.5 g/ml of propidium iodide (PI) were also added to the samples to confirm cell death. For PI fluorescence detection, the filter combination consisted of a 540/25 nm excitation filter, a 565 nm beam splitter, and a 605/55 nm band-pass emission filter. Fluorescence images were captured by a highly light sensitive thermo-electrically cooled charge-coupled device camera ORCAII-ER (Hamamatsu, Japan).

[0258] Statistical Analysis.

[0259] For statistical evaluation of data and curve fittings, Sigma plot software was used. The Student's t-tests were applied for statistical analysis.

[0260] Results.

[0261] Spectral Measurements.

[0262] The fluorescence excitation spectrum of cellular HAL-induced PpIX was measured in the suspension of Jurkat cells incubated with HAL in serum-free medium for 4 hours. The spectra of lights emitted by the lamps used in our laboratory and by the light source of the clinically used Therakos photopheresis system were measured under various settings (compact lamp, fluorescent tubes only, light transmitted through plastic, etc.).

[0263] From the comparison of the measured spectra (FIG. 1A) two conclusions can be drawn. First, the emission spectra of all the lamps partially overlap the excitation spectrum of cellular HAL-induced PpIX. Second, the light emitted by UV lamps depends on the settings (FIG. 1B).

[0264] Treatment with HAL and Light.

[0265] The fluorescence excitation spectrum of HAL-induced PpIX produced in cells has shown a considerable overlap with emission spectra of both blue as well as UV-A lamps used in the present study (FIG. 1A). The sensitivity of Jurkat cells to HAL-mediated PDT was therefore examined after irradiation of the cells by either blue or UV-A light. Ten or 20 s blue light illumination, a high enough dose typically used for photodynamic inactivation of leukemia and lymphoma cell lines; and 5 or 10 min UV-A irradiation, corresponding roughly to the time periods of cell exposure to UV-A light during clinical ECP, were tested. Cell proliferation, evaluated by MTS assay, was found to decrease in a concentration-dependent manner in all cases (FIG. 2A). Presence of 1.00 1V18-MOP in the samples during HAL-based PDT with blue light did not affect cell proliferation. The same results, within experimental error, were obtained when serum (FBS, final concentration of 10%) was added to the cells after UV-A irradiation (FIGS. 2B, C).

[0266] These results clearly show that HAL can be used in combination with the Therakos UV-A light to photoinactivate cells.

[0267] Treatment with 8-MOP and Light.

[0268] The photosensitivity of Jurkat cells to the combined treatment with 8-MOP and UV-A light was first tested in the absence of HAL. At both UV-A irradiation times tested (5 and 10 min), increasing 8-MOP concentration in the samples resulted in decreasing cell proliferation measured by MTS assay (FIG. 3). Since the emission spectrum of the blue lamp occurs far from the action spectrum of 8-MOP (e.g. [38]), activation of 8-MOP by the blue light has not been tested. On the contrary, the effect of 8-MOP is expected to be dependent on the spectral range of the emission spectrum of the UV-A lamp. Indeed, the ability of 1.00 M 8-MOP to sensitise the cells to photoinactivation, was significantly increased when the spectrum of the UV-A lamp was shifted by only few nm (FIG. 4).

[0269] The experiments clearly document that the effect of 8-MOP is highly dependent on spectral region covered by the UV-A light.

[0270] Treatment with the Combination of HAL, 8-MOP and Light.

[0271] Since both HAL and 8-MOP can generate an effect of cell photoinactivation after UV-A light irradiation, the possibility of using combination of the two drugs at reduced concentrations was tested. For this purpose several combinations of 8-MOP and HAL were explored (Table 1). A significantly lower cell proliferation could be achieved after the incubation of the cells with 0.10 M 8-MOP+1.55 M HAL as compared to corresponding concentrations of 8-MOP or HAL alone if followed by 10 min UV-A irradiation (FIG. 5). Similar effect could be achieved with the combination of 0.50 M 8-MOP+1.55 M HAL if followed by 5 min UV-A irradiation (FIG. 5). Qualitatively the same results were obtained when serum (10% FBS) were added to the samples after UV-A irradiation (not shown). A HAL concentration of 5.00 M was sufficient to decrease cell proliferation to minimum after light exposure, therefore, the effect of combination with 8-MOP could not be tested.

[0272] The data thus demonstrate the possibility of cell photoinactivation by the combination of HAL with 8-MOP, each given at concentrations lower than those necessary for the cell photoinactivation alone.

[0273] Dark Toxicity.

[0274] 8-MOP binds to DNA raising concerns about its toxicity. HAL may also be toxic when given systemically. Therefore, in each experiment, samples treated with the drugs but not with the light were included to check for the dark toxicity of the drugs. In general, there was no indication for dark toxicities of HAL (up to 11.00 M; FIG. 2, 6) or 8-MOP (up to 1.00 M; FIGS. 3, 4, 6) based on the data of MTS assay. Slightly lower cell proliferation rates were found when combinations of the drugs were used to treat the cells (FIG. 6).

[0275] Long Term Cell Survival.

[0276] All treatment regimes tested resulted in decreases in Jurkat cell proliferation. To check whether the decrease in cell proliferation correlated with long term cell death, a clonogenic assay in a semisolid medium was intended after the following treatments: 1.0 M MOP/10 min UV-A, 5.0 M HAL/10 min UV-A, 0.1 M MOP+1.55 M HAL/10 min UV-A, 7.4 M HAL/10 s blue light. Surprisingly, a week after the cell seeding, in none of the samples (including control, untreated ones) compact colonies could be found, although the colonies were expected to form in such special medium [39-41]. In the control sample, however, numerous living single cells were seen. After two weeks, spread and overlapping areas of high cell densities appeared in control samples, which covered almost the whole area of the dishes. No living cells were seen in the samples treated with UV-A. In some cases, occasional areas of dense cells, similar to those seen in controls, were visible in the samples treated with the combination of HAL with blue light (Table 2). This is in agreement with the results depicted in FIG. 2 showing that at the conditions (7.4 M HAL/10 s blue light), cell proliferation did not decrease completely. Consistent with the presence of living, metabolizing cells, a yellowish change of the medium colour was noticed in control samples (FIG. 7).

[0277] Cell Death Assessment.

[0278] All treatments tested in the present study (8-MOP/UV-A, HAL/blue light, HAL/UV-A, MOP+HAL/UV-A) could induce death of Jurkat cells in a drug or light dose-dependent manner. Twenty hours following the treatments, the cells were analyzed by fluorescence microscopy in order to check whether the modes of cell death differ depending on the treatment modality. There were considerable differences in the contents of apoptotic bodies present in the samples. While almost all the cells seemed to die by apoptosis after the treatment with 1.00 M 8-MOP followed by UV-A, no apoptotic bodies could be seen in the samples treated with 5.00 M HAL and 10 min UV-A irradiation (FIG. 8). The amount of apoptotic bodies among dead cells in the samples treated with HAL was dependent on the treatment regimen (Table 3).

Discussion.

[0279] In the present study, the inventors have tested HAL in combination with UV-A to inactivate T-cell lymphoma Jurkat cell line as an in vitro model of cutaneous T-cell lymphoma and compared the effect with those achieved under typically used regimens combining either UV-A irradiation with sensitization of cells with 8-MOP or blue light irradiation with treatment of cells with HAL.

[0280] Decrease in both proliferation and survival of Jurkat cells could be induced by the treatment with HAL and UV-A irradiation. This is not surprising due to a partial overlap between excitation spectrum of PpIX and the emission spectrum of UV-A lamp used in this study. The overlap is comparable with that between PpIX excitation spectrum and the emission spectrum of the used blue light lamp (FIG. 1A). The blue light lamp was shown to be effective for inactivation of cells of various origins, including leukemia and lymphoma cell lines after HAL-PDT [7, 8, 34-37]. Decrease in proliferation of Jurkat cells after the treatment with HAL and UV-A light is in agreement with results of another study, where the combination of ALA and UV-A was tested with T-cell lymphoma HUT-78 cell line for the purposes of PUVA treatment [42].

[0281] Depending on the treatment regimen used, differences could be seen in the mode of cell death. While only apoptosis was induced when cells were treated with 8-MOP and UV-A light, parameters such as HAL concentration, light source (UV-A or blue light) and irradiation time affected the mode of the death of Jurkat cells in the case of HAL-induced cell photoinactivation.

[0282] As discussed in several publications, the therapeutic effect of ECP in vivo is not caused by cell inactivation only, but additionally relies on the induction of immune response (for references see [43]). In this regard, it is important to mention that several publications discussed the significance of the mode of cell death for induction of immune effects [44-48]. It has been recognised that viable cells are able to discriminate apoptotic from necrotic targets via distinct cell surface receptors and such receptors can induce signalling events that differ for apoptotic compared with necrotic targets [49]. Our data show that apoptosis is induced by 8-MOP-UV-A and the clinical experience indicates that this mode of cell death is efficient to achieve a treatment effect of photophoresis. Apoptosis as well as necrosis could be induced by HAL-PDT in the present study and the question is whether appearance of necrotic cells would be beneficial for the treatment outcome. In this respect, immunopotentiation induced by PDT has been documented in numerous publications (reviewed in e.g. [14, 50]). Importantly, vaccines and lysates generated by exogenous photosensitiser-mediated PDT were shown to be more effective than those generated by means of UV and ionizing irradiations or a freeze-thaw technique [51-53]. Improved effect of PDTgenerated vaccines has been ascribed to the concurrent appearance of necrotic and apoptotic cells under PDT treatment protocols contrary to the presence of pure apoptotic or pure necrotic cells under UV or ionizing radiation protocols, respectively [51]. It should be mentioned, however, that under certain conditions, PDT was reported to cause immunosuppression as well (for references see [50]).

[0283] ECP is a recommended treatment of CTCL and there is fair evidence to support its use in GVHD [1]. However, many patients experience refractory disease, thus further development of the current ECP regime may hopefully be of benefit to patients.

[0284] Based on the data of the present study, the inventors concluded that HAL is effective for the photoinactivation of T-cell lymphoma Jurkat cell line after using a UV-A lamp with an emission spectrum similar to that of the light source used in the commercial Therakos photopheresis system. HAL-UV-A induced both apoptosis and necrosis of the Jurkat cells and may thus provide a potential option for enhanced efficacy of ECP. If HAL should be used for ECP, the treatment conditions need to be optimized in subjects with an intact immune system to achieve induction of a desirable immune response.

TABLE-US-00001 TABLE 1 Conditions of the cell treatment with the combination of HAL, 8-MOP and UV-A light. 8-MOP (M) HAL (M) UV-A (min) 0.10 1.55 10 0.10 5.00 10 0.50 1.55 5 0.50 5.00 5

TABLE-US-00002 TABLE 2 Number of high cell density areas formed in semisolid medium 2 weeks after treatment. Sample Number of colonies control (no treatment) many 1.00 M MOP/10 min UV-A 0.8 2.0 (1/6*) 5.00 M HAL/10 min UV-A 0 7.40 M HAL/10 s blue light 4.5 5.6 (4/6) 1.55 M HAL + 0.10 M MOP/10 min UV-A 0 *number of experiments when colonies were formed/total number of experiments

TABLE-US-00003 TABLE 3 Assessment of the mode of cell death after HAL-induced photoinactivation. (Quantitative evaluation was not possible due to the disintegration of apoptotic cells.) Drug Cell appearance 20 hours after concentration Irradiation treatment 1.55 M HAL 10 min UV-A mostly apoptotic + some alive, or mostly necrotic + some apoptotic* 5.00 M HAL 10 min UV-A only necrotic, no apoptotic 5.00 M HAL 10 s UV-A apoptotic + alive (similar proportions) 5.00 M HAL 10 s blue light mostly apoptotic + some alive 1.55 M HAL + 10 min UV-A almost only apoptotic + few alive 0.10 M 8-MOP *sample to sample variations

FIGURE LEGENDS

[0285] FIG. 1 A) Normalized fluorescence excitation spectra of HAL-induced PpIX in Jurkat cells and normalized emission spectra of blue and UV-A lamps. B) Spectra of emitted light by UV-A lamps under different settings (compact lamp used in our laboratory covered with plastic culture plate, fluorescent tubes of the lamp used in our laboratory, lamp of the clinically used photopheresis system).

[0286] FIG. 2 Photodynamic inactivation of Jurkat cells using HAL. A) blue light illumination in the absence (open circles) and the presence (full circles) of 1.00 M 8-MOP; a) control (no blue light), b) 10 s and c) 20 s blue light illumination. B) UV-A light irradiation by light with the spectrum shown in FIG. 1, fluorescent tubes only; () control (no irradiation), () 5 min, (.square-solid.) 10 min irradiation, C) same as B) but, FBS added to the samples (final concentration of 10%) after UV-A irradiation. Each data point represents an averageS.D. from at least 4 different cell samples.

[0287] FIG. 3 Photoinactivation of Jurkat cells using 8-MOP and UV-A light irradiation. () control (no irradiation), () 5 min, (.square-solid.) 10 min irradiation. The samples were irradiated by the compact lamp with the spectrum shown in FIG. 1. Each data point represents an averageS.D. from 6 different cell samples.

[0288] FIG. 4 Dependence of Jurkat cell photoinactivation by 8-MOP on UV-A light source. The cells were incubated with 1.00 M 8-MOP and samples were irradiated by lights with spectra shown in FIG. 1, fluorescent tubes only () and whole lamp (.square-solid.); (, ) controls (no irradiation). Each data point represents an averageS.D. from 6 different cell samples except from 10 min and 30 min time points, where only 4 and 2 samples, respectively were used.

[0289] FIG. 5 Photoinactivation of Jurkat cells by using combination of HAL with 8-MOP and irradiation by UV-A light. The data for 1.55 M HAL are shown. The samples were irradiated by the fluorescent tubes light with the spectrum shown in FIG. 1. The data are expressed relative to control. The bars represent an averageS.D. from 8 different cell samples.

[0290] FIG. 6 Dark toxicity (relative to control). The cells were treated with indicated drugs and doses, but the samples were not irradiated by light. The bars represent an averageS.D. from at least 6 different cell samples.

[0291] FIG. 7 Cell survival assay. Change in the color of the medium in control sample three weeks after seeding the cellsconsistent with the presence of living, metabolizing cells.

[0292] FIG. 8 Fluorescence images of Jurkat cells after different treatments showing nuclear morphology after staining with Hoechst 33342 (left column) and PI (middle column). Phase contrast images are shown in the right column. (A) control, (B) 0.10 M 8-MOP/UV-A, (C) 1.55 M HAL/UV-A, (D) 0.10 M 8-MOP+1.55 M HAL/UV-A, (E) 1.00 M 8-MOP/UV-A, (F) 5.00 M HAL/UV-A, (G) 5.00 M HAL/UV-A, (H) 5.00 M HAL/blue light. The samples were irradiated for 10 min (B-F) or 10 s (G, H) by lights with the spectra shown in FIG. 1, fluorescent tubes only (FIG. 1B) or blue lamp (FIG. 1A).

EXAMPLE 2

Evaluation of Ex Vivo Efficacy of ALA-ECP in Patients with cGvHD and CTCL

[0293] Optimization of experimental conditions for ALA-ECP was performed in Jurkat cells, varying ALA concentration (1 to 5 mM) and treatment time (1 to 4 hours) in the presence of 10% foetal bovine serum. The cells were then exposed to UVA for 10 min using an UV-A lamp with almost identical emission spectra to those of the UV-A lamp used in the existing commercial Therakos photopheresis system. The cell survival after the ALA/UVA treatment was measured by fluorescence microscopy with propidium iodide (PI) staining.

[0294] A suitable dose and incubation time of ALA was used in leukocyte samples collected from ECP patients. The dark toxicity of ALA at the concentration in question was evaluated, followed by the ALA/UVA-mediated killing efficiency before studies on the mechanism of its action (apoptosis/necrosis). The lymphocytes from patients were incubated with ALA under the optimized experimental conditions before exposure to the UVA lamp in the Therakos Photopheresis System at the same light dose as used in the ECP treatment. The cell survival of ALA dark toxicity, ALA/UVA-mediated killing efficiency and apoptosis/necrosis were measured with flow cytometry using combined labelling of CD4.sup.+ or CD8.sup.+ with Annexin V/PI staining.

[0295] The killing effects of 5-ALA (a porphyrin precursor) plus the UVA light irradiation on CD4.sup.+/CD8.sup.+ T lymphocytes collected from a Graft versus Host Disease (GvHD) patient who went to a standard photopheresis treatment was evaluated. The cells were exposed to ALA+UVA in the standard photopheresis machine, and percentage survival evaluated at various timepoints between 1 h and 20 h. The 8-MOP plus UVA used for the standard photopheresis was also included for comparison. The cell survival was measured with flow cytometry using a combined staining of CD4/CD8 antibody, Annexin-V and eFluor450.

[0296] Data for proliferative/activated CD4+ T-cells from one patient are shown in FIG. 1, and for CD8 T-cells in FIG. 2. The results show that the treatment with ALA plus UVA killed the CD4.sup.+ and CD8.sup.+ T-cells much more effectively than 8-MOP plus UV-A, particularly at a low light dose, ie. 5-min light illumination.

[0297] The inventors have so far treated the T-cells from 4 patients with cutaneous T-cell lymphoma or GvHD patients, with similar results, summarized in Table 4. It is clear that UVA irradiation alone can kill T-cells, suggesting that there is a need to improve this modality with non-toxic visible light source.

TABLE-US-00004 TABLE 4 1 hour (CD4+/CD8+) 20 hours (CD4+/CD8+) Control 5-ALA 8-MOP Control 5-ALA 8-MOP Patient-1 89.7/95.8 73.7/85.1 87.3/94.2 72.2/61.6 4.3/0.0 57.2/56.9 Patient-2 79.8/83.6 52.1/53.1 78.4/84.7 40.4/16.3 1.2/0.3 13.3/5.8 Patient-3 78.4/82.2 78.0/76.1 81.3/78.1 45.8/35.0 6.2/0.7 18.2/15.7 Patient-4 48.3/31.3 56.4/35.7 53.6/30.6 27.7/22.5 6.4/1.0 12.0/8.8

[0298] Data presented with the percentages of CD4.sup.+/CD8.sup.+ T-cell survival at 1 hour and 20 hours after treatment with UVA alone (control), UVA plus 5-ALA or UVA plus 8-MOP. The UVA light dose was 0.158 J/cm.sup.2.

FIGURE LEGENDS

[0299] FIG. 9. The killing effects of 5-ALA plus the UVA light irradiation on CD4.sup.+ T lymphocytes collected from a GvHD patient.

[0300] FIG. 10. The killing effects of 5-ALA plus the UVA light irradiation on CD8.sup.+ T lymphocytes collected from a GvHD patient.

EXAMPLE 3

[0301] Under the same conditions as said out in Example 1 above, experiments were performed in Karpas 299, another human T-cell lymphoma cell line different from the Jurkat cells used in Example 1 with similar results shown in FIGS. 11 and 12.

FIGURE LEGENDS

[0302] FIG. 11 Photodynamic inactivation of human T-cell lymphoma cell line (Karpas 299) using HAL plus UV-A exposure. () control (no irradiation), () 5 min, (.square-solid.) 10 min irradiation.

[0303] FIG. 12 Photodynamic inactivation of human T-cell lymphoma cell line (Karpas 299) using 8-MOP plus UV-A exposure. () control (no irradiation), () 10 min, (.square-solid.) 20 min irradiation.

EXAMPLE 4

Evaluation of 5-ALA Dark Toxicity on Human Leukocytes

[0304] The general safety of 5-ALA in absence of light-mediated activation (dark toxicity) was next evaluated. Blood samples from 10 GvHD or CTLC patients were treated with 10 mM 5-ALA at 37 C. in a 5% CO.sub.2 humidified incubator overnight (17-24 hours). Buffy coat samples from patients were directly harvested from the Therakos Photopheresis System during the standard photopheresis treatment. The cells incubated with the culture medium containing no 5-ALA were also included as a control in all individuals. The leukocytes were then labelled with antibodies for flow cytometry analysis of various subpopulations. The antibodies used were: CD45 PerCP-Cyanine5.5 for leukocytes, CD4 FITC for T helper cells, CD8 FITC for T cytotoxic cells and CD19 FITC for B-cells. The cells were also labelled with Fixable Viability Dye eFluor 450 for dead cells and Annexin V for apoptotic/dead cells. The different subpopulations of leukocytes were gated out and analyzed for cell viability. The cells that were negative for both the Fixable Viability Dye eFluor 450 and Annexin V were considered as viable cells.

[0305] The results are summarized in FIG. 13, showing different analysed fractions from the same treated patient blood samples. Note that for technical reasons, B-cell fractions were obtained from only 5 patient samples, while a whole blood cell (WBC) fraction was missing for one of the patient samples. Generally, no significant dark cytotoxic effect of 5-ALA was noted on the leukocytes studied, ata concentration (10 mM) and exposure time (17-24 hours) of 5-ALA in excess of that expected to be used in a clinical setting. It has to be noted though, that the concentration and exposure time of 5-ALA finally used in a clinical setting will depend on many factors and the specific circumstances of a case and will thus be subject to change.

REFERENCES

[0306] [1] Scarisbrick J J, Taylor P, Holtick U, et al. Photopheresis Expert Group. U.K. consensus statement on the use of extracorporeal photopheresis for treatment of cutaneous T-cell lymphoma and chronic graft-versus-host disease. Br J Dermatol 2008; 158:659-78. [0307] [2] Edelson R, Berger C, Gasparro F et al. Treatment of cutaneous T cell lymphoma by extracorporeal photochemotherapy. N Engl J Med 1987; 316: 297-303. [0308] [3] Worel N, Leitner G. Clinical results of extracorporeal photopheresis. Tansfus

[0309] Med Hemother 2012; 39: 254-62. [0310] [4] Gupta A K, Anderson T F. Psoralen photochemotherapy. J Am Acad Dermatol 1987; 17:703-34. [0311] [5] Peng Q, Berg K, Moan J, et al. Review: 5-Aminolevulinic acid-based photodynamic therapy: Principle and experimental research. Photochem Photobiol 1997a; 65:235-51. [0312] [6] Peng Q, Warloe T, Berg K, et al. Review: 5-Aminolevulinic acid-based photodynamic therapy: Clinical research and future challenges. Cancer 1997b; 79:2282-308. [0313] [7] Furre 1E, Mller M T, Shahzidi S, et al. Involvement of both caspase-dependent and -independent pathways in apoptotic induction by hexaminolevulinate-mediated photodynamic therapy in human lymphoma cells. Apoptosis 2006; 11:2031-42. [0314] [8] Furre I E, Shahzidi S, Luksiene Z, et al. Targeting PBR by hexaminolevulinate-mediated photodynamic therapy induces apoptosis through translocation of apoptosis-inducing factor in human leukemia cells. Cancer Res 2005; 65:11051-60. [0315] [9] Gaullier J M, Berg K, Peng Q, et al. The use of esters of 5-aminolevulinic acid to improve photodynamic therapy on cells in culture. Cancer Res 1997; 57:1481-6. [0316] [10] Peng Q, Moan J, Warloe T, et al. Build-up of esterified aminolevulinic-acidderivative-induced porphyrin fluorescence in normal mouse skin. J Photochem Photobiol B 1996; 34:95-6. [0317] [11] Rittenhouse-Diakun K, Van Leengoed H, Morgan J, et al. The role of transferrin receptor (CD71) in photodynamic therapy of activated and malignant lymphocytes using the heme precursor delta-aminolevulinic acid (ALA). Photochem Photobiol 1995; 61:523-8. [0318] [12] Hryhorenko E A, Rittenhouse-Diakun K, Harvey N S, et al. Characterization of endogenous protoporphyrin IX induced by delta-aminolevulinic acid in resting and activated peripheral blood lymphocytes by four-color flow cytometry. Photochem Photobiol 1998; 67:565-72. [0319] [13] Casas A, Perotti C, Fukuda H, del C Battle A M. Photodynamic therapy of activated and resting lymphocytes and its antioxidant adaptive response. Lasers Med Sci 2002; 17:42-50. [0320] [14] Castano A, Mroz P, Hamblin M R. Photodynamic therapy and anti-tumor immunity. Nature Reviews Cancer 2006; 6:535-45. [0321] [15] Gollnick S O, Brackett C M. Enhancement of anti-tumor immunity by photodynamic therapy. Immunol Res 2010; 46:216-26. [0322] [16] Warloe T, Peng Q, Heyerdahl H, et al. Photodynamic therapy with 5-aminolevulinic acid-induced porphyrins and DMSO/EDTA for basal cell carcinoma. SPIE 1995; 2371:226-235. [0323] [17] Christensen E, Skogvoll E, Viset T, et al. Photodynamic therapy with 5-aminolaevulinic acid, dimethylsulfoxide and curettage in basal cell carcinoma: a 6-year clinical and histological follow-up. J Eur Acad Dermatol Venereol 2009; 23:58-66. [0324] [18] Christensen E, Mork C, Skogvoll E. High and sustained efficacy after two sessions of topical 5-aminolaevulinic acid photodynamic therapy for basal cell carcinoma: a prospective, clinical and histological 10-year follow-up study. Br J Dermatol 2012; 166:1342-8. [0325] [19] Stummer W, Pichlmeier U, Meinel T, et al. Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomised controlled multicentre phase III trial. Lancet Oncol 2006; 7: 392-401 [0326] [20] Stummer W, Novotnt A, Stepp H, et al. Fluorescence-guided resection of glioblastoma multiforme by using 5-aminolevulinic acid-induced porphyrins: a prospectie study in 52 consecutive patients. J Neurosurg 2000; 93: 1003-1013. [0327] [21] Panciani P P, Fontanella M, Schatlo B, et al. Fluorescence and image guided resection in high grade glioma. Clin Neurol Neurosurg 2012; 114: 37-41 [0328] [22] Piccirillo S G M, Dietz S, Madhu B, et al. Fluorescence-guided surgical sampling of glioblastoma identifies phenotypically distinct tumour-initiating cell populations in the tumour mass and margin. Br J Cancer 2012; 107: 462-8 [0329] [23] Gross S A, Wolfsen H C. The role of photodynamic therapy in the esophagus. Gastrointest Endoscopy Clin N Am 2010; 20: 35-53 [0330] [24] Dunn J, Lovat L. Photodynamic therapy using 5-aminolevulinic acid for the treatment of dysplasia in Barrett's oesophagus. Expert Opin Pharmacother 2008; 9:851-8 [0331] [25] Menon D, Stafinski T, Wu H, et al. Endoscopic treatments for Barrett's esophagus: a systemic review of safety and effectiveness compared to esophagectomy. BMC Gastroenterology 2010; 10: [0332] [26] lshizuka M, Abe F, Sano Y, et al. Novel development of 5-aminolevulinic acid (ALA) in cancer diagnoses and therapy. Int Immunopharmacol 2011; 11: 358-365 [0333] [27] Regula J, Macrobert A J, Gorchein A, et al. Photosensitisation and photodynamic therapy of oesophageal, duodenal, and colorectal tumours using 5-aminolevulinic acid induced protoporphyrin IX: a pilot study. Gut 1995; 36: 67-75 [0334] [28] Sylantiev C, Schoenfeld N, Mamet R, et al. Acute neuropathy mimicking porphyria induced by aminolevulinic acid during photodynamic therapy. Muscle Nerve 2005; 31: 390-3 [0335] [29] Peng, Q, Evensen J F, Rimington C, et al. A comparison of different photosensitizing dyes with respect to uptake C3H-tumors and tissue of mice. Cancer Lett 1987; 36: 1-10 [0336] [30] Peng Q, Moan J, Warloe T, et al. Distribution and photosensitizing efficiency of porphyrins induced by application of exogenous 5-aminolevulinic acid in mice bearing mammary carcinoma. Int J Cancer 1992; 52: 433-43 [0337] [31] Warloe T, Peng Q, Moan J, et al. Photochemotherapy of multiple basal cell carcinoma with endogenous porphyrins induced by topical application of 5-aminolevulinic acid. In: Photodynamic therapy and biomedical lasers. Spinelli P, Dal Fante M, Marchesini R, editors. Excerpta Medica, Elsevier Science Publishers B.V., Amsterdam; 1992; pp. 449-53. [0338] [32] Warloe T, Peng, Q, Heyerdahl H, et al. Photodynamic therapy of human tubulo villous adenomas. SPIE 1995; 2325: 425-36 [0339] [33] Noodt, B B, Berg K, Stokke T, et al. Apoptosis induced by photodynamic therapy (PDT) with ALA. Br J Cancer 1996; 74: 22-9 [0340] [34] Shahzidi S, Stokke T, Soltani H, et al. Induction of apoptosis by hexaminolevulinate-mediated photodynamic therapy in human colon carcinoma cell line 320DM. J Environ Pathol Toxicol Oncol 2006; 25:159-171. [0341] [35] Shahzidi S, {hacek over (C)}underlikova B, Widlocha A, et al. Simultaneously targeting mitochondria and endoplasmic reticulum by photodynamic therapy induces apoptosis in human lymphoma cells. Photochem Photobiol Sci. 2011; 10: 1773-1782. [0342] [36] {hacek over (C)}underlikova B, Vasovi{hacek over (c)} V, Sieber F, et al. Hexaminolevulinate-mediated photodynamic purging of leukemia cells from bone marrow. Bone Marrow Transplant 2010; 45:1553-1561. [0343] [37] {hacek over (C)}underlikov B, Vasovi{hacek over (c)} V, Sieber F, et al. Hexaminolevulinate-mediated photodynamic purging of marrow grafts with murine breast carcinoma. Bone Marrow Transplant 2011; 46: 1118-1127. [0344] [38] Ortel B, Gange R W. An action spectrum for the elicitation of erythema in skin persistently sensistized by photobound 8-methoxypsoralen. J Invest Dermatol 1990; 94:781-5. [0345] [39] Galietta A, Gunby R H, Redaelli S, et al. NPM/ALK binds and phosphorylates the RNA/DNA-binding protein PSF in anaplastic large-cell lymphoma. Blood 2007; 110:2600-9. [0346] [40] Fakler M, Loeder S, Vogler M, et al. Small molecule XIAP inhibitors cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells and overcome BcI-2-mediated resistance. Blood 2009; 113:1710-22. [0347] [41] Nahimana A, Attinger A, Aubry D, et al. The NAD biosynthesis inhibitor AP0866 has potent antitumor activity against hematologic malignancies. Blood 2009; 113:3276-86. [0348] [42] Akita Y, Watanabe D, Yanagishita T, et al. The effect of psoralen plus ultraviolet

[0349] A in vitro in HUT-78 enhances by 5-aminolevulinic acid. Photodermatol Photoimmunol Photomed 2007; 23:95-7. [0350] [43] Broady R, Yu J, Levings M K. Pro-tolerogenic effects of photodynamic therapy with TH9402 on dendritic cells. J Clin Apheresis 2008; 23:82-91. [0351] [44] Melcher A, Gough M, Todryk S, Vile R. Apoptosis or necrosis for tumor immunotherapy: what's in a name? J Mol Med 1999; 77:824-33. [0352] [45] Cocco R E, Ucker D S. Distinct modes of macrophage recognition for apoptotic and necrotic cells are not specified exclusively by phosphatidylserine exposure. Mol Biol Cell 2001; 12:919-30. [0353] [46] Scheffer S R, Nave H, Korangy F, et al. Apoptotic, but not necrotic, tumor cell vaccines induce a potent immune response in vivo. Int J Cancer 2003; 103:105-11. [0354] [47] Basu S, Binder R J, Suto R, et al. Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-kB pathway. Int Immunol 2000; 12:1539-46. [0355] [48] Srivastava PK. Hypothesis: controlled necrosis as a tool for immunotherapy of human cancer. Cancer Immun 2003; 3:4. [0356] [49] Patel V A, Lee D J, Longacre-Antoni A, et al. Apoptotic and necrotic cells as sentinels of local tissue stress and inflammation: Response pathways initiated in nearby viable cells. Autoimmunity 2009; 42:317-21. [0357] [50] Chen W R, Huang Z, Korbelik M, et al. Photoimmunotherapy for cancer treatment. J Environ Pathol Toxicol Oncol 2006; 25:281-91. [0358] [51] Gollnick S, Vaughan L, Henderson B W. Generation of effective anti-tumor vaccines using photodynamic therapy. Cancer Res 2002; 62:1604-8. [0359] [52] Bae S-M, Kim Y-W, Kwak S-Y, et al. Photodynamic therapy-generated tumor cell lysates with CpG-oligodeoxynucleotide enhance immunotherapy efficacy in human papillomavirus 16 (E6/E7) immortalized tumor cells. Cancer Sci 2007; 98:747-52. [0360] [53] Korbelik M, Sun J. Photodynamic therapy-generated vaccine for cancer therapy. Cancer Immunol Immunother 2006; 55:900-9.