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 or part of it containing sad 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 said blood, the method comprising: contacting the patient's blood or part thereof with a 5-aminolevulinic acid so as to provide a concentration of 5-aminolevulinic acid between 1 and 10 mM in the blood or part thereof; and exposing the resulting blood or part thereof to light of a wavelength between 315 nm and 450 nm inclusive, wherein said patient is suffering from a cancer, a lymphocyte-mediated malignant or non-malignant disorder, a T-Cell-mediated disease, an autoimmune disease or is afflicted with a malignancy or has an immunological disease.

2. The method of claim 1, wherein the time between contacting said patient's blood or part thereof with said 5-aminolevulinic acid and the exposure to light of said wavelength is between 5 min and 480 min inclusive.

3. The method of claim 1, further comprising: contacting the blood or part thereof with a psoralen.

4. The method of claim 1, wherein the wavelength of the light is between 380 nm and 450 nm inclusive.

5. The method of claim 1, patient suffering from a cancer selected from the group consisting of: T-cell lymphoma, cutaneous T-cell lymphoma, erythrodermic cutaneous T-cell lymphoma; a haematological cancer, or a lymphocyte leukaemia.

6. The method of claim 1, wherein said patient is suffering from a graft versus host disease; a transplant rejection; an organ allograft rejection; or multiple sclerosis, systemic sclerosis, progressive systemic sclerosis (PSS), systemic lupus erythematosus (SLE), rheumatoid arthritis, juvenile onset diabetes mellitus, or type I diabetes mellitus.

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

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

9. The method of claim 1, wherein the concentration of 5-aminolevulinic acid (ALA), in said patient's blood or part thereof is between 0.1 and 10 mM or between 0.1 and 10 mg/kg body weight of the subject, in the absence of serum or in the presence of 5 to 20% serum; wherein the time between contacting said patient's blood or part thereof is with ALA and the exposure to light is between 30 min and 120 min, the wavelength of the light is between 315 nm and 1000 nm inclusive and the dose of light is between 0.01 and 20 J/cm.sup.2 inclusive.

10. A method of extracorporeal photophoresis treatment of a patient's blood or part of said blood, the method comprising: contacting the patient's blood or part thereof with a 5-aminolevulinic acid so as to provide a concentration of 5-aminolevulinic acid between 1 and 10 mM in the blood or part thereof; and exposing the resulting blood or part thereof to light of a wavelength between 315 nm and 450 nm inclusive, wherein said patient is suffering from graft versus host disease (GVHD), chronic graft versus host disease (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, and/or acute or chronic graft versus host disease with gastrointestinal/pulmonary involvement.

11. The method of claim 1, wherein the time between contacting said patient's blood or part thereof with said 5-aminolevulinic acid and the exposure to light of said wavelength is between 15 min and 240 min inclusive.

12. The method of claim 11, wherein the time between contacting said patient's blood or part thereof with said 5-aminolevulinic acid and the exposure to light of said wavelength is between 30 min and 120 min inclusive.

13. The method of claim 11, wherein the time between contacting said patient's blood or part thereof with said 5-aminolevulinic acid and the exposure to light of said wavelength is between 45 min and 75 min inclusive.

Description

FIGURES

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

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

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

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

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

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

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

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

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

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

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

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

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

(14) FIG. 14 (referring to Example 2): 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.

EXAMPLE 1

(15) Materials and Methods.

(16) Chemicals.

(17) 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 RPM 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.

(18) Cell Line.

(19) 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.

(20) Fluorescence Spectroscopy.

(21) 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.

(22) 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-2048x14-USB2, The Netherlands, respectively).

(23) Photodynamic and Psoralen/UV-A Treatments.

(24) 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.

(25) For the UV-A irradiation of samples, a home-made UV-A lamp (Sorensen 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).

(26) In Vitro Cell Proliferation Assay.

(27) 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).

(28) In Vitro Long Term Cell Survival Assay.

(29) 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% CO.sub.2.

(30) Assessment of Apoptotic Cells.

(31) Apoptotic cells were identified by fluorescence microscopy based on nuclear morphology after staining with 4.0 g/mI 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/mI 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).

(32) Statistical Analysis.

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

(34) Results.

(35) Spectral Measurements.

(36) 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.).

(37) 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).

(38) Treatment with HAL and Light.

(39) 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 UVA 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 M 8-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).

(40) These results clearly show that HAL can be used in combination with the Therakos UV-A light to photoinactivate cells.

(41) Treatment with 8-MOP and Light.

(42) 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 UVA 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).

(43) The experiments clearly document that the effect of 8-MOP is highly dependent on spectral region covered by the UV-A light.

(44) Treatment with the Combination of HAL, 8-MOP and Light.

(45) Since both HAL and 8-MOP can generate an effect of cell photoinactivation after UVA 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.

(46) 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.

(47) Dark Toxicity.

(48) 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).

(49) Long Term Cell Survival.

(50) 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 HALJ10 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).

(51) Cell Death Assessment.

(52) 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

(53) 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.

(54) 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].

(55) 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.

(56) 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 PDT-generated 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]).

(57) 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.

(58) 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.

(59) 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

(60) 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

(61) 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

(62) 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).

(63) 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), (.circle-solid.) 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 average S.D. from at least 4 different cell samples.

(64) FIG. 3 Photoinactivation of Jurkat cells using 8-MOP and UV-A light irradiation. () control (no irradiation), (.circle-solid.) 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 average S.D. from 6 different cell samples.

(65) 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 (.circle-solid.) and whole lamp (.square-solid.); (, ) controls (no irradiation). Each data point represents an average S.D. from 6 different cell samples except from 10 min and 30 min time points, where only 4 and 2 samples, respectively were used.

(66) 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 average S.D. from 8 different cell samples.

(67) 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 average S.D. from at least 6 different cell samples.

(68) 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.

(69) 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

(70) Evaluation of Ex Vivo Efficacy of ALA-ECP in Patients with cGvHD and CTCL.

(71) 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.

(72) 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.

(73) 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.

(74) 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 UVA, particularly at a low light dose, ie. 5-min light illumination.

(75) 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 FIG. 14. 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.

FIGURE LEGENDS

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

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

(78) FIG. 14. 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.

EXAMPLE 3

(79) 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

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

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

EXAMPLE 4

Evaluation of 5-ALA Dark Toxicity on Human Leukocytes

(82) 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.

(83) 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, at a 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

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