ES2927913A1 - Composition comprising a combination of the furanocoumarins psoralen and angelicin and its use in therapy (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Composition comprising a combination of the furanocoumarins psoralen and angelicin and its use in therapy. The present invention refers to a new composition that comprises a mixture of two furanocoumarins, specifically psoralen and angelicin, as well as the medical uses of said composition, more specifically its use for the treatment of immunologically based diseases, such as, for example, diseases autoimmune, mainly mediated by T lymphocytes, atopic hypersensitivities, Graft Versus Host Disease (GVHD), organ rejection in transplants or lymphomas, in combination with extracorporeal photopheresis (ECF). Additionally, the present invention refers to an ex vivo/in vitro procedure for the generation of apoptotic cells, preferably autologous, useful in FEC procedures. Therefore, the present invention falls within the technical field of medicine, more specifically, in the sector for the treatment of immune-based diseases, autoimmune diseases, atopic hypersensitivities and GVHD, among others. (Machine-translation by Google Translate, not legally binding)

Description

description

Composition comprising a combination of the furanocoumarins psoralen and angelicin and its use in therapy

technical field

The present invention refers to a new composition comprising a mixture of two furanocoumarins, specifically psoralen and angelicin, as well as the ^applications doctors of said composition, more specifically the ^use of said composition for the treatment of immunologically based diseases, such as, for example, autoimmune diseases, mainly mediated by T lymphocytes, atopic hypersensitivities, Graft-versus-Host Disease (GVHD), organ rejection in transplants

^either lymphomas. Additionally, the present invention relates to an ex vivo/in vitro method for the generation of apoptotic cells, preferably autologous, useful in extracorporeal photopheresis (ECP) procedures. Therefore, the present invention falls within the technical field of medicine, more specifically, the sector for the treatment of immunologically-based diseases, autoimmune diseases, atopic hypersensitivities and GVHD, among others.

state of the art

The term furanocoumarin is applied collectively to a large number of compounds that have a benzo-2-pyrone (coumarin) nucleus, with a furanic ring attached at positions 6 and 7 for linear furanocoumarins, and 7 and 8 for angular ones. These compounds are present in the plant families Leguminous, Moráceas, Meliáceas, Compositae, Solanáceas, Rutáceas, Umbellíferas and Apiáceas.

Since the discovery of these plant compounds, extensive research has been carried out on their therapeutic interest, as well as other structurally related biomolecules.

Furanocoumarins are being used clinically in the therapy called PUVA (from ^its acronym "psoralen" and "UVA light") and in the therapy called extracorporeal photopheresis (ECF). PUVA therapy is being used clinically for the treatment of skin diseases such as vitiligo ^or psoriasis (Saurat et al., Dermatologica. 1988, 177, ^218-224 ; Westerhof and Nieuweboer-Krobotova, Arch Dermatol.

1997, 133, 1525-1528) and consists of the application of total body irradiation to the patient with ultraviolet light (UVA) after the administration of furanocoumarin, a process that is repeated several times a week. The most commonly used furanocoumarins for this modality of photochemotherapy include 8-methoxypsoralen ^or 8-MOP (also called methoxalene ^or xanthotoxin), 5-methoxypsoralen ( ^or bergapten), angelicin ( ^or isopsoralen), 4,5'-dimethylangelicin and 4, 5',8-trimethylpsoralen. Some of the commercially used furanocoumarins ^used for PUVA are Oxsoralen-Ultra®, Meladinine®, Deltasoralen®, Geroxalen®, Methoxalen® and Trioxalen®, among others.

On the other hand, ECF is an immunomodulatory therapy that combines leukapheresis (extraction of the leukocyte fraction from the patient's blood) with phototherapy. After the separation of a plasma rich in leukocytes, said leukocytes are treated ex vivo/in vitro with a photosensitizing agent, such as furanocoumarin 8-MOP coupled with UVA radiation and subsequently, ^the leukocytes treated in this way are reinfused again. new to the patient. Currently, this treatment modality is considered a safe immunomodulatory therapy, with few side effects and multiple clinical applications, and is being used for the treatment of different diseases mediated by T lymphocytes, such as episodes of rejection of solid organ transplants (heart, kidney, etc.). , liver ^or lung), cutaneous T lymphoma, Graft-versus-Host Disease (GVHD) ^or different autoimmune diseases such as type 1 diabetes, Crohn's disease, systemic lupus erythematosus, multiple sclerosis ^or rheumatoid arthritis (Marques and Adamski J Clin Apheresis. 2014, 29 : 228-234), among others.

^Thus , for example, in the case of patients diagnosed with malignant blood diseases, allogeneic transplantation of hematopoietic progenitor cells (HSCT) is the treatment of choice. However, long- ^term results are limited by the high toxicity of the procedure, which causes significant transplant-related morbidity and mortality. Said morbidity and mortality is largely related to the development of GVHD, which occurs as a consequence of immunological aggression by infused lymphoid cells against ^the host's organs, mainly skin, liver, and intestine. Approximately 30-50% of the patients who receive ^HSCT from a family donor develop acute GVHD, although this percentage increases up to 40-80% in those patients receiving HSCT from an unrelated donor (Hurley et al. Biol Blood Marrow Transplant. 2003, 9(10): 610-615; Petersdorf et al. Blood. 1998, 92(10) : 3515-3520). In addition, up to 80% of patients with acute GVHD relapse after complete remission ^or are refractory to the first line of treatment, usually corticosteroids. Various studies have described that only 38% of patients are alive and in remission 6 weeks after receiving treatment with corticosteroids for GVHD (Arai et al. Biol Blood Marrow Transplant. 2002, 8(3): 155- 160). The lack of response to the first line of treatment conditions an even more serious prognosis and, although a high percentage of responses have been described with some drugs, in the long term the percentage of patients alive is only 5-15% due to recurrences ^or progression of GVHD ^or the development of infectious complications in the context of immunosuppressive treatment (Weisdorf et al. Blood. 1990, 75(4): 1024-1030; Przepiorka et al. Blood. 2000, 95(1): 83-89) .

ECF is an excellently tolerated immunomodulatory technique that has been shown to be effective in the treatment of GVHD, both acute and chronic, although it represents a second-line treatment. Although other furanocoumarins exist in nature, currently the ECF protocol is only being carried out through the administration of furanocoumarin 8-MOP as a photochemotherapeutic agent for the treatment of GVHD.

In this sense, various patents related to ECF based on the administration of furanocoumarin 8-MOP for the treatment of GVHD, atopic ^or autoimmune diseases, transplant rejection, etc. are known in the state of the art. The patent EP2443922B1 refers to an in vitro method of generating lymphocytes with regulatory activity (Treg) that comprises incubating autologous CD4+ leukocytes with autologous apoptotic mononuclear cells obtained from peripheral blood by a FEC procedure, using psoralen derivatives in said procedure. photoactivables, such as 8-MOP, and UVA light as a photoactivator. Said patent also describes the ^use of the Treg lymphocytes obtained for the treatment of a patient with GVHD. International patent application W09736581A1 discloses the treatment of mononuclear cell infections by means of FEC using 8-MOP and UVA light. The treated blood fraction is reinfused into the patient to modulate monocyte function and/or stimulate the patient's immune response against the infected cell population. In the international patent application WO03045979A2 it is made reference to treatment with apoptotic cells obtained, for example, from T-lymphocytes, from individuals predisposed to autoimmune ^or atopic disease , as well as from individuals with GVHD. As in previous disclosures, apoptotic cells are obtained by FEC and treated with 8-MOP ^or other derivatives thereof by substitution, and UVA radiation. Additionally, Zheng et al. (Zheng et al. Biochem Biophys Res Commun. 2010; 395: 540-546) describe a therapeutic strategy to prevent rejection of a heart transplant that comprises the application of CSF to the donor's immune cells. Leukocytes obtained from the donor's blood are treated with 8-MOP, after which the mixture is subjected to UVA radiation. These treated cells are exposed to the phagocytic action of the recipient's immature dendritic cells. The authors demonstrate that the dendritic cells that have incorporated the treated cells retain ^their immature phenotype with a low capacity to stimulate the "naive" T ^cells of the recipient and, when infused into the latter, promote survival after transplantation in the absence of treatment. with immunosuppressive drugs, also inducing the generation of CD4+CD25altoFoxp3+ regulatory T cells.

Despite the existence of such combination treatments of FEC and 8-MOP, there is still a need to develop new treatments that are more effective and/or with fewer side effects to treat patients having autoimmune diseases, mainly mediated by T lymphocytes, atopic hypersensitivities ^either GVHD, and for which existing treatments are not as effective as they could otherwise be, can have serious side effects, ^either are difficult to administer at levels where each treatment is administered by ^Yeah same.

description of the invention

The present document refers to a composition comprising the combination of two furanocoumarins, psoralen and angelicin, as well as the combination of psoralen and angelicin per se . Likewise, this document refers to the ^use of said composition ^either of said combination, preferably wherein said composition is a pharmaceutical composition, for the treatment of a wide variety of disorders ^either immune-based diseases, without limitation, autoimmune diseases preferably mediated by T lymphocytes, graft-versus-host disease (GVHD), solid organ and/or tissue transplantation, T-cell lymphoma, among others; where said treatment is a combination treatment with FEC, in which a patient's blood, ^either part of it, which is subjected to treatment with the composition ^either with the combination of the invention, it is exposed to light of a wavelength that activates the photoactivatable compounds that make up the composition and combination of the invention.

Therefore, in a first aspect, the present document refers to a composition, preferably a pharmaceutical composition, hereinafter referred to as the composition of the invention, comprising a combination of psoralen and angelicin. Alternatively, the present invention also relates to the combination of psoralen and angelicin per se , which will hereinafter also be referred to as the combination of the invention.

As mentioned above, the term furanocoumarin is applied collectively to a large number of compounds that have the benzo-2-pyrone (coumarin) nucleus, with a furanic ring attached at positions 6 and 7 for linear furanocoumarins, and 7 and 8 for the angular ones. These compounds are present in the plant families Leguminous, Moráceas, Meliáceas, Compositae, Solanáceas, Rutáceas, Umbellíferas and Apiáceas. Furanocoumarins have a mixed origin, where the coumarin nucleus derives from the shikimic acid pathway, passing through trans-cinnamic acid and ending with umbelliferone. The furanic ring comes from the acetate-mevalonate pathway, whose intermediate product, dimethylallylpyrophosphate, binds to umbelliferone to generate linear (commonly called psoralens) and angular (called angelicins) furanocoumarins.

Psoralen (C ^eleven h ⁶ EITHER ³ - Formula I) is structurally related to coumarin by the addition of a furan ring, and can be considered as a derivative of umbelliferone.

On the other hand the angelicina ^either isopsoralen (C ^eleven h ⁶ EITHER ³ - Formula II) can be considered, structurally, as a benzapyra-2-one fused with a furan moiety at position 7,8.

Both compounds, psoralen and angelicin, occur naturally in certain plant species of the Apiaceae, Leguminosae, and Fabaceae families, among others. ^Thus , the composition and combination of the invention can be prepared from the psoralen and angelicin compounds obtained commercially ^or by synthesis ^or from a purification process from the seeds of the plants that contain them. In a more preferred embodiment, the psoralen and angelicin comprising the pharmaceutical composition ^or combination of the present invention are obtained commercially.

The term "combination" "composition" ^either "Pharmaceutical composition", used interchangeably throughout this specification, refer to any substance used for prevention, diagnosis, relief, treatment ^either curing diseases in humans ^either in animals. The pharmaceutical composition of the invention can be used both alone and in combination with other compounds. ^either pharmaceutical compositions. Additionally, the terms composition, pharmaceutical composition, combination, and medicament are used interchangeably in this invention. In the context of the present invention the pharmaceutical composition ^either the medicament are characterized by comprising psoralen and angelicin in a therapeutically active amount. In the present invention, the expression "therapeutically effective amount" refers to the amount of the combination of psoralen and angelicin calculated to produce the desired effect and, in general, will be determined, in the case of a therapeutic composition, by the characteristics of the compounds, the route, form and frequency of administration thereof, and other factors, including the age, condition of the patient, as well as the severity of the alteration ^either disorder.

In a particular embodiment, psoralen can be present in the composition of the invention in a concentration range selected from any of the following: 50 to 700 ng/mL, 150 to 650 ng/mL, 200 to 600 ng/mL, 300 to 500 ng/mL, 200 to 400 ng/mL, and 250 to 350 ng/mL, more preferably 200 to 400 ng/mL and 250 to 350 ng/mL, most preferably between 280 to 320 ng/mL and still more preferably 300 ng/mL.

In another particular embodiment, angelicin can be present in the composition of the invention in a concentration range selected from any of the following: 50 to 1120 ng/mL, 150 to 1000 ng/mL, 200 to 900 ng/mL, 3250 to 800 ng/mL, 300 to 700 ng/mL, 350 to 600 ng/mL, 400 to 500 ng/mL, and 450 to 490 ng/mL, more preferably 400 to 500 ng/mL, even more preferably 450 at 490 ng/mL, and even more preferable 480 ng/mL.

In another more preferred embodiment, the ratio of psoralen and angelicin present in the composition of the invention is in a range from 1:1 to 1:3.4; preferably from 1:1 to 1:2, more preferably from 1:1.2 to 1:1.8, still more preferably from 1:1.5 to 1:1.6.

Alternatively, the concentrations and ratios of psoralen and angelicin in the composition of the invention are extrapolated in the same way to the ^use of both molecules in combination, without having to form part of the composition of the invention.

The term "vehicle", like the "excipient", refers to a substance that is used in the pharmaceutical composition ^or medicine to dilute any of the components of the present invention included in it up to a determined volume ^or weight. The function of the vehicle is to facilitate the incorporation of other elements, to allow a better dosage and administration ^or to give consistency and shape to the composition. When the presentation form is liquid, the pharmacologically acceptable vehicle is the diluent. The pharmaceutically ^acceptable vehicles that can be used in the pharmaceutical composition of the present invention are the vehicles known to those skilled in the art.

^Thus , in a particular embodiment, the composition of the invention also comprises one ^or more pharmaceutically acceptable excipients ^or vehicles.

The term "pharmacologically acceptable" ^either “pharmaceutically acceptable”, used interchangeably throughout this document, refers to the fact that the compound to which it refers is permitted and evaluated, that is, it is physiologically tolerable and safe, such that ^his administration does not cause harm to the organisms to which it is administered. Preferably, as used herein, the term "pharmaceutically acceptable" means that it has been approved by a state government regulatory agency. ^either federal ^either which is listed in the US Pharmacopeia ^or other generally recognized pharmacopoeia for ^{its use} in animals and more particularly in humans. Therefore, the composition of the invention is free of pyrogens and endotoxins.

In another particular embodiment, the composition of the invention is characterized in that it is an aqueous solution.

In another particular embodiment, the composition of the invention is characterized in that it is an injectable solution.

Psoralen and angelicin are photoactivatable compounds. ^either photoactive, indicating that in ^his natural state are inactive, but after being exposed to light of a specific wavelength are activated and exert ^his therapeutic function ^either desired effect on a target cell (ie, preferably a predetermined cellular change). For purposes of the present invention, said photoactivatable compounds are preferably activated by long-wavelength ultraviolet light (UVA) radiation, eg, at a wavelength within the range of 315 to 400 nm. Exposure to UVA light is carried out for a temporary duration sufficient to deliver approximately a 1-2 J/cm2 dose to the cell population. When activated, such photoactivatable pharmaceuticals can effect cellular changes including, but not limited to, apoptosis, redirection of metabolic pathways, upregulation of certain genes, downregulation of certain genes, secretion of cytokines, altering the responses of cytokine receptors, ^either combinations thereof.

A particular aspect includes the presence of a second drug in the composition of the invention. Said second medicine can be part of the composition ^either can be provided as a separate composition for administration at the same time ^either at different times.

In a second aspect, the present specification refers to the pharmaceutical composition of the invention for ^{its use} as a medication, preferably in combination with an ECF treatment. Alternatively, the present specification also refers to the combination of psoralen and angelicin, combination of the invention, for ^{its use} as a medication, preferably in combination with an ECF treatment.

In another aspect, the present specification refers to the pharmaceutical composition of the invention for ^{its use} in the prevention and/or treatment of a wide variety of disorders ^either immune-based diseases, without limitation, autoimmune diseases preferably mediated by T lymphocytes, GVHD, solid organ and/or tissue transplantation, T-cell lymphoma, among others, preferably in combination with ECF treatment. Alternatively, the present specification also refers to the combination of psoralen and angelicin, combination of the invention, for ^{its use} in the prevention and/or treatment of a wide variety of disorders ^either immune-based diseases, without limitation, autoimmune diseases preferably mediated by T lymphocytes, GVHD, solid organ and/or tissue transplantation, T-cell lymphoma, among others, preferably in combination with ECF treatment.

All the definitions mentioned throughout this document apply to both the second and third aspects of the present invention, as well as to the combination of psoralen and angelicin, combination of the invention.

For purposes herein, an extracorporeal photopheresis (ECP) procedure ^or treatment, also known as extracorporeal phototherapy, refers to a treatment of a cell population that has been subjected to a treatment with a compound ^or composition comprising at least a photoactivatable compound and UVA light, capable of activating said compound/composition. Preferably the cell population is from an organ ^or tissue; more preferably, the population of cells is a population of blood cells, more preferably, the population of cells is a population of peripheral blood mononuclear cells ( ^MNCs ). FEC is sometimes used to refer to a procedure in which a cell population has undergone an apoptosis-inducing procedure with UVA light in the presence of al least one photoactivatable agent, which for the purposes of the present invention refers to the components of the composition of the invention, psoralen and angelicin ^. Once said cell population is subjected to FEC treatment, it is reinfused back into the subject ^or patient.

For purposes herein, the immunological ^or immune-based diseases ^or disorders are preferably T cell-mediated autoimmune diseases, including, without limitation, disorders such as type 1 diabetes, Crohn's disease, systemic lupus erythematosus, psoriasis, multiple sclerosis, rheumatoid arthritis and thyroidosis, among others. Additionally, among the immunologically based diseases that benefit from treatment with the composition of the present invention are also graft-versus-host disease, solid organ and/or tissue transplants, such as ^{heart transplants} , kidney, liver, lung, bone marrow, etc., acute and chronic leukemias, ^I cutaneous T lymphomas and atopic diseases that include chronic inflammatory pathologies such as sarcoidosis, chronic inflammatory bowel disease and ulcerative colitis, among others.

^Thus , for the purposes of the present invention, the composition of the invention is used for the ex vivo/in vitro treatment of said pathologies, by means of a leukapheresis and phototherapy procedure. ^Thus , a blood sample extracted from a subject suffering from any of the pathologies mentioned above, in which the mononuclear cells are isolated, is placed in contact with the composition of the invention and subjected to UVA radiation to activate the psoralen and the angelicin of the composition of the invention. Subsequently, said leukocytes are reinfused into the subject again.

As shown in ^the Examples included herein, both at the in vitro and in vivo level, a significantly higher therapeutic efficacy of the treatment with the composition of the invention is demonstrated, which comprises the combination of psoralen and angelicin, compared to treatment exclusively with psoralen 8-MOP. All the results shown in ^the Examples ^show a greater in vitro effectiveness of the composition of the invention compared to 8-MOP, in terms of certain immunomodulatory functions, which confers a greater therapeutic potential of the ^use of the composition of the invention in combination with FEC. On the other hand, the in ^vivo results shown herein (Example 7) demonstrate that the composition of the The invention has a therapeutic efficacy greater than that of 8-MOP in the reversal of acute murine GVHD by means of FEC.

In another aspect, the present specification refers to an ex vivo/in vitro process for the generation of apoptotic cells, hereinafter, the ex vitro/in vitro process of the invention, wherein said process comprises:

a) Isolate the population of cells of interest from a biological blood sample isolated from a subject, preferably the cells of interest are mononuclear cells;

b) Incubating the cells isolated in step a) with the composition of the invention ^or with the combination of the invention;

^c ) Irradiate the cells from step b) with UVA light, and

d) Isolate the cells obtained in step ^c ).

This ex vivo/in vitro production procedure offers several advantages over drug-induced procedures known in the state of the art. It ^is important to emphasize that the possible toxic and unnatural effects of the drugs used are avoided. In addition, there are advantages to the ex vivo/in vitro generation of apoptotic cells over the in vivo use thereof including, without limitation, greater control over the number, activity, and function of these cells. This therapeutic control provides better treatment protocols to the patient.

For purposes herein, the term "apoptotic cells" includes cells that display, ^or will display, one ^or more phenotypic and/or functional properties that characterize the mechanism of apoptosis. An apoptotic cell can comprise any cell that is in the induction phase, the effector phase in which DNA is fragmented, ^or the phase of nuclear and cytoplasmic destruction.

T ^he terms "in vitro" and "ex vivo" refer to the fact that the method of the invention is performed outside of the subject ^'s body. That is, it is performed on a biological sample from a subject.

For purposes herein, the term "biological sample" may include, without limitation, whole blood ^{or its} derivatives such as serum, plasma, platelets, erythrocytes, and leukocytes. The sample may also include single cells ^or a plurality ^or fragments thereof. Preferably, the sample comes from blood serum ^or plasma, peripheral blood mononuclear cells (MNC ^s ) ^or culture supernatants of MNC ^s . More preferably, the biological sample is MNC ^s . The sample can be obtained from a patient by venous puncture ^or any other clinical procedure known in the state of the art.

A "cell population" generally includes a cell type found in blood. The term may include one ^either more types of blood cells, specifically, erythrocytes, platelets, and leukocytes. A cell population may comprise subtypes of leukocytes, eg, T lymphocytes, dendritic cells, B lymphocytes, etc. In a particular embodiment, a cell population can comprise a mixture ^either pool of cell types. Alternatively, a cell population may comprise a substantially purified type of cell, for example, T lymphocytes. ^either dendritic cells.

The terms "subject" ^{or "} patient" are used interchangeably and refer to an animal, preferably a mammal, and more preferably a human.

In step a) of the ex vitro/in vitro procedure of the invention, from a biological sample isolated from a subject, using procedures known to any person skilled in the art, the fraction ^or population of cells of interest is isolated. , which are preferably peripheral blood mononuclear cells, to be subsequently subjected to a specific treatment with the composition of the invention and UVA light, to obtain the apoptotic cells of the invention. Said procedure for obtaining apoptotic cells is an in vitro/ex vivo procedure, that is, it is a procedure that is carried out extracorporeally, outside the body of the subject, donor ^or recipient, and is compatible with said subject, donor, ^or receiver, respectively. Said cell population can be prepared from substantially any type of mammalian cell including cultured cell lines. For example, a cell population can be prepared from a cell type derived from the mammalian subject's own body ^or from an established cell line. Specifically, a cell population can be prepared from blood leukocytes compatible with that of the mammalian subject, more specifically, from the subject's own leukocytes, and even more specifically, from the subject's own T-lymphocytes. The cell population can be prepared extracorporeally prior to administration to the subject, donor, ^or recipient. ^Thus , in one embodiment, the biological sample of the subject's blood, of the Blood from the recipient, ^or from the blood of the donor can be withdrawn, for example by venipuncture, and at least a part of the mononuclear cells therein can be subjected extracorporeally to the conditions that induce apoptosis.

Once the peripheral blood sample is obtained, it must be treated to isolate the mononuclear cells. Any of the existing methods in the state of the art to isolate mononuclear cells from peripheral blood ^either from ^their Blood products can be used in the practice of the present invention, for example, by centrifugation in Ficoll-Hypaque (GE Healthcare Bio-Science), Percoll, sucrose, etc., as well as any other technique known and used by those of ordinary skill. in technique.

Once the mononuclear cells are isolated, proceed in step b) of the ex vitro/in vitro method of the invention to the incubation of said cells in the presence of the composition of the invention ^or the combination of the invention, which comprises the two photoactivatable compounds, psoralen and angelicin, and is simultaneously exposed to UVA light. The levels in ^terms of concentration and treatment time appropriate for the induction of apoptosis in the cell population and the type of treatment chosen to induce apoptosis, exposure to UVA light as indicated in the present invention, are easily determinable by those experts in the art. ^Thus , the cell population to which the composition of the invention ^or the combination of the invention has been added is treated with light of a wavelength that activates the photoactivatable compounds psoralen and angelicin. The treatment step that activates the composition of the invention is preferably carried out using long-wavelength ultraviolet (UVA) light, for example, at a wavelength within the range of 315 to 400 nm. Exposure to UVA light during photopheresis treatment is of sufficient duration in time to deliver approximately 1-2 J/cm2 to the cell population. The ECP apparatus useful in the invention includes any device used to carry out said therapy.

Methods for the detection and quantification of apoptosis are useful to determine the presence and level of apoptosis in the cell population subjected to treatment with the composition of the invention and irradiation with UVA light. The number of apoptotic cells in a cell population required to obtain clinical benefit in a subject may vary depending on the source of the cells, the condition of the subject, the age and weight of the subject and other relevant factors, which are readily determinable by known methods.

In a particular embodiment of the ex vitro/in vitro method of the invention, it is characterized in that the apoptotic cells are autologous.

In another particular embodiment of the ex vivo/in vitro method of the present invention, it is characterized in that the autologous apoptotic cells are leukocytes.

In another particular embodiment of the ex vivo/in vitro procedure of the present invention, it is characterized in that the incubation and irradiation steps are carried out during a time automatically calculated by the FEC photoactivator device and under sufficient conditions to generate apoptotic cells.

Another aspect of the present invention refers to apoptotic cells, hereinafter, apoptotic cell of the invention, obtained ^or obtainable by the ex vivo/in vitro production method of apoptotic cells of the invention.

In a preferred embodiment, the apoptotic cells of the invention are useful as a medicament. In another more preferred embodiment, the apoptotic cells of the invention are useful in the prevention and/or treatment of a wide variety of disorders. ^either immune-based diseases, without limitation, autoimmune diseases preferably mediated by T lymphocytes, graft-versus-host disease (GVHD), solid organ and/or tissue transplantation, T-cell lymphoma, among others, preferably in combination with a treatment of FEC, as described throughout this document.

Another aspect of the present invention refers to a method of prevention and/or treatment of a wide variety of disorders ^either immune-based diseases, without limitation, autoimmune diseases preferentially mediated by T lymphocytes, graft-versus-host-disease (GVHD), solid organ and/or tissue transplantation, T-cell lymphoma, preferably in combination with ECF treatment, as and as described throughout this document, in a subject, hereinafter, prevention and/or treatment method of the present invention, wherein said method comprises:

a) Contacting a biological sample isolated from said subject with the composition ^either the combination of the invention;

b) Irradiate the biological sample from step a) with a photoactivator device, as described throughout this document, and

^c ) Reinfuse the irradiated isolated biological sample from step b) into said subject.

In a preferred embodiment of the prevention and/or treatment method of the present invention, the isolated biological sample is as defined herein. Preferably it is a biological sample isolated from blood, and more preferably a biological sample comprising peripheral blood mononuclear cells.

All terms used in the prevention and/or treatment method of the present invention have been previously described throughout this document and apply mutatis mutandis to said prevention and/or treatment method of the invention.

Throughout the description and claims the word "comprises" and ^its variants are not intended to exclude other technical characteristics, additives, components ^or steps. For those skilled in the art, other objects, advantages, and features of the invention will emerge in part from the description and in part from the practice of the invention. The following ^examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

brief description of the figures

Figure 1. Shows the structural formulas of the furanocoumarins psoralen and angelicin, components of the PSA composition of the present description. Both compounds are classified as furanocoumarins, psoralen with linear structure and angelicin with angular structure.

Figure 2. Shows the analysis of the induction of apoptosis of human peripheral blood mononuclear cells (MNC ^s ) after undergoing treatment with furanocoumarins 8-MOP and PSA (psoralen and angelicin - composition of the invention) and UVA light. MNCs were ^incubated with the indicated concentrations of each furanocoumarin in combination with UVA light (2 J/cm2). After incubation for 48 hours at 37 ^qC , the total percentages of apoptotic cells were measured (percentage of cells annexin-V+ 7AAD- (early apoptosis) plus the percentage of annexin-V+ 7AAD+ (late apoptosis) cells by flow cytometry. The data ^represent the mean ± standard deviation of 12 independent experiments. The percentage of apoptosis was significantly increased, *** ^p < 0.001, compared to the levels of apoptosis achieved after treatment with 8-MOP and UVA light in each condition, respectively. The X axis indicates the concentration of psoralen expressed in ng/mL, both for the case of 8-MOP, and for the concentration of said compound in the PSA composition of the invention. Note that the psoralen:angelylcin ratio for the tested composition shown in the present figure is 1:1.6.

Figure 3. Shows the analysis of in vitro treatment with different furanocoumarins and UVA light on the proliferation of MNC ^s after mixed cultures with allogeneic myeloid dendritic cells (DC ^s ). The ^MNCs treated with the different furanocoumarins (300 ng/mL of 8-MOP ^or PSA (composition of the invention: 300 ng/mL of psoralen and 480 ng/mL of angelicin)) and UVA light (2 J/cm2) were used as responder cells in mixed cultures of leukocytes in the presence of immature DC ^s ( ^í DC ^s ) (A) ^or mature DC ^s (mDCs) (B) stimulators and previously irradiated to prevent ^their proliferation. After co-cultivation for 5 days at 37 ^q C, the proliferation of the ^MNCs was determined by incorporation of bromo-deoxyuridine (BrdU). As a control, basal proliferation of irradiated DCs ^{or mDCs} , ^or of unstimulated ^MNCs with DCs, ^is also shown. The data ^represent the mean ± standard deviation of 3 independent experiments performed in triplicate for each experimental condition. The level of proliferation of MNC ^s was significantly decreased compared to MNC ^s not previously treated with any furanocoumarin (-PSOR), ** ^p < 0.01, ^or significantly decreased compared to MNC ^s previously treated with 8-MOP , ^p < 0.05, respectively.

Figure 4. Shows the analysis of the levels of pro-inflammatory and anti-inflammatory cytokines obtained in co-cultures of ^í DCs ^{or mDCs} with allogeneic ^MNCs pre-treated with the different furanocoumarins (300 ng/mL of 8-MOP ^or PSA (composition of the invention: 300 ng/mL of psoralen and 480 ng/mL of angelicin)) and UVA light (2 J/cm2). The supernatant of mixed cultures of ^í DCs ^{or mDCs} and ^MNCs treated with the different furanocoumarins and UVA light was collected and analyzed to determine the levels of different pro-inflammatory cytokines (IFNy, IL-2 and IL-12p70) (A) and anti-inflammatory (IL-10 and TGFp) (B) using ELISA kits. Data ^are shown as the percent change (mean ± standard deviation) of the concentration of each cytokine relative to control, which was normalized to 100%, and were obtained in three independent experiments, each performed in triplicate. The level of each cytokine decreased significantly, * ^p <0.05, ** ^p <0.01, *** ^p <0.001 ^or significantly increased, ^#p <0.05, ##p<0.01, ### ^p <0.001, in comparison with conditions in the absence of furanocoumarins (-PSOR), respectively. The level of each cytokine was significantly decreased, ^ap < 0.05, ^aap < 0.01, ^aaap < 0.001 ^or significantly increased , 5p < 0.05, ^555p < 0.001, compared to 8-MOP treatment, respectively.

Figure 5. Shows the analysis of the level of migration of ^í DC ^s (A) ^or mDCs (B) after ^their co-cultivation with MNC ^s pretreated with the different furanocoumarins (300 ng/mL of 8-MOP ^or PSA (composition of the invention). : 300 ng/mL of psoralen and 480 ng/mL of angelicin)) and UVA light (2 J/cm2) in response to the chemokine CCL21. The ^í DC ^{or mDCs} co-cultured with MNC ^s pretreated with the different furanocoumarins and UVA light were subjected to migration assays in response to the chemokine CCL21 ^or in response to culture medium without chemokine as negative control (Medium). The migration level of ^í DCs ^{or mDCs} was analyzed by flow cytometry. The data ^represent the mean ± standard deviation obtained in four independent experiments performed in triplicate. The level of migration of DC ^s in response to CCL21 was significantly increased, ♦♦♦ ^p < 0.001. The level of migration was significantly increased, ### ^p < 0.001 ^or significantly decreased, *** ^p < 0.001 compared to ^í DCs ^{or mDCs} co-cultured with MNCs without furanocoumarin (-PSOR) pretreatment, respectively. Furthermore, the level of migration of ^í DCs ^{or mDCs} was significantly increased, 5 ^p < 0.05 ^or significantly decreased, ^aaap < 0.001, compared to 8-MOP treatment, respectively.

Figure 6. Shows the analysis by means of flow cytometry of the expression level of co-stimulatory and maturation molecules of DC ^s , MHC class II and chemokine receptors in ^í DCs ^{or mDCs} after ^their co-cultivation with allogeneic MNC ^s pretreated with furanocoumarins (300 ng/mL of 8-MOP ^or PSA (composition of the invention: 300 ng/mL of psoralen and 480 ng/mL of angelicin)) and UVA light (2 J/cm2). (A) The ^í DCs ^{or mDCs} were co-cultured in mixed cultures with ^MNCs together with the MNCs pretreated with furanocoumarins and UVA light and were subsequently analyzed for the expression of costimulatory and maturational molecules characteristic of myeloid ^DCs ( CD80, CD83 and CD86) and major histocompatibility complex (MHC) class II (HLA-DR) by flow cytometry. For the analysis of the different cell populations, the ^í DC ^s and mDCs with positive expression of the CD45, BDCA-1 and HLA-DR markers were selected. For the ^{í DCs} (left column), the numbers inserted in the histograms represent the percentage of BDCA-1+ CD83low0 cells, while for the mDCs (middle columns), the Percentages represent the BDCA-1+ cell population with high expression of CD80, CD83, and CD86, respectively. For HLA-DR expression analysis (right column), the numbers inserted into the histograms represent the mean fluorescence intensity values of the population of BDCA-1+ HLA-DRalt0 mDCs. (B) Dotted band diagrams show CCR7 chemokine receptor expression in ^{í DCs} (left panel) ^{or mDCs} (right panel) and positive for CD45, BDCA-1 and HLA-DR expression after being co-cultured with ^MNCs pretreated with the different furanocoumarins and UVA light. The numbers ^inserted in the panels represent the mean fluorescence intensity values of CCR7 expression for each condition. The data ^shown are representative of three independent experiments.

Figure 7. Shows the analysis by means of flow cytometry of the levels of regulatory T cells (Treg) induced after co-cultivation with ^í DC ^s previously co-cultivated with MNC ^s pretreated with furanocoumarins (300 ng/mL of 8-MOP ^or PSA (composition of the invention: 300 ng/mL of psoralen and 480 ng/mL of angelicin)) and UVA light (2 J/cm2). Allogeneic MNC ^s pretreated with furanocoumarins and UVA light were co-cultured with ^í DC ^s for 5 days at 37 ^q C. Subsequently, the same ^í DC ^s were washed and co-cultivated with autologous CD3+ T cells to analyze the level of differentiation towards Treg cells. (A) The numbers inserted in the histograms represent the percentages of CD3+ CD4+ CD25+ CD127- ^{F ox} P3+ Treg cells ^or (B) CD3+ CD8+ CD25+ F ^ox P3+ Treg cells, respectively.

The data ^shown are representative of three independent experiments.

Figure 8. Shows the percentage of survival and clinical grade of mice with acute GVHD and treated by FEC with 8-MOP (300 ng/mL), PSA (composition of the invention: 300 ng/mL of psoralen and 480 ng/mL of angelicin) and UVA light (2 J/cm2), ^or without treatment with furanocoumarins (-PSOR). (A) C57BL/ ^6J strain mice were transplanted with bone marrow cells and allogeneic splenocytes from BALB/ ^c strain mice to induce acute GVHD. Subsequently, the transplanted C57BL/6J mice were treated with four weekly infusions of splenocytes isolated from different cohorts of C57BL/6J mice untreated (-PSOR) ^or treated by FEC with the different furanocoumarins (8-MOP ^or PSA) and light. GRAPE. Survival of C57BL/6J mice with acute GVHD was significantly increased compared to the untreated (-PSOR) group of animals, *** ^p < 0.001, ^or compared to those treated with 8-MOP, ^{at p} < 0, 05, respectively. As a control, the percentage of survival of the C57BL/6J animals only irradiated (Irradiation Control) ^or transplanted only with bone marrow cells (MO transplant control). Survival curves are represented as Kaplan-Meier estimates and analyzed using the Mantel-Cox statistical test. (B) The clinical grade of acute GVHD for each experimental group was calculated using a clinical scoring system consisting of five individual variables (weight loss, posture, activity, coat texture and skin integrity, maximum index=10). The clinical grade of GVHD was significantly decreased compared to the group of untreated (-PSOR) animals, *** ^p < 0.001, ^or compared to those treated with 8-MOP, ^{at p} < 0.05, respectively. The different ^experimental groups were constituted with a total of 16 animals/group and show the results of 10 independent experiments.

Figure 9. Shows images of the histopathological damage associated with acute GVHD in the main target organs of the disease (skin, liver and intestine) of the different groups of animals treated by FEC with 8-MOP (300 ng/mL), PSA (composition of the invention: 300 ng/mL of psoralen and 480 ng/mL of angelicin) and UVA light (2 J/cm2),

^or untreated with furanocoumarin (-PSOR) by staining with hematoxylin and eosin (H&E). The level of histopathological damage was evaluated by an expert pathologist in distant areas of skin, liver and intestine samples from the animals of the different experimental groups on day 30 post-transplant. The images shown of H&E staining of the different organs (magnification ^x 100) are representative of five animals per experimental group.

examples

Next, the invention will be illustrated by means of tests carried out by the inventors, in which the effectiveness of the product of the invention is revealed. The following ^examples serve to illustrate the invention and should not be considered as limiting its scope.

materials and methods

Cells and reagents used.

^MNCs were isolated from heparinized human peripheral blood, obtained from healthy volunteers after signing informed consent, using a Ficoll-Paque density gradient (Sigma-Aldrich, St. ^Louis , Missouri, United States). The committee Ethics from the Hospital Clínico Universitario Virgen de la Arrixaca (Murcia) approved ^the protocols used to obtain and treat human samples. MNCs were grown in ^RPMI1640 culture medium (Gibco Invitrogen, Paisley, Scotland) supplemented with penicillin/streptomycin (PAA Laboratories, Pasching, Austria), L-glutamine (PAA Laboratories), and 10% fetal bovine serum (FBS) ( Gibco Invitrogen) (complete culture medium). ^Monocytes were purified from human peripheral blood samples using a human monocyte enrichment cocktail (StemCell Technologies, Grenoble, France) following the manufacturer's protocol.

For immature myeloid dendritic cell differentiation ( ^í DC ^s ), monocytes were resuspended at ^{1 x} 10 6 cells/ml and cultured in complete culture medium containing 50 ng/ml GM-CSF (Invitrogen) and 25 ng/ml IL-4 (Invitrogen) in 6-well flat bottom culture plates. The cytokines GM-CSF and IL-4 were added to the culture every 3 days, for 7 days. On day 7, more than 95% of the cells were CD14- CD11 ^c + CD1a+ BDCA-1+.

Mature myeloid dendritic cells (mDCs) were obtained from ^í DC ^s cultured for 6 days as described above and after ^their subsequent stimulation with 200 ng/ml lipopolysaccharide (LPS) (Sigma-Aldrich) for an additional 24 hours.

For the in vitro ^induction assays of regulatory CD4+ and CD8+ T cells, CD3+ T cells from the MNC fraction of ^peripheral blood samples were purified using anti-human CD3 antibodies conjugated to magnetic microspheres (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer's instructions.

In vitro treatment with the different furanocoumarins and UVA light.

The ^MNCs were incubated in PBS supplemented with different concentrations of furanocoumarins (see examples below), specifically with the combination of furanocoumarins of the invention (PSA), and with 8-MOP, which is the furanocoumarin used clinically. Said treatment was carried out in the dark at room temperature and the ^MNCs were subsequently irradiated with UVA light using a UVA irradiation chamber (Dr. Grobel UV-Elektroník GmbH, Ettlingen, Germany) at a final dose of 2 J/cm2 and measured with a radiometer equipped with a UVA radiation detector (Dr.

Grobel UV-Elektronik GmbH). The UVA light dose and the concentration of furanocoumarins used were equivalent to those used in clinical ECP procedures. After treatment with furanocoumarins and UVA light, the ^MNCs were resuspended in complete culture medium.

Untreated control ^MNCs were incubated with PBS without furanocoumarins and irradiated with UVA light under the same conditions. All of the furanocoumarins used in the examples described here, 8-MOP, psoralen, and angelicin, were obtained from Therakos (Uvadex™, Pennsylvania, USA) and Sigma-Aldrich, respectively.

Analysis by flow cytometry.

Cells were incubated in the dark at 40C for 30 minutes with fluorescence-conjugated monoclonal antibodies specific for the surface markers CDIa, CD14, HLA-DR (eBioscience, San Diego, California, United States), CD3, CD8, CD25, CD80. , CD83, CD86, CD127, CD197 (CCR7) (BioLegend, San Diego, California, USA), CD1 ^c (BDCA-1), CD11 ^c (Miltenyi Biotec), CD4 (Beckman Coulter, Fullerton, California, USA) and CD45 (Immunostep, Salamanca, Spain).

For intracellular staining for the F ^ox P3 transcription factor, cells were previously fixed and permeabilized using the PerFix-nc kit (Beckman Coulter) and allophycocyanin-conjugated anti-FoxP3 monoclonal antibody (eBioscience).

After staining with antibodies and washing, cells were acquired using a Beckman Coulter Navios flow eithometer and finally, they were analyzed with the Kaluza (Beckman Coulter) and Infinieyt (Cytognos, Salamanca, Spain) analysis programs. Basal non-specific fluorescence of the cells was measured using isotype and fluorochrome specific monoclonal antibodies.

For the detection of apoptosis in ^MNCs after treatment with furanocoumarins and UVA light, annexin-V and phyeoerythrin-conjugated 7-AAD reagents (BD Bioscienees, San Diego, California, United States) were used following the manufacturer's instructions.

Cell migration assays in transwell type chambers.

The ^MNCs were incubated with the different furanocoumarins and irradiated with UVA light as described above. Subsequently, these MNCs were cultured at 37qC with ^í DCs ^or allogeneic mDCs in complete culture medium in a 5:1 ratio. After 5 days of co-cultivation, iDC ^or mDC were isolated and resuspended in RPMI1640 culture medium supplemented with 0.5% bovine serum albumin (BSA) (Sigma-Aldrich) (migration medium) and subjected to assays. migration through a polycarbonate filter with a 5 .^m pore size ( transwells ; Corning Costar, Cambridge, Massachusetts, United States) in 24-well culture plates. iDC ^or mDC were added to the upper chamber of the transwell (3 ^x 10 5 cells in 100 ql of migration medium) while the lower chamber contained 200 ng/ml of the chemokine CCL21 (Invitrogen) in 600 ql of migration medium ^or migration medium only as spontaneous migration control.

The number of cells that migrated to the lower chamber after incubation for 3 hours at 37qC was determined in a flow cytometer (Beckrnan Coulter Navios). Cell migration was calculated as the percentage of cells initially added to the upper chamber of each transwell.

Cell proliferation assays and cytokine secretion analysis.

The MNC ^s previously treated with the different furanocoumarins and UVA light, and the allogeneic ^í DC ^{or mDCs} were used in mixed lymphocyte cultures (MLC ^s ) as responder and stimulator cells, respectively, in a 5:1 ratio for 5 days at 37 ^q C. To prevent proliferation of the stimulatory iDCs ^{or mDCs} , they were previously irradiated in a Yxlon Smart 200E X-ray irradiator (Yxlon International GmbH, Hamburg, Germany) with a total dose of 30 Gy.

Cell proliferation was determined using a bromodeoxyuridine (BrdU) ELISA colorimetric kit (Roche Diagnostics, Mannheim, Germany) based on the measurement of BrdU incorporation during DNA synthesis in proliferating cells. Briefly, BrdU labeling reagent was added to the wells 16 hours prior to the determination. Subsequently, the cells were fixed, the DNA denatured and incubated with a peroxidase-conjugated anti-BrdU antibody. After washing and addition of the substrate, the absorbance at 450 nm was measured with a Tecan Infinite M200Pro plate reader (Tecan, Mannedorf, Switzerland). All experiments were performed in triplicate.

To quantify the cytokines released in the ^MLCs , culture supernatants were collected on day 5 before the addition of BrdU and frozen at -80oC until ^further analysis. The levels of human IFNy, IL-2, IL-12 ^p70 , IL-10 and ^TGFp were measured using specific ELISA kits (eBioscience), following the instructions and protocols recommended by the manufacturer.

In vitro induction of regulatory CD4+ and CD8+ T cells.

MNC ^s previously treated with the different furanocoumarins and UVA light were cultured in MLC with ^allogeneic DC ^s in complete culture medium at 37 ^oc for 5 days in a 5:1 ratio in 96-well round bottom culture plates. Subsequently, the same ^DCs were ^isolated and cultured with autologous CD3+ T cells at 37 ^o C for an additional 5 days in a 1:5 ratio. Finally, levels of induced CD4+ and CD8+ regulatory T cells were measured by flow cytometry as described above.

Murine model of acute GVHD,

Allogeneic hematopoietic progenitor cell transplantation ^or allogeneic HSCT with complete mismatch in the murine H2 major histocompatibility system was performed by transplanting bone marrow cells and splenocytes from donor BALB/ ^c mice (H2d haplotype) into recipient mice (H2b haplotype). A 10-week-old boy previously irradiated with a potentially lethal dose of 10 Gy divided into two 5 Gy doses spaced 24 hours apart and at an intensity of 1 Gy/minute (days -1 and 0). On day 0, recipient C57BL/6J mice were transplanted intravenously with 1 ^x 107 bone marrow cells and 1.5 ^x 107 splenocytes from donor BALB/ ^c mice to induce acute GVHD.

To carry out the treatment of the different groups of animals by FEC, on days 7, 14, 21 and 28 post-transplant ^{I the} animals received an additional intravenous infusion of 1 ^x 107 splenocytes pretreated with the different furanocoumarins (8-MOP (300ng/mL) ^either PSA (composition of the invention: 300 ng/mL of psoralen and 480 ng/mL of angelicin) and UVA light (2 J/cm2), which were previously isolated from different cohorts of C57BL/6J mice transplanted in the same conditions and on day 12 post-transplant. The control group of animals not treated by FEC (-PSOR) received the same weekly dose of splenocytes pretreated with UVA light but without furanocoumarins. Post-transplant survival of the animals was monitored daily, while the clinical grade of GVHD was assessed using a scoring system consisting of five individual parameters (weight loss, posture, activity, coat texture, and skin integrity). Each individual parameter is given a score between 0 and 2, with the maximum total score being a value of 10.

T he different experimental groups ( ^-PSOR , 8-MOP and PSA) consisted of a total of 16 animals. The ^survival data and clinical grade of acute GVHD were analyzed using the Mantel-Cox and Student's T tests, respectively.

The histopathological analysis of acute GVHD was performed in at least two areas distant from the main target organs of the disease, mainly liver, intestine (colon) and skin.

The following criteria were used for skin assessment: grade 0 (normal), grade I (vacuolar degeneration of cells of the basal layer of the epidermis), grade II (spongiosis and presence of individual scattered apoptotic cells in the basal epidermis), grade III (separation of the dermal-epithelial junction) and grade IV (diffuse and severe ulceration, extensive destruction of the epidermis).

In the case of the liver, it was as follows: grade 0 (normal), grade I (epithelial damage and destruction of <25% of the bile ducts), grade II (epithelial damage and destruction of 25-49% of the bile ducts), grade III (epithelial damage and destruction of 50-74% of the bile ducts) and grade IV (epithelial damage and destruction of >75% of the bile ducts).

For intestine, the following assessment was used: grade 0 (normal), grade I (presence of individual scattered apoptotic cells and inflammatory cell infiltrate at the base of intestinal crypts), grade II (flattening of intestinal villi, presence of burst crypts). ), grade III (focal mucosal ulceration) and grade IV (diffuse and severe mucosal ulceration).

Statistic analysis.

Data ^were statistically analyzed using one-way analysis of variance (ANOVA), followed by Bonferroni post hoc correction for multiple comparisons. For all studies, a p value less than 0.05 was considered significant. All values were expressed as mean ± standard deviation. Survival curves were represented as Kaplan-Meier estimates and analyzed using the Mantel-Cox statistical test. The computer programs GraphPad Prism (GraphPad Software, San Diego, California, United States) and Microsoft Excel (Microsoft, Redmond, Washington, United States) were used for the analyses.

Example 1, Analysis of the apoptotic activity of the composition of the invention

As mentioned throughout this document, furanocoumarins are compounds that induce apoptosis after irradiation with UVA light. Bearing this in mind, the in vitro effectiveness of the mixture of psoralen and angelicin furanocoumarins (PSA) of the invention in the induction of MNC ^s apoptosis, obtained as previously described, was analyzed. A mixture of psoralen and angelicin (PSA) was used in a ratio of 1:1.6 and ^its effectiveness was compared with that shown by the compound 8- mop.

The ^MNCs were incubated in vitro in the presence of furanocoumarins in a range of concentrations that goes from 0 ng/ml to 700 ng/ml of 8-MOP, more specifically concentrations of 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 300 ng/mL, 400 ng/mL, 500 ng/mL, 600 ng/mL, and 700 ng/mL of 8-MOP; and in the case of treatment with the PSA composition of the invention, the tested doses of psoralen and angelicin varied from 0 ng/mL to 700 ng/mL of psoralen and from 0 ng/mL to 1120 ng/mL of angelicin, always maintaining a 1:1.6 ratio between both molecules (see Figure 2) so that the concentrations of both components tested in the composition of the invention were: 50 ng/mL psoralen 80 ng/mL angelicin, 100 ng/mL psoralen 160 ng /mL angelicin, 200 ng/mL psoralen 320 ng/mL angelicin, 300 ng/mL psoralen 480 ng/mL angelicin, 400 ng/mL psoralen 640 ng/mL angelicin, 500 ng/mL psoralen 800 ng/mL of angelicin, 600 ng/mL psoralen 960 ng/mL angelicin, and 700 ng/mL psoralen 1120 ng/mL angelicin. sayings Treatments were carried out in combination with UVA light irradiation (2 J/cm2 dose calculated automatically by the UVA light irradiator). After 48 hours of culture, the total percentages of apoptotic cells by annexin-V and 7-AAD binding (annexin-V+/7AAD- (early apoptosis) and annexin-V+/7AAD+ (late apoptosis)) were determined by cytometry. flow, as previously explained.

The results obtained show that the incubation of ^{MNC s with} 8-MOP and UVA light (at a dose of 2 J/cm2) produced a dose-dependent progressive increase in the percentage of apoptotic cells, from a basal value of apoptosis after 48 hours from 34% to a maximum value of 59.7% at the concentration of 500 ng/ml. However, the PSA mixture of furanocoumarins of the invention was more efficient in inducing apoptosis than 8-MOP, obtaining a percentage of apoptotic cells of 72% with the PSA composition of the invention that comprises a concentration of 500 ng/mL of psoralen plus 800 ng/mL angelicin, with significant differences with 8-MOP from a concentration of 50 ng/mL psoralen plus 80 ng/mL angelicin (*** p<0.001) (Figure 2).

Example 2. Analysis of MNC ^s proliferation after mixed culture with allogeneic DC ^s

It ^is known that binding of furanocoumarins to DNA prevents the proliferative response of human T cells in response to mitogenic ^or antigenic stimulation. To analyze the ability of the PSA furanocoumarin mixture of the invention to induce the tolerogenic function of dendritic cells in culture, mixed cultures of immature ( ^íDCs ) ^or mature (mDCs) dendritic cells with ^previously treated allogeneic MNCs were carried out ^. in vitro with the PSA mixture of the invention (composition of the invention: 300 ng/mL of psoralen and 480 ng/mL of angelicin) ^or with 8-MOP (300 ng/mL) together with UVA light (2 J/cm2) , ^or no treatment (only UVA light, -PSOR).

The allogeneic DCs ^{or mDCs} were previously irradiated with X-rays (30 Gy) to prevent ^their proliferation. After that, the incorporation of bromo-deoxyuridine (BrdU) into the DNA of the cells in the proliferative state was determined.

As can be seen in Figure 3, the results obtained show that the absence of treatment with furanocoumarin (-PSOR), and after ^{its allogeneic} stimulation with ^í DCs ^{or mDCs} , gives rise to a high proliferation of the ^MNCs . However, MNC ^s pre-treated with 8-MOP ^or PSA plus UVA light and ^co - cultured with ^í DC ^{or mDCs} showed a significant reduction in ^their proliferation compared to untreated MNC ^s (** ^p <0.01). When mDCs were used as proliferation-stimulating cells, this degree of inhibition was higher and statistically significant when the ^MNCs were treated with PSA and UVA light compared to that obtained with 8-MOP ( ^ap <0.05). The data ^represent the mean ± standard deviation of 3 independent experiments, analyzing each condition in triplicate.

Example 3. Analysis of cytokine concentrations after co-cultivation of allogeneic DC ^s and MNC ^s pretreated with PSA and UVA light.

In order to analyze ^{the soluble} factors that could be involved in ^{the immunomodulatory} mechanisms and in the induction of peripheral tolerance after ECF, the levels of different pro-inflammatory and anti-inflammatory cytokines were determined ⁱⁿ mixed cultures of ^MNCs pretreated with 8 -MOP (300 ng/mL) ^or PSA (composition of the invention: 300 ng/mL of psoralen and 480 ng/mL of angelicin) plus UVA light (2 J/cm2) in an allogeneic context with DCs ^{or mDCs} .

The supernatants of said co-cultures were collected and used for the quantification of ^the levels of the pro-inflammatory cytokines IFNy, IL-2 and IL-12 and the anti-inflammatory cytokines IL-10 and ^TGFp by means of ELISA techniques. as previously described.

The secretion of pro-inflammatory cytokines IFNy, IL-2 and IL-12 was significantly lower when the MNCs from the co-culture with ^í DCs ^{or mDCs} were pretreated with 8-MOP ^or PSA and UVA light with respect to ^the levels detected in absence of treatment with furanocoumarin (-PSOR) (* ^p <0.05, ** ^p <0.01 ^or *** ^p <0.001, respectively), said production being significantly lower when I ^os cultures were treated with PSA in locus of 8-MOP ( ^{a p} < 0.05, ^{aa p} < 0.01 ^{or aaa p} < 0.001, respectively) (Figure 4).

In contrast, the detected levels of the anti-inflammatory cytokines TGFp and IL-10 were significantly higher when ^{MNCs were} pretreated with PSA . relative to untreated MNCs ^{(#p < 0.05, ## p < 0.01, or ### p < 0.001 , respectively} ), ^or with PSA relative to 8-MOP (5 ^p < 0.05 ^or 555 ^p <0.001, respectively) (Figure 4). The data show the mean ± standard deviation of the concentration of each cytokine normalized with respect to the control ( ^-PSOR ) of 3 independent experiments, analyzing each condition in triplicate.

Example 4. Migration of DC ^s after ^{their co} - ^cu I ^{íív o} with MNC ^s pretreated with PSA and UVA light in response to the chemokine CCL21

For a better understanding of the mechanisms involved in the acquisition of peripheral tolerance by means of FEC, we analyzed whether the in vitro co-culture of ^í DCs ^{or mDCs} together with MNCs ^pretreated with furanocoumarins and UVA light could alter ^their migratory capacity in response to FEC. CCL21 chemokine.

The ^í DC s ^{or mDCs} were collected after ^their co-cultivation for 5 days with the MNC ^s pretreated with the different furanocoumarins and UVA light and, then, they were used in in vitro migration assays in transwell in response to the chemokine CCL21 during 3 hours at 37 ^q C, using culture medium without chemokine as spontaneous migration control.

The results shown in Figure 5 show that the DC ^s that were previously co-cultured with MNC ^s without treatment with furanocoumarin did not show an increase in migration in response to the ^CCL21 chemokine, while those that were in contact with ^{MNC s} previously pretreated with 8-MOP ^or PSA and UVA light showed a significant increase in ^their migratory ability (### ^p <0.001). Additionally, PSA treatment resulted in a significantly greater increase in migration than 8-MOP treatment (5 ^p < 0.05).

On the contrary, the migration of mDCs in response to CCL21, which increased significantly compared to when the chemokine was absent, decreased significantly after ^their co-cultivation with ^MNCs pretreated with furanocoumarins and UVA light (***p<0.001). , being significantly lower with PSA compared to 8-MOP ( ^{aaa p} <0.001) (Figure 5). Migration was quantified by flow cytometry, and data represented as the mean ± standard deviation of 4 independent experiments, analyzing each condition in duplicate.

These results show that treatment with the PSA mixture of the invention is more effective than treatment with 8-MOP, specifically with respect to the ability of tolerogenic DCs to migrate ^{to lymph} nodes in response to chemokines, as well as the ability to decrease the migration of mDCs, which have an immunostimulatory function of the effector T response.

Example 5. Expression analysis of co-stimulatory ^or maturation molecules, MHC class II and chemokine receptors in DC ^s after ^their co-cultivation with allogeneic MNC ^s pretreated with PSA and UVA light.

To assess whether the MNC ^s pretreated with the combination of PSA furanocoumarins of the invention and UVA light could affect the generation of DC ^s with a tolerogenic phenotype after ^their co -cultivation, the expression profile of markers of maturation ^o with costimulatory function characteristic of DC ^s , both in DC ^s and ^mDCs , said markers being CD80, CD83, CD86 and the class II major histocompatibility complex (MHC) molecule HLA-DR.

For the analysis of the different cell populations, the ^íDCs and the ^mDCs were selected for ^their positivity for the CD45, BDCA-1 and HLA-DR markers. In the case of the ^í DC ^s , the percentage of the population with the BDCA-1 CD83baj0 phenotype was analyzed, while in the mDCs the percentages ^or mean fluorescence intensity of the BDCA-1 CD80alt0, BDCA-1 CD83alt0, BDCA-1 CD86alt0 and BDCA-1+ HLA-DRalt0.

On the other hand, the analysis of the expression of the CCR7 chemokine receptor was carried out in the populations of ^í DCs ^{or mDCs} with the CD45+ BDCA-1+ HLA-DR+ phenotype. The data ^show a representative result from a total of three independent experiments.

Figure 6A shows that the phenotype of the ⁱ DC ^s after ^co -cultivation with MNC ^s pretreated with PSA and UVA light did not show significant changes in the expression of CD80, CD86 ^or HLA-DR, although there was an accumulation of ^í DC ^s expressing low levels of CD83. Although it is described that the ^í DC ^s express low levels of CCR7, receptor of the chemokines CCL19 and CCL21 and involved in the migration of the DC ^s to In the lymph ^nodes , after ^their co-culture with ^MNCs pretreated with furanocoumarins and UVA light, this expression was increased, an increase that was greater in the case of treatment with PSA than with 8-MOP (Figure 6B).

In addition, a decrease in the percentage of mDCs expressing high levels of CD80, CD83, CD86 and HLA-DR was observed, which was somewhat more pronounced with PSA than with 8-MOP, as well as a decrease in the mean fluorescence intensity of the HLA-DR and CCR7 expression, which was more pronounced with PSA treatment than with 8-MOP (Figure 6).

^Taken together, these results suggest that ^after contact with ^MNCs pretreated with ^{furanocoumarins} , these results suggest that DCs maintain ^their immature phenotype and acquire a more migratory behavior in response to chemokines as a consequence of increased expression of ^their CCR7 receptor, while mDCs lose part of ^their costimulatory and antigen-presenting molecules, as well as ^their migratory potential to secondary lymphoid organs.

Example 6. Induction of Treg cells after co-cultivation with DC ^s previously co-cultivated with MNC ^s pretreated with the composition of the invention and UVA light.

Recent studies have shown that the infusion of apoptotic cells in a FEC procedure increases ^the percentages of regulatory T cells, this being the most clinically relevant fact. Therefore, it was analyzed whether the ^í DC ^s that had previously been in contact with apoptotic allogeneic ^MNCs after treatment with the combination of PSA furanocoumarins of the invention and UVA light were capable of inducing in vitro, more efficiently than 8-MOP, the differentiation of different subpopulations of regulatory T cells. For this, the levels of regulatory T cells with the phenotype CD3+CD4+CD25+ ^CD127 -F ^ox P3+ and CD3+CD8+CD25+F ^ox P3+ were analyzed by flow cytometry. The data ^show a representative result from a total of three independent experiments.

The results shown in Figure 7 show that the ^DCs previously ^co -cultured ^{with MNCs} pretreated with PSA and UVA light stimulated an increase in ^the levels of regulatory ^CD4 T cells, from a baseline value of 1.76% in absence of treatment with furanocoumarin (-Psor) at a value of 4.95% with 8-MOP, and significant, at 6.69% with PSA. Using the same co-culture protocol, it was observed that the ^í DC ^s that had previously been co-cultured with MNC ^s apoptotic by the action of furanocoumarins and UVA light were capable of inducing the differentiation of CD8 T cells with a regulatory phenotype, from a baseline from 0.02% in the absence of furanocoumarin treatment to a value of 0.27% with 8-MOP and 0.22% with PSA.

Taken together, these results suggest that the PSA furanocoumarin mixture of the invention is more effective in vitro than 8-MOP in terms of certain immunomodulatory functions, which confers greater therapeutic potential on ECF.

Example 7. Therapeutic procedure for performing ECF using apoptotic cells generated after their treatment with PSA and u v a light.

The development of the ex vivo procedure for the generation of apoptotic cells for their subsequent application to the FEC method was carried out in an experimental murine model of acute GVHD, based on previous studies (Gatza et al ^. I., Blood 112 , 2008, 1515-1521; Marques and Adarnski, J Clin Apheresis. 2014, 29, 228-234). Acute murine GVHD was performed as previously described.

To carry out the analysis of the post-transplant survival curve, as well as the clinical grade of acute GVHD, on day 30 post-transplant ^{I the} mice of ^{I the} different groups were sacrificed and ^their different organs (skin, liver and intestine ) were fixed with paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin.

T ^he results shown in Figure 8 show that the treatment by FEC with 8-MOP ^or with the PSA combination of the invention gave rise to a significant increase in survival and a reduction in the clinical grade of acute GVHD compared to the group from animals not treated with furanocoumarins (-PSOR) (***p<0.001). On the other hand, I ^os animals treated weekly with the PSA combination of the invention showed a significant increase in ^their survival compared to I ^os treated with 8-MOP, obtaining a survival of 68.75% compared to 37.5% with 8-MOP on day 45 post-transplant ( ^{a p} <0.05).

The images shown in Figure 9 are representative of a total of 5 animals per experimental group (magnification: 100 ^x ). The histopathological grade of acute GVHD was determined blindly in different distant areas of each organ by an experienced pathologist, following previously published criteria (Gatza et al., Blood 112, 2008, 1515-1521; Hill et al., Blood 1997, 90, 3204-3213). As shown in said figure, treatment by FEC with 8-MOP ^or with the PSA combination of the invention gave rise to a decrease in the histopathological grade of acute GVHD compared to the group of untreated animals (-PSOR), still being lower with PSA than with 8-MOP. In the case of the skin, animals not treated with furanocoumarin (-PSOR) show a grade IV acute GVHD characterized by separation of the dermal-epithelial junction and extensive destruction of the epidermis, while those treated by FEC with 8- MOP

^either PSA only scattered apoptotic cells are observed in the basal layer of the epidermis and some degree of spongiosis (grade ll-lll ^either grade II, respectively). In the liver, untreated animals (-PSOR) show almost total destruction of all bile ducts and high epithelial leukocyte infiltration (grade IV), while animals treated by FEC with 8-MOP ^either PSA shows less destruction of bile ducts and less leukocyte infiltration (grade lll-lV ^either grade III, respectively). Finally, the intestine of untreated animals (-PSOR) shows massive destruction of the mucosa, with denudation and atrophy of the villi (grade Vl), while animals treated by FEC with 8-MOP ^either PSA show some degree of atrophy and scattered apoptotic cells at the base of the intestinal crypts (grade lll ^either grade II, respectively). Table 1 shows a summary of the histopathological findings obtained in the different experimental groups.

Table 1. Histopathological grade of GVHD.

These in vivo results demonstrate that the PSA composition of the invention, comprising psoralen and angelicin, has greater therapeutic efficacy than 8-MOP in the reversal of acute murine GVHD by means of FEC.

Claims (15)

claims 1. Composition comprising a combination of psoralen and angelicin. 2. Composition according to claim 1, characterized in that the psoralen concentration is in a range between 50 to 700 ng/mL, and the angelicin concentration is in a range between 50 to 1120 ng/mL. Composition according to any of claims 1 to 2, characterized in that the ratio of psoralen and angelicin is in a range from 1:1 to 1:3.4. 4. Composition according to any of claims 1 to 3, characterized in that it additionally comprises one ^either plus pharmaceutically acceptable excipients. Composition according to any of claims 1 to 4, characterized in that it is an aqueous solution. Composition according to any of claims 1 to 5, characterized in that it is an injectable solution. Composition according to any of claims 1 to 6, characterized in that it is a pharmaceutical composition. 8. Composition according to any of claims 1 to 7, for ^{its use} as medicine. 9. Composition according to any of claims 1 to 7, for ^{its use} in the treatment of immunologically based diseases, by means of extracorporeal photopheresis. 10. Composition for ^{its use} according to claim 9, wherein the immunologically based diseases are selected from the list consisting of autoimmune diseases mediated by T lymphocytes, solid organ and/or tissue transplant rejection episodes, cutaneous T lymphomas, graft-versus-host disease and atopic diseases. 11. Composition for ^{its use} according to claim 10, wherein the T cell-mediated autoimmune diseases are selected from the list consisting of type 1 diabetes, Crohn's disease, systemic lupus erythematosus, psoriasis, multiple sclerosis, rheumatoid arthritis, and thyroidosis; and atopic diseases preferably include inflammatory diseases selected from the list consisting of sarcoidosis, chronic inflammatory bowel disease, and ulcerative colitis. 12. Ex vivo/in vitro procedure for the generation of apoptotic cells comprising: a) Isolate the population of mononuclear cells from a biological sample of blood isolated from a subject, b) Incubating the mononuclear cells from the previous step with a composition according to any one of claims 1 to 7; ^c ) Irradiate the cells of step b) with a photoactivator device. d) Isolating the irradiated cells from step ^c ). The method according to claim 12, wherein the apoptotic cells are autologous. The method according to any of claims 12 to 13, wherein the apoptotic cells are leukocytes. The method according to any of claims 12 to 14, wherein the photoactivator device irradiates UVA light.