WO2019207582A1 - Use of cb2 inhibitors for lymphocyte recovery - Google Patents

Use of cb2 inhibitors for lymphocyte recovery Download PDF

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WO2019207582A1
WO2019207582A1 PCT/IL2019/050458 IL2019050458W WO2019207582A1 WO 2019207582 A1 WO2019207582 A1 WO 2019207582A1 IL 2019050458 W IL2019050458 W IL 2019050458W WO 2019207582 A1 WO2019207582 A1 WO 2019207582A1
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inhibitor
expression
activity
lymphocytopenia
cannabinoid receptor
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French (fr)
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Osnat HAZAN
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Hadasit Medical Research Services And Development Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/76—Undefined extracts from plants

Definitions

  • the present invention in some embodiments thereof, relates to CB2 inhibitors and, more particularly, but not exclusively, to the use of same for lymphocyte recovery.
  • Lymphocytopenia is the condition of having an abnormally low level of lymphocytes in the blood. Lymphopenia can be a major problem in patients with solid tumors treated with radiation and chemotherapy, in patients recovering from hematopoietic stem cell transplantation (HSCT), and in other medical conditions. Lymphopenia makes patients susceptible to various infections. An association between treatment-related lymphopenia (TRL) and decreased survival was demonstrated in patients.
  • TRL treatment-related lymphopenia
  • HSCT is a well-established treatment for malignant and non-malignant hematological diseases. Allogeneic transplantation comes with the risk of Graft versus Host Disease (GVHD), a major cause of morbidity and mortality in HSCT patients. In addition, the toxicity of the conditioning protocol that precedes HSCT impairs innate and adaptive immunity, making transplanted patients very susceptible to both common and rare infections. The early post- engraftment period is categorized by a progressive recovery of cell mediated immunity, however, full reconstitution of the hematological components may take years.
  • GVHD Graft versus Host Disease
  • Cannabis contains numerous molecules, including more than 60 chemical compounds classified as cannabinoids and the different sub-strains vary in their cannabinoid content.
  • cannabinoids Two cannabinoids have been the focus of most of the studies examining medical uses: D9 tetrahydrocannabinol (THC) and cannabidiol (CBD).
  • THC and some of the other cannabinoids mediate their actions primarily through the Gi protein-coupled seven transmembrane cannabinoid receptors (members of the G-protein coupled receptor (GPCR) family): Cannabinoid receptor 1 (CB 1), which is mainly expressed in the brain and to some extent in peripheral tissues such as the immune tissues and Cannabinoid receptor type 2 (CB2), which is expressed at high density in immune cells.
  • CB2 is primarily expressed by cells and progenitors of the immune system, it is expressed at very low levels by non-hematopoietic cells in the brain, is conserved between species and has a role in suppressing immune cell function [Turcotte C, et al. Cell Mol Life Sci.
  • CBD has a very weak affinity to CB1 and CB2 (5).
  • PPAR-g nuclear receptor- Peroxisome Proliferator-Activated Receptor gamma
  • both fyto and the endogenous cannabinoids possess a wide range of anti-inflammatory properties as they induce the production of anti inflammatory cytokines such as IL-4, IL-5 and IL-10, and affect the differentiation and function of several immune cells (8, 9).
  • cytokines such as IL-4, IL-5 and IL-10
  • cannabinoid receptor signaling in the biology of hematopoietic stem and progenitor cells has also been reported (10, 11).
  • a method of treating a lymphocytopenia in a subject in need thereof, wherein the lymphocytopenia is not associated with hematopoietic stem cell transplantation comprising administering to the subject a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby treating the lymphocytopenia.
  • a method of treating a lymphocytopenia associated with hematopoietic stem cell transplantation in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby treating the lymphocytopenia associated with hematopoietic stem cell transplantation.
  • a method of increasing lymphocyte count in a human subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby increasing the lymphocyte count.
  • an ex vivo method of enhancing lymphocyte recovery comprising ex vivo contacting hematopoietic stem and progenitor cells with an effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby enhancing lymphocyte recovery.
  • the method further comprises administering the hematopoietic stem and progenitor cells to a subject in need thereof.
  • the lymphocytopenia is not associated with transplantation of a solid organ or tissue.
  • the lymphocytopenia is associated with transplantation of a solid organ or tissue.
  • the lymphocytopenia is associated with administration of an anti-cancer therapy.
  • the lymphocytopenia is associated with administration of a chemotherapeutic agent, a radiation therapy or an immunotherapy.
  • the subject has a disease or condition selected from the group consisting of a malignant disease, a non-malignant tumor, an infection, an autoimmune disease, an aplastic anemia and a nuclear or radiation exposure.
  • the hematopoietic stem cell transplantation is used to treat a disease or condition selected from the group consisting of a hematologic malignancy, an aplastic anemia or an immune deficiency.
  • the hematopoietic stem cell transplantation is obtained from a source selected from the group consisting of bone marrow stem cells, peripheral blood stem cells and cord blood stem cells.
  • the hematopoietic stem cell transplantation comprises T cell depleted hematopoietic stem cells.
  • the inhibitor of the CB2 is administered to the subject prior to, concomitantly with and/or subsequent to the hematopoietic stem cell transplantation.
  • the hematopoietic stem cell transplantation is autologous with the subject. According to some embodiments of the invention, the hematopoietic stem cell transplantation is non-autologous with the subject.
  • the subject is monitored for graft versus host disease.
  • the inhibitor of the CB2 is a genome editing agent or an RNA silencing agent.
  • the inhibitor of the CB2 is an antagonist or an inverse agonist.
  • the inhibitor is selected from the group consisting of a small molecule, an antibody and an aptamer.
  • the inhibitor e.g. antagonist or inverse agonist
  • the inhibitor is selected from the group consisting of SR144528, AM630, JTE-907, COR170, GPla, SCH336, RO6957022 and AM 1241.
  • the lymphocytopenia comprises T lymphocytopenia, B lymphocytopenia and/or NK lymphocytopenia.
  • the lymphocytopenia is determined when a complete blood count of the subject shows a lymphocyte count lower than the age-appropriate reference.
  • the subject is a human subject.
  • FIGs. 1A-E are graphs illustrating the influence of pure CBD/THC and cannabis extracts on lymphocyte activation.
  • Figure 1A Proliferation of CFSE stained, CD3 activated, murine splenocytes was analyzed using flow cytometry analysis. Summary of a minimum of 6 independent experiments is provided. Results are expressed as mean. Quantification of IL-l7a ( Figure 1B), IL-10 (Figure 1C), TNF-a ( Figure 1D) and IL-5 (Figure 1E) secretion was performed using enzyme-linked immunosorbent assay on culture medium of activated cells. Results are expressed as mean + SEM. Data are summarized from a minimum of 5 independent experiments p value * ⁇ 0.05; ** ⁇ 0.001; *** ⁇ 0.0001.
  • FIGs. 1F-G are graphs illustrating that THC and CBD affect lymphocyte activation by different mechanisms.
  • Figure 1F Proliferation of CFSE stained, CD3 activated, splenocytes from CB2 knockout mice was analyzed using flow cytometry analysis. Summary of a minimum of 3 independent experiments is provided. Results are expressed as mean. Of note, the differences of CBD, THC BDS (Botanical Drug Substance) and CBD BDS as compared to control are significant starting from 3 pg/ml. The differences of THC as compared to control are significant starting from 10 pg/ml.
  • Figure 1G The influence of PPARy antagonist, GW9662, on CBD’s effect on lymphocyte activation.
  • FIGs. 2A-E illustrate administration of cannabis/cannabinoids in a syngeneic BMT model.
  • Figure 2A Recipient C57BL/6 mice received lethal whole-body irradiation and were reconstituted with 8 x 10 6 donor C57BL/6 bone marrow cells. Cannabis/cannabinoids were administered IP every other day, for two weeks from the day of transplantation. Blood samples for CBC were obtained once a week. Lymphocyte counts in pure cannabinoid treated groups (Figure 2B) and BDS treated groups ( Figure 2C) are presented. Average counts at different time points (left) and day 21 after transplantation counts (right). Platelet counts in pure cannabinoid treated groups ( Figure 2D) and BDS treated groups ( Figure 2E), day 14 after transplantation. Data are summarized from a minimum of 3 independent experiments p value * ⁇ 0.05; ** ⁇ 0.001; *** ⁇ 0.0001.
  • FIGs. 3A-C are graphs illustrating the role of CB2 in lymphocyte recovery.
  • Figure 3 A Recipient C57BL/6 mice underwent syngeneic BMT. CB2 inverse agonist SR144528, was administered IP once a day for one week from the day of transplantation. Mice were monitored for weight loss. Blood samples were obtained once a week. Average lymphocyte counts are provided. The average percent of initial weight during the experiment is provided. No signs of toxicity were evident.
  • Figure 3B Syngeneic BMT from CB2 KO donor mice to C57BL/6 WT mice. Average counts at different time points (left) and day 21 after transplantation counts (right).
  • FIGs. 4A-C illustrate cannabis/cannabinoids administration for GVHD prophylaxis.
  • Figure 4A Recipient BALB/c mice received lethal whole-body irradiation and were reconstituted with 8 x 10 6 donor C57BL/6 bone marrow cells and 2 x 10 6 spleen cells. Cannabis/cannabinoids were administered IP every other day, for two weeks from the day of transplantation. The clinical condition of the mice was evaluated for up to 67 days after transplantation.
  • Figure 4B Survival curve. Of note, the differences between control and THC BDS as well as CBD BDS are significant.
  • Figure 4C Average GVHD score (Days 15-26). Of note, the differences between THC BDS / CBD BDS and control group are significant.
  • FIGs. 5A-B are graphs illustrating the influence of pure CBD/THC and cannabis extracts on lymphocyte activation. Proliferation of CFSE stained, CD3 activated, lymphocytes was analyzed using flow cytometry analysis. Summary of a minimum of 3 independent experiments is provided. Results are expressed as mean.
  • Figure 5A Balb/C murine splenocytes
  • Figure 5B Human PBMC.
  • FIGs. 6A-B are graphs illustrating the influence of pure CBD/THC and cannabis extracts on lymphocyte activation.
  • the levels of cytokines ( Figure 6A - IL-17a and Figure 6B - IL-10) in the culture medium of CD3 activated, lymphocytes were analyzed using ELISA assay. Summary of a minimum of 3 independent experiments are provided. Results are expressed as mean.
  • FIGs. 7A-B illustrate CB2 KO mice (CNR /_ ).
  • Figure 7A PCR results for CNR gene.
  • FIGs. 8A-C are graphs illustrating the influence of TRP antagonists on CBD’s effect on lymphocyte activation. Proliferation of CFSE stained, CD3 activated, murine splenocytes was analyzed using flow cytometry analysis.
  • Figure 8A A967079 antagonist to TRPA1.
  • Figure 8B BCTC antagonist to TRPV1.
  • Figure 8C GSK2193874 antagonist to TRPV4. Summary of a minimum of 2 independent experiments is provided. Results are expressed as mean + SEM. p value - samples were compared to activated splenocytes + CBD (right) or activated splenocytes (left).
  • FIGs. 9A-B illustrate blood chimerism in allogeneic transplanted mice.
  • Figure 9A example for FACS analysis of blood cells stained with anti-H2Db and anti-H2Dd antibodies.
  • Figure 9B Average blood chimerism is provided. Data are summarized from 2 independent experiments.
  • FIGs. 10A-D are graphs illustrating the role of CB2 in lymphocyte recovery in a model of treatment-induced lymphocytopenia without bone marrow transplantation. C57BL/6 and CB2 KO mice received non-lethal 6 Gy whole -body irradiation.
  • Figures 10A-B Average blood counts (CBC) at different time points ( Figure 10B) and day 21 after irradiation treatment ( Figure 10A).
  • the present invention in some embodiments thereof, relates to CB2 inhibitors and, more particularly, but not exclusively, to the use of same for lymphocyte recovery.
  • Hematopoietic stem cell transplantation is a well-established treatment for malignant and non-malignant hematological diseases.
  • the toxicity of the conditioning protocol which precedes HSCT impairs innate and adaptive immunity, making transplanted patients very susceptible to various infections.
  • the early post-engraftment period is categorized by progressive recovery of cell mediated immunity.
  • full reconstitution of the hematological components may take years.
  • THC mediates its action primarily through the cannabinoid receptors: CB1, which is mainly expressed in the brain and to some extent in peripheral tissues such as the immune tissues, and CB2, which is expressed at high density in immune cells.
  • CB1 cannabinoid receptors
  • CB2 cannabinoid receptors
  • THC cannabinoid treatment
  • CBD cannabinoid treatment
  • the present inventor has compared the influence of pure THC, pure CBD and high THC/high CBD cannabis extracts treatment in murine bone marrow transplantation (BMT) models. It is shown that all of the tested cannabinoid treatments reduced lymphocyte proliferation in vitro (Example 1 of the examples section which follows) as well as lymphocyte recovery in vivo (Example 3 of the examples section which follows).
  • BMT murine bone marrow transplantation
  • CBD and THC affect lymphocyte proliferation, they utilize different signal transduction pathways to cause these effects.
  • THC signals through CB2 receptor CBD signals through PPAR-g (Example 2 of the examples section which follows).
  • CB2 was found to be directly associated with inhibition of lymphocyte recovery, as shown that using CB2 knock-out mice as a syngeneic transplant model, improved lymphocyte recovery (Example 4 of the examples section which follows).
  • CB2 inverse agonist improved lymphocyte recovery after syngeneic bone marrow transplantation (Example 4 of the examples section which follows).
  • a method of treating a lymphocytopenia in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby treating the lymphocytopenia.
  • a method of treating a lymphocytopenia in a subject in need thereof, wherein the lymphocytopenia is not associated with hematopoietic stem cell transplantation comprising administering to the subject a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby treating the lymphocytopenia.
  • a method of treating a lymphocytopenia associated with hematopoietic stem cell transplantation in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby treating the lymphocytopenia associated with hematopoietic stem cell transplantation.
  • a method of increasing lymphocyte count in a human subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby increasing the lymphocyte count.
  • the term“treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • the term "subject” or“subject in need thereof’ refers to a mammal, preferably a human being, male or female at any age or gender that has lymphocytopenia.
  • the subject has a lymphocytopenia due to a disorder or a pathological or undesired condition, state, or syndrome, or due to a therapeutic modality all of which cause acute or chronic lymphocytopenia. Examples of such disorders are provided further below.
  • lymphocytopenia refers to the condition of having an abnormally low level of lymphocytes (T lymphocytes, B lymphocytes, and/or natural killer cells) in the blood.
  • Lymphocytopenia is typically determined when a complete blood count (CBC) of the subject shows a lymphocyte count lower than the age- appropriate reference. Accordingly, in adults (i.e. over 20 years old), lymphocytopenia refers to a count of less than 1,000 lymphocytes per microliter of blood. In children lymphocyte counts vary with age. For children up to 5 months old, lymphocytopenia refers to a count of less than 2,000 lymphocytes per microliter of blood. For children of age 6 months to 2 years, lymphocytopenia refers to a count of less than 4,000 lymphocytes per microliter of blood. For children of age 2-3 years, lymphocytopenia refers to a count of less than 3,000 lymphocytes per microliter of blood.
  • lymphocytopenia refers to a count of less than 2,000 lymphocytes per microliter of blood.
  • lymphocytopenia refers to a count of less than 1,500 lymphocytes per microliter of blood.
  • the lymphocytopenia comprises T lymphocytopenia (i.e. abnormally low level of T lymphocytes, e.g. CD3+ cells, CD2+ cells, CD8+ cells, CD4+ cells, a/b T cells and/or g/d T cells).
  • T lymphocytopenia i.e. abnormally low level of T lymphocytes, e.g. CD3+ cells, CD2+ cells, CD8+ cells, CD4+ cells, a/b T cells and/or g/d T cells).
  • the lymphocytopenia comprises B lymphocytopenia (i.e. abnormally low level of B lymphocytes).
  • the lymphocytopenia comprises NK lymphocytopenia (i.e. abnormally low level of natural killer cells).
  • the methods of the present invention can also be used to increase lymphocyte counts in a subject having low lymphocyte counts and not having lymphocytopenia (e.g. adults having lymphocyte counts higher than 1,000 lymphocytes per microliter of blood but lower than 2000 lymphocytes per microliter of blood).
  • lymphocytopenia e.g. adults having lymphocyte counts higher than 1,000 lymphocytes per microliter of blood but lower than 2000 lymphocytes per microliter of blood.
  • the lymphocyte counts in the subject increase by at least about 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 100 % or more, as compared to the lymphocyte counts prior to treatment.
  • Lymphocyte counts can be determined by any method known in the art, such as by standard blood test (e.g. complete blood counts (CBC)).
  • standard blood test e.g. complete blood counts (CBC)
  • Lymphocytopenia may be caused by disease progression (e.g. cancer, infection, autoimmune disease) or by disease treatment (e.g. ultraviolet A irradiation, steroid treatment, chemotherapy, or hematopoietic stem cell transplantation) as discussed below.
  • disease progression e.g. cancer, infection, autoimmune disease
  • disease treatment e.g. ultraviolet A irradiation, steroid treatment, chemotherapy, or hematopoietic stem cell transplantation
  • lymphocytopenia is associated with various diseases and conditions.
  • lymphocytopenia may be associated with a malignant disease, with a non- malignant tumor, with an infection, with an autoimmune disease, with aplastic anemia, with an immune deficiency and/or with a nuclear or radiation exposure.
  • lymphocytopenia is associated with a non-malignant tumor or lesion (benign tumor that is not a malignant).
  • the non-malignant tumor includes, but is not limited to, an adenoma, a lipoma, a fibroma and a myoma.
  • lymphocytopenia is associated with a malignant disease (also termed cancer).
  • the malignant disease is a solid tumor or tumor metastasis.
  • the malignant disease is a carcinoma, a lymphoma, a blastoma, a sarcoma, and a leukemia.
  • the malignant disease is a hematological malignancy.
  • the hematological malignancy comprises a leukemia or a lymphoma.
  • Exemplary hematological malignancies include, but are not limited to, leukemia [e.g., acute lymphatic, acute lymphoblastic, acute lymphoblastic pre-B cell, acute lymphoblastic T cell leukemia, acute - megakaryoblastic, monocytic, acute myelogenous, acute myeloid, acute myeloid with eosinophilia, B cell, basophilic, chronic myeloid, chronic, B cell, eosinophilic, Friend, granulocytic or myelocytic, hairy cell, lymphocytic, megakaryoblastic, monocytic, monocytic- macrophage, myeloblastic, myeloid, myelomonocytic, plasma cell, pre-B cell, promyelocytic, subacute, T cell, lymphoid neoplasm, predisposition to myeloid malignancy, acute nonlymphocytic leukemia, T-cell acute lymphocytic
  • the malignant disease is a myeloproliferative disease, such as but not limited to, Solid tumors, Benign Meningioma, Mixed tumors of salivary gland, Colonic adenomas; Adenocarcinomas, such as Small cell lung cancer, Kidney, Uterus, Prostate, Bladder, Ovary, Colon, Sarcomas, Liposarcoma, myxoid, Synovial sarcoma, Rhabdomyosarcoma (alveolar), Extraskeletel myxoid chonodro sarcoma, Ewing's tumor; Other cancer types include Testicular and ovarian dysgerminoma, Retinoblastoma, Wilms' tumor, Neuroblastoma, Malignant melanoma, Mesothelioma, Myeloma, Colorectal cancer, Breast cancer, Skin cancer, Pancreatic cancer, Prostate cancer, Esophageal cancer and Ovarian
  • lymphocytopenia is associated with an immune deficiency, including but not limited to, severe combined immunodeficiency syndromes (SCID), X-linked agammaglobulinemia (XLA), common variable immunodeficiency (CVID), and AIDS.
  • SCID severe combined immunodeficiency syndromes
  • XLA X-linked agammaglobulinemia
  • CVID common variable immunodeficiency
  • AIDS AIDS
  • lymphocytopenia is associated with a viral, a bacterial, a parasitic, or a fungal infection.
  • infections include, but are not limited to, AIDS, viral hepatitis, tuberculosis, typhoid fever, histoplasmosis, influenza or malaria.
  • lymphocytopenia is associated with an autoimmune disease, such as but not limited to, psoriasis or lupus.
  • lymphocytopenia is associated with a nuclear or radiation exposure e.g. ionizing radiation including, but not limited to, cosmic, alpha, beta, gamma or X- rays, or neutrons.
  • Nuclear or radiation exposure typically refers to large doses of ionizing radiation (e.g. 0.25 - 4 Gy) which can lead to Acute Radiation Syndrome (ARS) i.e. radiation poisoning.
  • ARS Acute Radiation Syndrome
  • lymphocytopenia is associated with treatment of the disease or disorder. Accordingly, lymphocytopenia may be instigated directly by the disease or condition (e.g. immune deficiency, hematopoietic malignancy, infection, radiation exposure). Additionally or alternatively, lymphocytopenia may be instigated by a treatment modality associated with the disease or condition (e.g. chemotherapy, immunotherapy, radiation therapy, immunosuppression, transplantation of a cell or tissue graft), as discussed below.
  • a treatment modality associated with the disease or condition e.g. chemotherapy, immunotherapy, radiation therapy, immunosuppression, transplantation of a cell or tissue graft
  • lymphocytopenia is associated with a hematopoietic stem cell transplantation employed for treatment of a malignancy (e.g. hematological malignancy, e.g. leukemia or lymphoma), aplastic anemia, immune deficiency (e.g. SCID), infection (e.g. viral infection) or autoimmune disease.
  • a malignancy e.g. hematological malignancy, e.g. leukemia or lymphoma
  • aplastic anemia e.g. SCID
  • infection e.g. viral infection
  • lymphocytopenia is associated with an anti-cancer drug.
  • Anti-cancer drugs include, but are not limited to Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Ce
  • Additional antineoplastic agents include those disclosed in Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner), and the introduction thereto, 1202-1263, of Goodman and Gilman's "The Pharmacological Basis of Therapeutics", Eighth Edition, 1990, McGraw-Hill, Inc. (Health Professions Division).
  • lymphocytopenia is associated with chemotherapy, immunotherapy or radiation therapy.
  • chemotherapy which may cause lymphocytopenia include, but are not limited to, Abarelix, Actinomycin D, Aldesleukin, Aldesleukin, Alemtuzumab, Alitretinoin, Allopurinol, Altima (Pemetrexed), Altretamine, Amifostine, Anastrozole, Arsenic trioxide, Asparaginase, Azacitidine, Bevacuzimab, Bexarotene, Bleomycin, Bortezomib, Busulfan, Calusterone, Capecitabine, Carboplatin, Carmustine, Celecoxib, Cetuximab, Cisplatin, Cladribine, Clofarabine, Cyclophosphamide, Cytarabine, dacarbazine, Dactinomycin, Darbepoetin alfa, Darbepoetin alfa, Daunorubi
  • Exemplary immunotherapy which may cause lymphocytopenia include, but are not limited to, monoclonal antibodies (e.g. Alemtuzumab, Trastuzumab, Ibritumomab tiuxetan, Brentuximab vedotin, Ado-trastuzumab emtansine, Denileukin diftitox, Blinatumomab, Bevacizumab and Cetuximab), Immune checkpoint inhibitors (e.g. Pembrolizumab, Nivolumab, Cemiplimab, Atezolizumab, Avelumab, Durvalumab, Ipilimumab), interleukins (e.g. IL-2), interferons (e.g. IFN- alfa, IFN-beta, IFN-gamma), immunomodulating drugs (e.g. Thalidomide, Lenalidomide, and Pomalidomide).
  • lymphocytopenia is associated with irradiation therapy.
  • lymphocytopenia is associated with total body irradiation
  • the TBI comprises a single or fractionated irradiation dose within the range of 0.25-0.5 Gy, 0.25-1 Gy, 0.25-2.5 Gy, 0.25-5 Gy, 0.25-10 Gy, 0.5-1 Gy, 0.5- 1.5 Gy, 0.5-2.5 Gy, 0.5-5 Gy, 0.5-7.5 Gy, 0.5-10 Gy, 0.5-15 Gy, 1-1.5 Gy, 1-2 Gy, 1-2.5 Gy, 1-3 Gy, 1- 3.5 Gy, 1-4 Gy, 1-4.5 Gy, 1-1.5 Gy, 1-7.5 Gy, 1-10 Gy, 2-3 Gy, 2-4 Gy, 2-5 Gy, 2-6 Gy, 2-7 Gy, 2- 8 Gy, 2-9 Gy, 2-10 Gy, 3-4 Gy, 3-5 Gy, 3-6 Gy, 3-7 Gy, 3-8 Gy, 3-9 Gy, 3-10 Gy, 4-5 Gy, 4-6 Gy, 4-7 Gy, 4-8 Gy, 4-9 Gy, 4-10 Gy, 5-6 Gy, 5-7 Gy, 5-8 Gy, 5-9 Gy, 5-10 Gy, 6-7 Gy, 6-8 Gy, 6-9 Gy, 6-10 Gy, 7-8 Gy,
  • lymphocytopenia is associated with total lymphoid irradiation (TLI, i.e. exposure of all lymph nodes, the thymus, and spleen).
  • TKI total lymphoid irradiation
  • the TLI comprises an irradiation dose within the range of 0.25-0.5 Gy, 0.25-1 Gy, 0.25-2.5 Gy, 0.25-5 Gy, 0.25-10 Gy, 0.5-1 Gy, 0.5-1.5 Gy, 0.5-2.5 Gy, 0.5-5 Gy, 0.5-7.5 Gy, 0.5-10 Gy, 0.5-15 Gy, 1-1.5 Gy, 1-2 Gy, 1-2.5 Gy, 1-3 Gy, 1-3.5 Gy, 1-4 Gy,
  • lymphocytopenia is associated with partial body irradiation (e.g. specific exposure of the lungs, kidney, brain etc.).
  • the partial body irradiation comprises an irradiation dose within the range of 0.25-0.5 Gy, 0.25-1 Gy, 0.25-2.5 Gy, 0.25-5 Gy, 0.25-10 Gy, 0.5-1 Gy, 0.5- 1.5 Gy, 0.5-2.5 Gy, 0.5-5 Gy, 0.5-7.5 Gy, 0.5-10 Gy, 0.5-15 Gy, 1-1.5 Gy, 1-2 Gy, 1-2.5 Gy, 1-3
  • Gy 1-3.5 Gy, 1-4 Gy, 1-4.5 Gy, 1-1.5 Gy, 1-7.5 Gy, 1-10 Gy, 2-3 Gy, 2-4 Gy, 2-5 Gy, 2-6 Gy, 2-7 Gy, 2-8 Gy, 2-9 Gy, 2-10 Gy, 3-4 Gy, 3-5 Gy, 3-6 Gy, 3-7 Gy, 3-8 Gy, 3-9 Gy, 3-10 Gy, 4-5 Gy, 4- 6 Gy, 4-7 Gy, 4-8 Gy, 4-9 Gy, 4-10 Gy, 5-6 Gy, 5-7 Gy, 5-8 Gy, 5-9 Gy, 5-10 Gy, 6-7 Gy, 6-8 Gy, 6-9 Gy, 6-10 Gy, 7-8 Gy, 7-9 Gy, 7-10 Gy, 8-9 Gy, 8-10 Gy, 10-12 Gy, 10-15 Gy, 10-20 Gy, 10-30 Gy, 10-40 Gy, 10-50 Gy, 0.5-20 Gy, 0.5-30 Gy, 0.5-40 Gy or 0.5-50 Gy.
  • lymphocytopenia is associated with administration of an immunosuppressive drug.
  • Immunosuppressive drugs include, but are not limited to, Calcineurin Inhibitors, Antiproliferative agents, mTOR inhibitors and Steroids.
  • immunosuppressive drugs include, but are not limited to, Tacrolimus (also referred to as FK-506 or fujimycin, trade names: Prograf, Advagraf, Protopic), Mycophenolate Mofetil, Mycophenolate Sodium, Prednisone, methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine ( sulphas alazopyrine), gold salts, D- penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE), etanercept, TNF.alpha.
  • Tacrolimus also referred to as FK-506 or fujimycin, trade names: Prograf, Advagraf, Protopic
  • Mycophenolate Mofetil Mycophenolate Sodium
  • Prednisone methotrexate
  • cyclophosphamide
  • NSAIDs Non-Steroidal Anti-Inflammatory Drug
  • NSAIDs include, but are not limited to acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, tramadol, rapamycin (sirolimus) and rapamycin analogs (such as CCI-779, RAD001, AP23573). These agents may be administered individually or in combination.
  • lymphocytopenia is associated with a combination of any of the above described therapeutic protocols (e.g. chemotherapeutic agent and TBI, chemotherapeutic agent and TLI, antibody immunotherapy and chemotherapeutic agent, etc.).
  • therapeutic protocols e.g. chemotherapeutic agent and TBI, chemotherapeutic agent and TLI, antibody immunotherapy and chemotherapeutic agent, etc.
  • the lymphocytopenia is not associated with transplantation of a cell or tissue graft.
  • the lymphocytopenia is not associated with transplantation of hematopoietic stem cells.
  • lymphocytopenia is associated with transplantation of a cell or tissue graft.
  • the phrase“cell or tissue graft” refers to a bodily cell (e.g. a single cell or a group of cells) or tissue (e.g. solid tissues or soft tissues, which may be transplanted in full or in part).
  • tissue e.g. solid tissues or soft tissues, which may be transplanted in full or in part.
  • Exemplary tissues which may be transplanted according to the present teachings include, but are not limited to, liver, pancreas, spleen, kidney, heart, lung, skin, intestine and lymphoid/hematopoietic tissues (e.g. lymph node, Peyer’s patches, thymus or bone marrow).
  • Exemplary cells which may be transplanted according to the present teachings include, but are not limited to, immature hematopoietic cells including stem cells.
  • the present invention also contemplates transplantation of whole organs (e.g. solid organs), such as for example, kidney, heart, lung, liver, pancreas,
  • the cell or tissue graft comprises co-transplantation of more than one cell or tissue type (e.g. co-transplantation of hematopoietic stem cells and a solid organ or tissue e.g. kidney, liver, lung etc. or co-transplantation of a number of solid organs or tissues e.g. liver and intestine, lung and heart, etc.).
  • a cell or tissue type e.g. co-transplantation of hematopoietic stem cells and a solid organ or tissue e.g. kidney, liver, lung etc.
  • co-transplantation of a number of solid organs or tissues e.g. liver and intestine, lung and heart, etc.
  • the hematopoietic stem cells and the solid organ or obtained from the same donor are hematopoietic stem cells and the solid organ or obtained from the same donor.
  • the cell or tissue graft does not comprise hematopoietic stem cells.
  • the cell or tissue graft comprises hematopoietic stem cells. According to one embodiment, the cell or tissue graft comprises hematopoietic progenitor cells.
  • the hematopoietic stem cell transplantation is used to treat a disease or condition selected from the group consisting of a hematologic malignancy, an aplastic anemia, a viral infection, or an immune deficiency.
  • hematopoietic stem cells refers to a hematopoietic tissue or cell preparation comprising precursor hematopoietic cells (e.g. immature hematopoietic cells).
  • tissue/cell preparation includes or is derived from a biological sample, for example, bone marrow, mobilized peripheral blood (e.g. mobilization of CD34 cells to enhance their concentration), cord blood (e.g. umbilical cord), fetal liver, yolk sac and/or placenta.
  • purified CD34+ cells or other hematopoietic stem cells such as CD131+ cells can be used in accordance with the present teachings, either with or without ex-vivo expansion.
  • Hematopoietic stem cells typically have the capacity to self-renew (i.e. expand) and to differentiate into progenitor cells, which are more developmentally committed to a cell line than are stem cells, but which are nevertheless undifferentiated or immature in comparison to those cells that have differentiated and matured into specialized cells.
  • Progenitor cells may be identified by expression of a cell marker including, but not limited to, CD45, CD34, CD38, SCA1, CD59, CD90/Thyl and/or IL-3 R alpha.
  • hematopoietic stem and progenitor cells may relate to hematopoietic stem cells, to progenitor cells or to a combination of these cells.
  • hematopoietic stem cells can be committed to a particular line of differentiation, e.g. common myeloid progenitors (CMP), common lymphoid progenitors (CLP), Colony-Forming Unit-Granulocyte/Erythrocyte/Macrophage/Megakaryocyte (CFU-GEMM).
  • CMP common myeloid progenitors
  • CLP common lymphoid progenitors
  • CFU-GEMM Colony-Forming Unit-Granulocyte/Erythrocyte/Macrophage/Megakaryocyte
  • CFU-GM colony forming unit-granulocyte-monocyte
  • CFU-E colony forming unit-erythrocyte
  • BFU-E burst forming unit-erythrocyte
  • CFU-G colony forming unit- granulocyte
  • CFU-eo colony forming unit-eosinophil
  • CFU-Meg colony forming unit-megakaryocyte
  • Hematopoietic stem cells are capable of differentiating into any of granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages, dendritic cells) and lymphocytes (common lymphoid progenitors, pre-B, pro-B, mature B, pre-T, pro-B, mature T and NKT lymphocytes and NK cells).
  • granulocytes e.g., promyelocytes, neutrophils, eosinophils, basophils
  • erythrocytes e.g., reticulocytes, erythrocytes
  • thrombocytes e.g.
  • the hematopoietic stem cells are human hematopoietic stem cells.
  • the progenitor cells are human progenitor cells.
  • the hematopoietic stem cells comprise T cell depleted hematopoietic stem cells, i.e. a population of hematopoietic cells which are depleted of T lymphocytes.
  • the T cell depleted hematopoietic stem cells may include e.g. CD34+, CD33+ and/or CD56+ cells.
  • the T cell depleted hematopoietic stem cells may be depleted of CD3+ cells, CD2+ cells, CD8+ cells, CD4+ cells, a/b T cells and/or g/d T cells.
  • the hematopoietic stem cells comprise T cell depleted G-CSF mobilized blood cells enriched for CD34 + immature hematopoietic cells.
  • the method may be effected using a cell or tissue graft which is syngeneic or non-syngeneic (e.g. allogeneic or xenogeneic) with the subject.
  • a cell or tissue graft which is syngeneic or non-syngeneic (e.g. allogeneic or xenogeneic) with the subject.
  • syngeneic cells refer to cells which are essentially genetically identical with the subject or essentially all lymphocytes of the subject.
  • Examples of syngeneic cells include cells derived from the subject (also referred to in the art as“autologous”), from a clone of the subject, or from an identical twin of the subject.
  • non-syngeneic cells refer to cells which are not essentially genetically identical with the subject or essentially all lymphocytes of the subject, such as allogeneic cells or xenogeneic cells.
  • allogeneic refers to cells which are derived from a donor who is of the same species as the subject, but which is substantially non-clonal with the subject. Typically, outbred, non-zygotic twin mammals of the same species are allogeneic with each other. It will be appreciated that an allogeneic cell may be HLA identical, partially HLA identical or HLA non identical (i.e. displaying one or more disparate HLA determinant) with respect to the subject.
  • xenogeneic refers to a cell which substantially expresses antigens of a different species relative to the species of a substantial proportion of the lymphocytes of the subject. Typically, outbred mammals of different species are xenogeneic with each other.
  • the subject is a human being and the cells are from a human origin (e.g. autologous or non-autologous).
  • a human origin e.g. autologous or non-autologous
  • the subject is a human being and the cells are from a xenogeneic origin (e.g. porcine origin).
  • a xenogeneic origin e.g. porcine origin
  • the cell or tissue graft of the present invention may be obtained from a prenatal organism, postnatal organism, an adult or a cadaver donor. Moreover, depending on the application needed, the cell or tissue graft may be naive or genetically modified. Determination of the type of cell or tissue graft to be used is well within the ability of one of ordinary skill in the art. Furthermore, any method known in the art may be employed to obtain a cell or tissue graft (e.g. for transplantation).
  • hematopoietic stem cells or progenitor cells may be obtained by collection of bone marrow from the rear pelvic bones.
  • hematopoietic stem cells or progenitor cells may be obtained by collecting peripheral blood (e.g. peripheral blood stem cells (PBSCs)) from a donor following a cell immobilization protocol (e.g. following administration of granulocyte colony stimulating factor (G-CSF)).
  • PBSCs peripheral blood stem cells
  • G-CSF granulocyte colony stimulating factor
  • heparin or citrate or by apheresis, a procedure in which the peripheral blood of an individual is passed through an apparatus, yielding a predominant constituent (e.g. mononuclear cells), and returning the other constituents to the subject's circulation.
  • a predominant constituent e.g. mononuclear cells
  • cells may be obtained by in-vitro or ex-vivo culture of cells. It will be appreciated that the cells of the invention may be of fresh or frozen (e.g., cryo-preserved) preparations.
  • hematopoietic stem cells or progenitor cells may be collected from cord blood.
  • Transplanting the cell or tissue into the subject may be effected in numerous ways, depending on various parameters, such as, for example, the cell or tissue type; the type, stage or severity of the recipient's disease (e.g. organ failure); the physical or physiological parameters specific to the subject; and/or the desired therapeutic outcome.
  • various parameters such as, for example, the cell or tissue type; the type, stage or severity of the recipient's disease (e.g. organ failure); the physical or physiological parameters specific to the subject; and/or the desired therapeutic outcome.
  • graft rejection and graft versus host disease GVHD
  • the method typically comprises conditioning the subject prior to transplantation under conditioning protocols, e.g. reduced intensity conditioning (RIC), such pre-transplant conditioning protocol may lead to lymphocytopenia.
  • conditioning protocols e.g. reduced intensity conditioning (RIC)
  • RIC reduced intensity conditioning
  • the conditioning protocol comprises a total body irradiation (TBI), total lymphoid irradiation (TLI), partial body irradiation (e.g. specific exposure of the lungs, kidney, brain etc.), myeloablative conditioning and/or non-myeloablative conditioning, e.g. with different combinations including, but not limited to, co-stimulatory blockade, chemotherapeutic agent and/or antibody immunotherapy.
  • the conditioning comprises a combination of any of the above described conditioning protocols (e.g. chemotherapeutic agent and TBI, co- stimulatory blockade and chemotherapeutic agent, antibody immunotherapy and chemotherapeutic agent, etc.).
  • conditioning agents which may be used to condition the subject and which can cause lymphocytopenia include, without limitation, irradiation and pharmacological agents.
  • lymphocytopenia examples include myelotoxic drugs (e.g. busulfan, dimethyl mileran, melphalan and thiotepa), lymphocytotoxic drugs (e.g. lymphocytotoxic antibodies) and immunosuppressant drugs (discussed in detail above).
  • myelotoxic drugs e.g. busulfan, dimethyl mileran, melphalan and thiotepa
  • lymphocytotoxic drugs e.g. lymphocytotoxic antibodies
  • immunosuppressant drugs discussed in detail above.
  • the method may further comprise conditioning the subject with an immunosuppressive regimen prior to, concomitantly with, or following transplantation of the cell or tissue transplant. It will be appreciated that the immunosuppressive regime may lead to lymphocytopenia.
  • immunosuppressive regimens include administration of immunosuppressive drugs and/or immunosuppressive irradiation (both of which are discussed above).
  • the method is effected by administering to the subject a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity.
  • inhibitor of a cannabinoid receptor CB2 expression and/or activity refers to a natural or synthetic compound which acts as an inhibitor of CB2 gene expression (at the DNA or RNA level) and/or as an inhibitor of CB2 receptor activity (at the protein level).
  • CB2 receptor refers to the cannabinoid receptor type 2.
  • the CB2 receptor is human CB2 receptor: an exemplary CB2 amino acid sequence is provided in GenBank accession number NP_00l832, and an exemplary CB2 nucleotide sequence is provided in GenBank accession number NM_00l84l.
  • RNA silencing agents e.g., RNA silencing agents
  • protein level e.g., aptamers, small molecules and inhibitory peptides, antagonists, enzymes that cleave the polypeptide, antibodies and the like
  • aptamers small molecules and inhibitory peptides, antagonists, enzymes that cleave the polypeptide, antibodies and the like
  • Down regulation of expression and/or activity may be either transient or permanent.
  • the downregulation is transient.
  • down regulating expression refers to the absence of mRNA and/or protein, as detected by RT-PCR or Western blot, respectively.
  • down regulating expression refers to a decrease in the level of mRNA and/or protein, as detected by RT-PCR or Western blot, respectively.
  • the reduction may be by at least a 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % or at least 99 % reduction.
  • down regulating activity refers to the absence of CB2 activity (e.g. lack of activation of factors in the CB2 cell signaling pathway, such as inhibition of adenylyl cyclase activity and reduced cAMP levels, or phosphorylation of the inhibitory Tyr-505 of the leukocyte-specific protein tyrosine kinase (Lck)), as detected e.g. by ELISA or Western Blot.
  • CB2 activity e.g. lack of activation of factors in the CB2 cell signaling pathway, such as inhibition of adenylyl cyclase activity and reduced cAMP levels, or phosphorylation of the inhibitory Tyr-505 of the leukocyte-specific protein tyrosine kinase (Lck)
  • down regulating activity refers to a decrease in the level of CB2 activity (e.g. reduction in the activation of factors in the CB2 cell signaling pathway, such as inhibition of adenylyl cyclase activity and reduced cAMP levels, or phosphorylation of the inhibitory Tyr-505 of the leukocyte-specific protein tyrosine kinase (Lck)), as detected e.g. by ELISA or Western Blot.
  • CB2 activity e.g. reduction in the activation of factors in the CB2 cell signaling pathway, such as inhibition of adenylyl cyclase activity and reduced cAMP levels, or phosphorylation of the inhibitory Tyr-505 of the leukocyte- specific protein tyrosine kinase (Lck)
  • the reduction may be by at least a 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % or at least 99 % reduction.
  • Non-limiting examples of compounds capable of inhibiting a cannabinoid receptor CB2 expression are described hereinbelow.
  • Down-regulation at the nucleic acid level is typically effected using a nucleic acid agent, having a nucleic acid backbone, DNA, RNA, mimetics thereof or a combination of same (also referred to herein as a polynucleotide agent).
  • the nucleic acid agent may be encoded from a DNA molecule or provided to the cell per se.
  • the agent is typically administered to hematopoietic cells (e.g. immune cells) expressing the cannabinoid receptor CB2.
  • RNA silencing refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression] mediated by RNA molecules which result in the inhibition or "silencing" of the expression of a corresponding protein-coding gene.
  • RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.
  • RNA silencing agent refers to an RNA which is capable of specifically inhibiting or “silencing" the expression of a target gene.
  • the RNA silencing agent is capable of preventing complete processing (e.g., the full translation and/or expression) of an mRNA molecule through a post-transcriptional silencing mechanism.
  • RNA silencing agents include non-coding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated.
  • Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs.
  • the RNA silencing agent is capable of inducing RNA interference.
  • the RNA silencing agent is capable of mediating translational repression.
  • the RNA silencing agent is specific to the target RNA (e.g., cannabinoid receptor CB2) and does not cross inhibit or silence other targets or a splice variant which exhibits 99% or less global homology to the target gene, e.g., less than 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% global homology to the target gene; as determined by PCR, Western blot, Immunohistochemistry and/or flow cytometry.
  • target RNA e.g., cannabinoid receptor CB2
  • RNA interference refers to the process of sequence- specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs).
  • RNA silencing agents that can be used according to specific embodiments of the present invention.
  • DsRNA, siRNA and shRNA - The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer.
  • Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs).
  • Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes.
  • the RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single- stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex.
  • RISC RNA-induced silencing complex
  • some embodiments of the invention contemplate use of dsRNA to downregulate protein expression from mRNA.
  • dsRNA longer than 30 bp are used.
  • some embodiments of the invention contemplate use of siRNA to downregulate protein expression from mRNA.
  • siRNA refers to small inhibitory RNA duplexes (generally between 18-30 base pairs) that induce the RNA interference (RNAi) pathway.
  • RNAi RNA interference
  • siRNAs are chemically synthesized as 21 mers with a central 19 bp duplex region and symmetric 2-base 3 '-overhangs on the termini, although it has been recently described that chemically synthesized RNA duplexes of 25-30 base length can have as much as a lOO-fold increase in potency compared with 21 mers at the same location.
  • RNA silencing agent of some embodiments of the invention may also be a short hairpin RNA (shRNA).
  • RNA agent refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
  • the number of nucleotides in the loop is a number between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11. Some of the nucleotides in the loop can be involved in base-pair interactions with other nucleotides in the loop.
  • RNA silencing agents suitable for use with some embodiments of the invention can be effected using any method known in the art such as disclosed in PCT publication no. W02010137020, incorporated herein by reference in its entirety.
  • Potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (www(dot)ncbi(dot)nlm(dot)nih(dot)gov/BLAST/). Putative target sites which exhibit significant homology to other coding sequences are filtered out.
  • sequence alignment software such as the BLAST software available from the NCBI server (www(dot)ncbi(dot)nlm(dot)nih(dot)gov/BLAST/).
  • Qualifying target sequences are selected as template for siRNA synthesis.
  • Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55 %.
  • Several target sites are preferably selected along the length of the target gene for evaluation.
  • a negative control is preferably used in conjunction.
  • Negative control siRNA preferably includes the same nucleotide composition as the siRNAs but lack significant homology to the genome.
  • a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.
  • siRNAs directed against cannabinoid receptor CB2 can be the siRNA commercially available from Origene or from Santa Cruz Biotechnology (SCBT).
  • RNA silencing agent of some embodiments of the invention need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides.
  • RNA silencing agent may be a miRNA.
  • miRNA refers to a collection of non-coding single- stranded RNA molecules of about 19-28 nucleotides in length, which regulate gene expression. miRNAs are found in a wide range of organisms (viruses. fwdarw .humans) and have been shown to play a role in development, homeostasis, and disease etiology.
  • the target sites in the mRNA may be in the 5' UTR, the 3' UTR or in the coding region.
  • multiple miRNAs may regulate the same mRNA target by recognizing the same or multiple sites.
  • the presence of multiple miRNA binding sites in most genetically identified targets may indicate that the cooperative action of multiple RISCs provides the most efficient translational inhibition.
  • miRNAs may direct the RNA induced silencing complex (RISC) to downregulate gene expression by either of two mechanisms: mRNA cleavage or translational repression.
  • the miRNA may specify cleavage of the mRNA if the mRNA has a certain degree of complementarity to the miRNA. When a miRNA guides cleavage, the cut is typically between the nucleotides pairing to residues 10 and 11 of the miRNA.
  • the miRNA may repress translation if the miRNA does not have the requisite degree of complementarity to the miRNA. Translational repression may be more prevalent in animals since animals may have a lower degree of complementarity between the miRNA and binding site.
  • miRNA mimic refers to synthetic non-coding RNAs that are capable of entering the RNAi pathway and regulating gene expression. miRNA mimics imitate the function of endogenous miRNAs and can be designed as mature, double stranded molecules or mimic precursors (e.g., or pre-miRNAs). miRNA mimics can be comprised of modified or unmodified RNA, DNA, RNA-DNA hybrids, or alternative nucleic acid chemistries (e.g., LNAs or 2'-0,4'-C-ethylene-bridged nucleic acids (ENA)).
  • nucleic acid chemistries e.g., LNAs or 2'-0,4'-C-ethylene-bridged nucleic acids (ENA)
  • the length of the duplex region can vary between 13-33, 18-24 or 21-23 nucleotides.
  • the miRNA may also comprise a total of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides.
  • the sequence of the miRNA may be the first 13-33 nucleotides of the pre-miRNA.
  • the sequence of the miRNA may also be the last 13-33 nucleotides of the pre-miRNA.
  • Preparation of miRNAs mimics can be effected by any method known in the art such as chemical synthesis or recombinant methods.
  • contacting cells with a miRNA may be effected by transfecting the cells with e.g. the mature double stranded miRNA, the pre-miRNA or the pri-miRNA.
  • the pre-miRNA sequence may comprise from 45-90, 60-80 or 60-70 nucleotides.
  • the pri-miRNA sequence may comprise from 45-30,000, 50-25,000, 100-20,000, 1,000- 1,500 or 80-100 nucleotides.
  • suitable miRNAs directed against cannabinoid receptor CB2 can be the miRNA available from Origene.
  • Antisense - Antisense is a single stranded RNA designed to prevent or inhibit expression of a gene by specifically hybridizing to its mRNA. Downregulation of a cannabinoid receptor CB2 can be effected using an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding cannabinoid receptor CB2.
  • the first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells, while the second aspect is design of an oligonucleotide which specifically binds the designated mRNA within cells in a way which inhibits translation thereof.
  • the prior art teaches of a number of delivery strategies which can be used to efficiently deliver oligonucleotides into a wide variety of cell types [see, for example, Jaaskelainen et al. Cell Mol Biol Lett. (2002) 7(2):236-7; Gait, Cell Mol Life Sci. (2003) 60(5):844-53; Martino et al. J Biomed Biotechnol. (2009) 2009:410260; Grijalvo et al. Expert Opin Ther Pat. (2014) 24(7):80l- 19; Falzarano et al, Nucleic Acid Ther. (2014) 24(l):87-l00; Shilakari et al. Biomed Res Int. (2014) 2014: 526391; Prakash et al. Nucleic Acids Res. (2014) 42(l3):8796-807 and Asseline et al. J Gene Med. (2014) 16(7-8): 157-65] .
  • Nucleic acid agents can also operate at the DNA level. Downregulation of cannabinoid receptor CB2 can also be achieved by inactivating the gene (e.g., cannabinoid receptor CB2) via introducing targeted mutations involving loss-of function alterations (e.g. point mutations, deletions and insertions) in the gene structure, typically referred to as genome editing. Exemplary methods used to introduce nucleic acid alterations to a gene of interest and agents for implementing same include engineered endonucleases, meganucleases, ZFNs and TALENs, CRISPR-Cas system and Site-Specific Recombinases.
  • the phrase“loss-of-function alterations” refers to any mutation in the DNA sequence of a gene (e.g., cannabinoid receptor CB2) which results in downregulation of the expression level and/or activity of the expressed product, i.e., the mRNA transcript and/or the translated protein.
  • a gene e.g., cannabinoid receptor CB2
  • Non-limiting examples of such loss-of-function alterations include a missense mutation, i.e., a mutation which changes an amino acid residue in the protein with another amino acid residue and thereby abolishes the enzymatic activity of the protein; a nonsense mutation, i.e., a mutation which introduces a stop codon in a protein, e.g., an early stop codon which results in a shorter protein devoid of the enzymatic activity; a frame-shift mutation, i.e., a mutation, usually, deletion or insertion of nucleic acid(s) which changes the reading frame of the protein, and may result in an early termination by introducing a stop codon into a reading frame (e.g., a truncated protein, devoid of the enzymatic activity), or in a longer amino acid sequence (e.g., a readthrough protein) which affects the secondary or tertiary structure of the protein and results in a non functional protein, devoid of the enzymatic activity of
  • loss-of-function alteration of a gene comprises both alleles of the gene.
  • the agent capable of downregulating a cannabinoid receptor CB2 is an antibody or antibody fragment capable of specifically binding cannabinoid receptor CB2.
  • the antibody specifically binds at least one epitope of a cannabinoid receptor CB2.
  • epitopic determinants refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • antibody as used in this invention includes intact molecules as well as functional fragments thereof (such as Fab, F(ab')2, Fv, scFv, dsFv, or single domain molecules such as VH and VF) that are capable of binding to an epitope of an antigen.
  • Suitable antibody fragments for practicing some embodiments of the invention include a complementarity-determining region (CDR) of an immunoglobulin light chain (referred to herein as “light chain”), a complementarity-determining region of an immunoglobulin heavy chain (referred to herein as“heavy chain”), a variable region of a light chain, a variable region of a heavy chain, a light chain, a heavy chain, an Fd fragment, and antibody fragments comprising essentially whole variable regions of both light and heavy chains such as an Fv, a single chain Fv (scFv), a disulfide- stabilized Fv (dsFv), an Fab, an Fab’, and an F(ab’)2.
  • CDR complementarity-determining region
  • light chain referred to herein as “light chain”
  • a complementarity-determining region of an immunoglobulin heavy chain referred to herein as“heavy chain”
  • variable region of a light chain a variable region of a heavy chain
  • CDR complementarity-determining region
  • VH VH
  • CDR H2 or H2 CDR H3 or H3
  • VL VL
  • the identity of the amino acid residues in a particular antibody that make up a variable region or a CDR can be determined using methods well known in the art and include methods such as sequence variability as defined by Rabat et al. (See, e.g., Rabat et ah, 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C.), location of the structural loop regions as defined by Chothia et al.
  • variable regions and CDRs may refer to variable regions and CDRs defined by any approach known in the art, including combinations of approaches.
  • Fv defined as a genetically engineered fragment consisting of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) expressed as two chains;
  • scFv single chain Fv
  • dsFv disulfide-stabilized Fv
  • Fab a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme papain to yield the intact light chain and the Fd fragment of the heavy chain which consists of the variable and CH1 domains thereof;
  • Fab’ a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin, followed by reduction (two Fab’ fragments are obtained per antibody molecule);
  • F(ab’)2 a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin (i.e., a dimer of Fab’ fragments held together by two disulfide bonds); and
  • Single domain antibodies or nanobodies are composed of a single VH or VL domains which exhibit sufficient affinity to the antigen.
  • Antibody fragments according to some embodiments of the invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)].
  • Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker.
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et ah, Nature, 321:522-525 (1986); Riechmann et ah, Nature 332:323-327 (1988); Verhoeyen et ah, Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)].
  • the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boemer et al., J. Immunol., 147(1):86-95 (1991)].
  • human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
  • the antibody is a humanized antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is a humanized monoclonal antibody.
  • the cannabinoid receptor CB2 targeting antibodies are commercially available from e.g. Abcacm, OriGene, Santa Cruz Biotechnology (SCBT), Antibodies -online .
  • the cannabinoid receptor CB2 targeting antibody is H60 (discussed in detail in Savonenko et al., PLoS One. 2015; 10(6): e0l296l8).
  • aptamer refers to double stranded or single stranded RNA molecule that binds to specific molecular target, such as a protein.
  • Various methods are known in the art which can be used to design protein specific aptamers. The skilled artisan can employ SELEX (Systematic Evolution of Ligands by Exponential Enrichment) for efficient selection as described in Stoltenburg R, Reinemann C, and Strehlitz B (Biomolecular engineering (2007) 24(4):381-403).
  • cannabinoid receptor CB2 Another agent capable of downregulating cannabinoid receptor CB2 would be any molecule which binds to and/or cleaves cannabinoid receptor CB2. Such molecules can be a small molecule, cannabinoid receptor CB2 antagonists, or cannabinoid receptor CB2 inhibitory peptide.
  • the agent capable of downregulating cannabinoid receptor CB2 binds to at least one residue in transmembrane domain 3, 4 and/or 5 of cannabinoid receptor CB2.
  • the agent capable of downregulating cannabinoid receptor CB2 binds to at least one residue in extracellular loop 2 of cannabinoid receptor CB2.
  • the agent capable of downregulating cannabinoid receptor CB2 binds to at least one residue in transmembrane domain 4-extracellular loop 2-transmembrane domain 5 region of cannabinoid receptor CB2.
  • the agent capable of downregulating cannabinoid receptor CB2 binds to at least one residue in the transmembrane domain of cannabinoid receptor CB2 through hydrogen bonds and/or aromatic and hydrophobic interactions.
  • the agent capable of downregulating cannabinoid receptor CB2 binds to residue Ser 161 .
  • the agent capable of downregulating cannabinoid receptor CB2 binds to residue Ser 165 .
  • the agent capable of downregulating cannabinoid receptor CB2 binds to residue Cys 175 .
  • the agent capable of downregulating cannabinoid receptor CB2 binds to any or all of residues Ser 180 -Phe 183 .
  • the inhibitor of cannabinoid receptor CB2 activity is a CB2 receptor antagonist.
  • the CB2 receptor antagonist may be a true antagonist or inverse agonist, and includes any chemical entity that, upon administration to a subject, results in inhibition or down- regulation of a biological activity associated with activation of the CB2 receptor in the subject.
  • Such CB2 receptor antagonist includes any agent that can block CB2 receptor activation or any of the downstream biological effects of CB2 receptor activation.
  • a CB2 receptor antagonist can act by occupying the ligand binding site or a portion thereof of the CB2 receptor, thereby making the receptor inaccessible to its natural ligand so that its normal biological activity is prevented or reduced.
  • Exemplary inverse agonists which may be used according to the methods of the invention include, but are not limited to, SR144528, AM630, JTE-907, COR170, GPla, SCH336, RO6957022, AM1241 and Sch.4l43l9.
  • the inhibitor of cannabinoid receptor CB2 activity is SR144528.
  • the inhibitor of cannabinoid receptor binds to CB2 with a higher affinity compared to cannabinoid CB1 receptor (e.g. at least a 500-fold higher affinity).
  • the inhibitor of CB2 is a small molecule having an IC50 for CB2 ⁇ 8 mM, e.g. having an IC50 for CB2 in a range of 0.1 - 8 mM, e.g. 1 - 8 pM).
  • the agent capable of decreasing an activity or expression of cannabinoid receptor CB2 downregulates an activity or expression of cannabinoid receptor CB2 in hematopoietic cells (e.g. immune cells).
  • the methods of some embodiments of the invention can be performed within a subject ⁇ i.e., in vivo), within cells derived from a subject ⁇ i.e., ex vivo or in vitro) or within a hematopoietic stem cell line ⁇ i.e., in vitro).
  • an ex vivo method of enhancing lymphocyte recovery comprising ex vivo contacting hematopoietic stem and/or progenitor cells with an effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby enhancing lymphocyte recovery.
  • the method of ex vivo enhancing lymphocyte recovery is effected by contacting hematopoietic stem and/or progenitor cells with an inhibitor of a cannabinoid receptor CB2 expression and/or activity. Contacting may be effected for several minutes (e.g. about 5, 10, 15, 20, 30, 40, 50, 60, 70, 80 or 90 minutes) or for several hours (e.g. about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24hours).
  • the cell culture may further comprise tissue culture medium, supplemented with appropriate supplements e.g. L-glutamine, antibiotics, etc. according to known procedures (e.g., as described in the ATCC protocols).
  • appropriate supplements e.g. L-glutamine, antibiotics, etc. according to known procedures (e.g., as described in the ATCC protocols).
  • the hematopoietic stem and/or progenitor cells may be stored (e.g. for later use), expanded in culture or administered to a subject in need thereof (as discussed in detail herein).
  • the inhibitor of a cannabinoid receptor CB2 expression and/or activity of some embodiments of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
  • a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the inhibitor of a cannabinoid receptor CB2 expression and/or activity accountable for the biological effect.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • compositions of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (inhibitor of a cannabinoid receptor CB2 expression and/or activity) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., lymphocytopenia) or prolong the survival of the subject being treated.
  • a therapeutically effective amount means an amount of active ingredients (inhibitor of a cannabinoid receptor CB2 expression and/or activity) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., lymphocytopenia) or prolong the survival of the subject being treated.
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
  • Animal models such as immunocompetent mice, immunodeficient mice (e.g. SCID mice) or animal models for hematopoietic cancer (e.g. a B-CLL animal model such as the NOD-SCID mouse chimera as described previously by Shimoni A et al.
  • a model for human B-chronic lymphocytic leukemia in human/mouse radiation chimera: evidence for tumor-mediated suppression of antibody production in low-stage disease. Blood. 1997; 89:2210-2218 can be used to determine therapeutic efficacy of the agents of the present invention in vivo.
  • Dosage amount and interval may be adjusted individually to provide the active ingredient at a sufficient amount to induce or suppress the biological effect (minimal effective concentration, MEC).
  • MEC minimum effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
  • a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
  • Such notice for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
  • the method further comprises administering to the subject lymphocytes such as donor lymphocyte infusion (DLI).
  • the lymphocyte infusion includes T cells (e.g. CD8+ T cells, CD4+ T cells, B cells, monocytes, macrophages, and/or NK cells).
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • the term“treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • Cannabis Sativa and Indika extracts with high content in THC or CBD were supplied by Cannabliss (Cannabliss, Israel).
  • THC-BDS/CBD BDS i.e. THC-BDS/CBD BDS, respectively
  • Cannabliss Cannabliss, Israel
  • the THC, CBN and CBD contents of the extracts were quantified against a commercial THC, CBN and CBD standards (Izun Pharma, Israel).
  • SR144528 - a CB2 receptor antagonist was purchased from Abeam.
  • A967079 - a TRPA1 Receptor antagonist and BCTC - a TRPV1 Receptor antagonist were purchased from Alomone Labs, Israel.
  • GSK2193874 - a TRPV4 antagonist was purchased from SIGMA- ALDRICH, Israel.
  • GW9662 - a PPARy antagonist was purchased from Enzo Life Sciences, New York, USA.
  • mice Female 8- to l l-week-old C57BL/6 and BALB/c mice were purchased from Envigo, Jerusalem, Israel and CB2 knockout mice (CNR /_ ) were bred in the specific pathogen-free (SPF) facility of the Authority of Biological and Biomedical Models at the Hebrew University of Jerusalem. The study was approved by the Institutional Animal Care and Use Committee of the Hebrew University of Jerusalem in accordance with national laws and regulations for the protection of animals. Mice were housed under specific pathogen-free (SPF) conditions.
  • SPF pathogen-free
  • C57BL/6 or CB2 knockout mice underwent lethal whole-body irradiation by single exposure to 10 Gy and were reconstituted with 8 x 10 6 donor C57BL/6 or CB2 knockout BM cells the following day.
  • Cannabis extracts/cannabinoids (5 mg/kg) were administered intraperitoneally (IP), from the day of transplantation, every other day, for two weeks. Mice were monitored for weight loss. Once a week, blood was collected from the mice tail into Ethylenediaminetetraacetic acid (EDTA) coated capillary tubes. A complete blood count (CBC) with differentials was performed using a validated BC-2800Vet Auto Hematology Analyzer (Mindray).
  • mice underwent lethal whole-body irradiation by single exposure to 8 Gy and were reconstituted with 8 x 10 6 donor C57BL/6 BM cells and 2 x 10 6 spleen cells the following day.
  • Cannabis extracts/cannabinoids (5 mg/kg) were administered intraperitoneally (IP), from the day of transplantation, every other day, for two weeks.
  • IP intraperitoneally
  • mice were monitored daily for weight loss, diarrhea, ruffled skin, and survival.
  • GVHD score based on all of the aforementioned factors (rated on a scale of 0-4), was calculated as previously described (32).
  • mice underwent non-lethal whole -body irradiation by single exposure to 6 Gy. Once a week, blood was collected from the mice tail into EDTA coated capillary tubes. CBC with differentials was performed using a validated BC-2800Vet Auto Hematology Analyzer (Mindray). Lymphocyte activation assays
  • CFSE carboxyfluorescin diacetate succinimidyl ester
  • CBD is a stronger inhibitor of in vitro lymphocyte activation, as compared to THC
  • Cannabis extracts with high content of CBD or THC were named CBD BDS/ THC BDS (Botanical Drug Substance). These extracts were used, in addition to the pure cannabinoids, for two reasons: first, most of the patients are currently treated with cannabis based treatment and not with pure cannabinoids. Second, it was suggested that the combination of cannabinoids with other active molecules in the plant may have better results in medical use (known as the entourage effect) (15).
  • FIGS 1B-E Shown in Figures 1B-E are the results of 3 pg/ml treatment with pure cannabinoids and 10 pg/ml treatment with the cannabis extracts, containing approximately 30 % of the designated cannabinoid.
  • the results for IL-17 and IL-10 after treatment with various other concentrations are illustrated in Figure 6A-B.
  • CBD and THC have an inhibitory effect on lymphocyte activation, associated with reduction in the secretion of the inflammatory IL-17 cytokine and an elevation in the secretion of the regulatory cytokine IL-10.
  • THC and CBD affect lymphocyte activation by different mechanisms
  • CB2 knock-out mice CB2 knock-out mice
  • Figure 7A splenocytes extracted from CB2 KO mice were used ( Figure 7A) in a CFSE lymphocyte proliferation assay, similar to the assay in Figure 1A.
  • the inhibitory effect of THC BDS was maintained.
  • Cannabinoid treatment alters hematologic rehabilitation after bone marrow transplantation
  • a syngeneic transplantation model was utilized.
  • C57BL/6 mice underwent lethal whole-body irradiation and were reconstituted with 8 x 10 6 donor C57BL/6 BM cells the following day ( Figure 2A).
  • 5 mg/kg of Cannabis extracts/pure cannabinoids/vehicle were administered intraperitoneally (IP), from the day of transplantation, every other day, for two weeks. Once a week, starting one week after transplantation, blood was collected from the mice tail and CBC with differentials was performed.
  • IP intraperitoneally
  • CB2 receptor has an inhibitory effect on lymphocytes recovery
  • CB2 KO mice were used as donors/acceptors in BMT experiments.
  • the normal blood counts of CB2 KO female mice are similar to the WT C57BL/6 counts ( Figure 7B).
  • C57BL/6 mice underwent lethal whole-body irradiation and were reconstituted with 8 x 10 6 donor CB2 KO or C57BL/6 BM cells the following day.
  • a significantly higher lymphocyte count was found in the group that received CB2 KO transplant as compared to control, starting from the second week after transplantation (Figure 3B).
  • lymphocyte counts were not significantly different (Figure 3C).
  • mice underwent whole-body irradiation followed by allogeneic BMT from C57BL/6 donor mice. 5 mg/kg of Cannabis extracts/pure cannabinoids/vehicle were administered IP, from the day of transplantation, every other day, for two weeks (Figure 4A). Mice chimerism was not affected by the treatment ( Figures 9A-B). In this model, both CBD BDS and THC BDS significantly improved survival ( Figure 4B, right), while pure cannabinoids had a significant but smaller effect (Figure 4B, left). Moreover, GVHD scores were significantly lower in mice receiving cannabis extracts (Figure 4C).
  • CB2 has an inhibitory function in lymphocyte recovery in a model of treatment-induced lymphocytopenia without BMT
  • CB2 has an inhibitory role in lymphocyte recovery in treatment-induced lymphocytopenia which is not associated with BMT.
  • CBD Cannabidiol
  • Cannabis sativa The Plant of the Thousand and One Molecules. Front Plant Sci. 2016

Abstract

A method of treating a lymphocytopenia in a subject in need thereof is disclosed. The method comprising administering to the subject a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby treating the lymphocytopenia. A method of treating a lymphocytopenia associated with hematopoietic stem cell transplantation is also disclosed. A method of increasing lymphocyte count in a human subject is also disclosed.

Description

USE OF CB2 INHIBITORS FOR FYMPHOCYTE RECOVERY
RELATED APPLICATION
This application claims the benefit of priority from U.S. Provisional Patent Application No. 62/661,112 filed on 23 April 2018, the contents of which are incorporated herein by reference in their entirety.
FIELD AND BACKGROUND OF THE INVENTION
The present invention, in some embodiments thereof, relates to CB2 inhibitors and, more particularly, but not exclusively, to the use of same for lymphocyte recovery.
Lymphocytopenia, or lymphopenia, is the condition of having an abnormally low level of lymphocytes in the blood. Lymphopenia can be a major problem in patients with solid tumors treated with radiation and chemotherapy, in patients recovering from hematopoietic stem cell transplantation (HSCT), and in other medical conditions. Lymphopenia makes patients susceptible to various infections. An association between treatment-related lymphopenia (TRL) and decreased survival was demonstrated in patients.
HSCT is a well-established treatment for malignant and non-malignant hematological diseases. Allogeneic transplantation comes with the risk of Graft versus Host Disease (GVHD), a major cause of morbidity and mortality in HSCT patients. In addition, the toxicity of the conditioning protocol that precedes HSCT impairs innate and adaptive immunity, making transplanted patients very susceptible to both common and rare infections. The early post- engraftment period is categorized by a progressive recovery of cell mediated immunity, however, full reconstitution of the hematological components may take years.
In recent years, numerous publications have suggested the potential of cannabis-based medicines for the treatment of various conditions (4). Among the patients who can benefit from such treatment are HSCT patients, who often suffer from nausea and chronic pain. Cannabis contains numerous molecules, including more than 60 chemical compounds classified as cannabinoids and the different sub-strains vary in their cannabinoid content. Two cannabinoids have been the focus of most of the studies examining medical uses: D9 tetrahydrocannabinol (THC) and cannabidiol (CBD). THC and some of the other cannabinoids mediate their actions primarily through the Gi protein-coupled seven transmembrane cannabinoid receptors (members of the G-protein coupled receptor (GPCR) family): Cannabinoid receptor 1 (CB 1), which is mainly expressed in the brain and to some extent in peripheral tissues such as the immune tissues and Cannabinoid receptor type 2 (CB2), which is expressed at high density in immune cells. CB2 is primarily expressed by cells and progenitors of the immune system, it is expressed at very low levels by non-hematopoietic cells in the brain, is conserved between species and has a role in suppressing immune cell function [Turcotte C, et al. Cell Mol Life Sci. (2016) 73(23):4449-4470]. CBD has a very weak affinity to CB1 and CB2 (5). Several reports demonstrated CBD signaling through non cannabinoid receptor mechanisms, such as TRP channels and the nuclear receptor- Peroxisome Proliferator-Activated Receptor gamma (PPAR-g) (6, 7).
In addition to their effect on the nervous system, both fyto and the endogenous cannabinoids possess a wide range of anti-inflammatory properties as they induce the production of anti inflammatory cytokines such as IL-4, IL-5 and IL-10, and affect the differentiation and function of several immune cells (8, 9). The involvement of cannabinoid receptor signaling in the biology of hematopoietic stem and progenitor cells has also been reported (10, 11).
Additional background art includes PCT publication nos. WO/2004/113320, WO/2007/038036, WO/2012/116277; Morales et al., Expert Opinion on Therapeutic Patents (2016) 26:7, 843-856 and Pandey et al., J Pharm and Exper Therap (2011) 338(3): 819-828.
SUMMARY OF THE INVENTION
According to an aspect of some embodiments of the present invention there is provided a method of treating a lymphocytopenia in a subject in need thereof, wherein the lymphocytopenia is not associated with hematopoietic stem cell transplantation, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby treating the lymphocytopenia.
According to an aspect of some embodiments of the present invention there is provided a method of treating a lymphocytopenia associated with hematopoietic stem cell transplantation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby treating the lymphocytopenia associated with hematopoietic stem cell transplantation.
According to an aspect of some embodiments of the present invention there is provided a method of increasing lymphocyte count in a human subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby increasing the lymphocyte count.
According to an aspect of some embodiments of the present invention there is provided a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity for use in treating a lymphocytopenia in a subject in need thereof, wherein the lymphocytopenia is not associated with hematopoietic stem cell transplantation. According to an aspect of some embodiments of the present invention there is provided a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity for use in treating a lymphocytopenia associated with hematopoietic stem cell transplantation in a subject in need thereof.
According to an aspect of some embodiments of the present invention there is provided an ex vivo method of enhancing lymphocyte recovery, the method comprising ex vivo contacting hematopoietic stem and progenitor cells with an effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby enhancing lymphocyte recovery.
According to some embodiments of the invention, the method further comprises administering the hematopoietic stem and progenitor cells to a subject in need thereof.
According to some embodiments of the invention, the lymphocytopenia is not associated with transplantation of a solid organ or tissue.
According to some embodiments of the invention, the lymphocytopenia is associated with transplantation of a solid organ or tissue.
According to some embodiments of the invention, the lymphocytopenia is associated with administration of an anti-cancer therapy.
According to some embodiments of the invention, the lymphocytopenia is associated with administration of a chemotherapeutic agent, a radiation therapy or an immunotherapy.
According to some embodiments of the invention, the subject has a disease or condition selected from the group consisting of a malignant disease, a non-malignant tumor, an infection, an autoimmune disease, an aplastic anemia and a nuclear or radiation exposure.
According to some embodiments of the invention, the hematopoietic stem cell transplantation is used to treat a disease or condition selected from the group consisting of a hematologic malignancy, an aplastic anemia or an immune deficiency.
According to some embodiments of the invention, the hematopoietic stem cell transplantation is obtained from a source selected from the group consisting of bone marrow stem cells, peripheral blood stem cells and cord blood stem cells.
According to some embodiments of the invention, the hematopoietic stem cell transplantation comprises T cell depleted hematopoietic stem cells.
According to some embodiments of the invention, the inhibitor of the CB2 is administered to the subject prior to, concomitantly with and/or subsequent to the hematopoietic stem cell transplantation.
According to some embodiments of the invention, the hematopoietic stem cell transplantation is autologous with the subject. According to some embodiments of the invention, the hematopoietic stem cell transplantation is non-autologous with the subject.
According to some embodiments of the invention, the subject is monitored for graft versus host disease.
According to some embodiments of the invention, the inhibitor of the CB2 is a genome editing agent or an RNA silencing agent.
According to some embodiments of the invention, the inhibitor of the CB2 is an antagonist or an inverse agonist.
According to some embodiments of the invention, the inhibitor is selected from the group consisting of a small molecule, an antibody and an aptamer.
According to some embodiments of the invention, the inhibitor (e.g. antagonist or inverse agonist) is selected from the group consisting of SR144528, AM630, JTE-907, COR170, GPla, SCH336, RO6957022 and AM 1241.
According to some embodiments of the invention, the lymphocytopenia comprises T lymphocytopenia, B lymphocytopenia and/or NK lymphocytopenia.
According to some embodiments of the invention, the lymphocytopenia is determined when a complete blood count of the subject shows a lymphocyte count lower than the age-appropriate reference.
According to some embodiments of the invention, the subject is a human subject.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced. In the drawings:
FIGs. 1A-E are graphs illustrating the influence of pure CBD/THC and cannabis extracts on lymphocyte activation. (Figure 1A) Proliferation of CFSE stained, CD3 activated, murine splenocytes was analyzed using flow cytometry analysis. Summary of a minimum of 6 independent experiments is provided. Results are expressed as mean. Quantification of IL-l7a (Figure 1B), IL-10 (Figure 1C), TNF-a (Figure 1D) and IL-5 (Figure 1E) secretion was performed using enzyme-linked immunosorbent assay on culture medium of activated cells. Results are expressed as mean + SEM. Data are summarized from a minimum of 5 independent experiments p value * < 0.05; ** < 0.001; *** < 0.0001.
FIGs. 1F-G are graphs illustrating that THC and CBD affect lymphocyte activation by different mechanisms. (Figure 1F) Proliferation of CFSE stained, CD3 activated, splenocytes from CB2 knockout mice was analyzed using flow cytometry analysis. Summary of a minimum of 3 independent experiments is provided. Results are expressed as mean. Of note, the differences of CBD, THC BDS (Botanical Drug Substance) and CBD BDS as compared to control are significant starting from 3 pg/ml. The differences of THC as compared to control are significant starting from 10 pg/ml. (Figure 1G) The influence of PPARy antagonist, GW9662, on CBD’s effect on lymphocyte activation. Proliferation of CFSE stained, CD3 activated, murine splenocytes was analyzed using flow cytometry analysis. Summary of a minimum of 8 independent experiments is provided. Results are expressed as mean + SEM. p value - samples were compared to activated splenocytes + CBD (right) or activated splenocytes (left) p value * < 0.05; ** < 0.001; *** < 0.0001.
FIGs. 2A-E illustrate administration of cannabis/cannabinoids in a syngeneic BMT model. (Figure 2A) Recipient C57BL/6 mice received lethal whole-body irradiation and were reconstituted with 8 x 106 donor C57BL/6 bone marrow cells. Cannabis/cannabinoids were administered IP every other day, for two weeks from the day of transplantation. Blood samples for CBC were obtained once a week. Lymphocyte counts in pure cannabinoid treated groups (Figure 2B) and BDS treated groups (Figure 2C) are presented. Average counts at different time points (left) and day 21 after transplantation counts (right). Platelet counts in pure cannabinoid treated groups (Figure 2D) and BDS treated groups (Figure 2E), day 14 after transplantation. Data are summarized from a minimum of 3 independent experiments p value * < 0.05; ** < 0.001; *** < 0.0001.
FIGs. 3A-C are graphs illustrating the role of CB2 in lymphocyte recovery. (Figure 3 A) Recipient C57BL/6 mice underwent syngeneic BMT. CB2 inverse agonist SR144528, was administered IP once a day for one week from the day of transplantation. Mice were monitored for weight loss. Blood samples were obtained once a week. Average lymphocyte counts are provided. The average percent of initial weight during the experiment is provided. No signs of toxicity were evident. (Figure 3B) Syngeneic BMT from CB2 KO donor mice to C57BL/6 WT mice. Average counts at different time points (left) and day 21 after transplantation counts (right). (Figure 3C) Syngeneic BMT from C57BL/6 WT donor mice to CB2 KO mice. Average counts at different time points (left) and day 21 after transplantation counts (right). Data are summarized from a minimum of 3 independent experiments p value * < 0.05; ** < 0.001; *** < 0.0001.
FIGs. 4A-C illustrate cannabis/cannabinoids administration for GVHD prophylaxis. (Figure 4A) Recipient BALB/c mice received lethal whole-body irradiation and were reconstituted with 8 x 106 donor C57BL/6 bone marrow cells and 2 x 106 spleen cells. Cannabis/cannabinoids were administered IP every other day, for two weeks from the day of transplantation. The clinical condition of the mice was evaluated for up to 67 days after transplantation. (Figure 4B) Survival curve. Of note, the differences between control and THC BDS as well as CBD BDS are significant. (Figure 4C) Average GVHD score (Days 15-26). Of note, the differences between THC BDS / CBD BDS and control group are significant.
FIGs. 5A-B are graphs illustrating the influence of pure CBD/THC and cannabis extracts on lymphocyte activation. Proliferation of CFSE stained, CD3 activated, lymphocytes was analyzed using flow cytometry analysis. Summary of a minimum of 3 independent experiments is provided. Results are expressed as mean. (Figure 5A) Balb/C murine splenocytes, (Figure 5B) Human PBMC.
FIGs. 6A-B are graphs illustrating the influence of pure CBD/THC and cannabis extracts on lymphocyte activation. The levels of cytokines (Figure 6A - IL-17a and Figure 6B - IL-10) in the culture medium of CD3 activated, lymphocytes were analyzed using ELISA assay. Summary of a minimum of 3 independent experiments are provided. Results are expressed as mean.
FIGs. 7A-B illustrate CB2 KO mice (CNR /_). (Figure 7A) PCR results for CNR gene. (Figure 7B) Complete Blood counts (CBC) of CB2KO and C57B1/6, age matched, female mice n=9 per group. Results are expressed as mean + SEM.
FIGs. 8A-C are graphs illustrating the influence of TRP antagonists on CBD’s effect on lymphocyte activation. Proliferation of CFSE stained, CD3 activated, murine splenocytes was analyzed using flow cytometry analysis. (Figure 8A) A967079 antagonist to TRPA1. (Figure 8B) BCTC antagonist to TRPV1. (Figure 8C) GSK2193874 antagonist to TRPV4. Summary of a minimum of 2 independent experiments is provided. Results are expressed as mean + SEM. p value - samples were compared to activated splenocytes + CBD (right) or activated splenocytes (left).
FIGs. 9A-B illustrate blood chimerism in allogeneic transplanted mice. (Figure 9A) example for FACS analysis of blood cells stained with anti-H2Db and anti-H2Dd antibodies. (Figure 9B) Average blood chimerism is provided. Data are summarized from 2 independent experiments. FIGs. 10A-D are graphs illustrating the role of CB2 in lymphocyte recovery in a model of treatment-induced lymphocytopenia without bone marrow transplantation. C57BL/6 and CB2 KO mice received non-lethal 6 Gy whole -body irradiation. (Figures 10A-B) Average blood counts (CBC) at different time points (Figure 10B) and day 21 after irradiation treatment (Figure 10A). (Figure 10C) Average Lymphocytes percentage in C57BL/6 and CB2 KO mice post non-lethal irradiation. (Figure 10D) granulocytes percentage in C57BL/6 and CB2 KO mice post non-lethal irradiation. Data are summarized from 2 independent experiments p value ** < 0.001; *** < 0.0001.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The present invention, in some embodiments thereof, relates to CB2 inhibitors and, more particularly, but not exclusively, to the use of same for lymphocyte recovery.
The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
Hematopoietic stem cell transplantation (HSCT) is a well-established treatment for malignant and non-malignant hematological diseases. However, the toxicity of the conditioning protocol which precedes HSCT impairs innate and adaptive immunity, making transplanted patients very susceptible to various infections. The early post-engraftment period is categorized by progressive recovery of cell mediated immunity. However, full reconstitution of the hematological components may take years.
Two cannabinoids have been subjects of most of the studies examining medical uses: THC and CBD. THC mediates its action primarily through the cannabinoid receptors: CB1, which is mainly expressed in the brain and to some extent in peripheral tissues such as the immune tissues, and CB2, which is expressed at high density in immune cells. Although evidence has been collected regarding the influence of cannabis and cannabinoids on the immune system, the effect of these drugs on rehabilitation of the hematologic system with or without HSCT remains unknown.
While reducing the present invention to practice, the present inventor has uncovered an association between cannabinoid treatment (THC or CBD) and inhibition of lymphocyte reconstitution. THC related inhibition of lymphocyte recovery was mediated via CB2 receptor and in turn the use of a CB2 antagonist significantly improved lymphocyte reconstitution.
As is shown herein below and in the Examples section which follows, the present inventor has compared the influence of pure THC, pure CBD and high THC/high CBD cannabis extracts treatment in murine bone marrow transplantation (BMT) models. It is shown that all of the tested cannabinoid treatments reduced lymphocyte proliferation in vitro (Example 1 of the examples section which follows) as well as lymphocyte recovery in vivo (Example 3 of the examples section which follows). However, while both CBD and THC affect lymphocyte proliferation, they utilize different signal transduction pathways to cause these effects. Thus, while THC signals through CB2 receptor, CBD signals through PPAR-g (Example 2 of the examples section which follows). Moreover, CB2 was found to be directly associated with inhibition of lymphocyte recovery, as shown that using CB2 knock-out mice as a syngeneic transplant model, improved lymphocyte recovery (Example 4 of the examples section which follows). Likewise, the use of CB2 inverse agonist improved lymphocyte recovery after syngeneic bone marrow transplantation (Example 4 of the examples section which follows). These results were specific for lymphocytes, as other hematopoietic cell populations including monocytes, granulocytes and platelets where not significantly affected by these treatments (Example 3 of the examples section which follows). Similar results were evident for lymphocyte recovery in the absence of BMT, where it was illustrated that CB2 knock-out mice had a significantly faster lymphocyte reconstitution evident 9 days after radiation therapy (Figures 10A-B). In addition, faster normalization of lymphocytes/granulocyte ratio was evident in CB2 KO mice as compared to control mice undergoing the same treatment (Figures 10C-D). Taken together, these results substantiate the use of CB2 inhibitors for lymphocyte reconstitution in subjects undergoing lymphocytopenia associated and not associated with transplantation.
Thus, according to one aspect of the present invention there is provided a method of treating a lymphocytopenia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby treating the lymphocytopenia.
According to one aspect, there is provided a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity for use in treating a lymphocytopenia in a subject in need thereof.
According to one aspect, there is provided a method of treating a lymphocytopenia in a subject in need thereof, wherein the lymphocytopenia is not associated with hematopoietic stem cell transplantation, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby treating the lymphocytopenia.
According to one aspect, there is provided a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity for use in treating a lymphocytopenia in a subject in need thereof, wherein the lymphocytopenia is not associated with hematopoietic stem cell transplantation.
According to one aspect, there is provided a method of treating a lymphocytopenia associated with hematopoietic stem cell transplantation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby treating the lymphocytopenia associated with hematopoietic stem cell transplantation.
According to one aspect, there is provided a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity for use in treating a lymphocytopenia associated with hematopoietic stem cell transplantation in a subject in need thereof.
According to one aspect, there is provided a method of increasing lymphocyte count in a human subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby increasing the lymphocyte count.
As used herein, the term“treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
As used herein, the term "subject" or“subject in need thereof’ refers to a mammal, preferably a human being, male or female at any age or gender that has lymphocytopenia. Typically the subject has a lymphocytopenia due to a disorder or a pathological or undesired condition, state, or syndrome, or due to a therapeutic modality all of which cause acute or chronic lymphocytopenia. Examples of such disorders are provided further below.
The term "lymphocytopenia" as used herein refers to the condition of having an abnormally low level of lymphocytes (T lymphocytes, B lymphocytes, and/or natural killer cells) in the blood.
Lymphocytopenia is typically determined when a complete blood count (CBC) of the subject shows a lymphocyte count lower than the age- appropriate reference. Accordingly, in adults (i.e. over 20 years old), lymphocytopenia refers to a count of less than 1,000 lymphocytes per microliter of blood. In children lymphocyte counts vary with age. For children up to 5 months old, lymphocytopenia refers to a count of less than 2,000 lymphocytes per microliter of blood. For children of age 6 months to 2 years, lymphocytopenia refers to a count of less than 4,000 lymphocytes per microliter of blood. For children of age 2-3 years, lymphocytopenia refers to a count of less than 3,000 lymphocytes per microliter of blood. For children of age 4-9 years, lymphocytopenia refers to a count of less than 2,000 lymphocytes per microliter of blood. For children of age 10-20 years, lymphocytopenia refers to a count of less than 1,500 lymphocytes per microliter of blood.
According to one embodiment, the lymphocytopenia comprises T lymphocytopenia (i.e. abnormally low level of T lymphocytes, e.g. CD3+ cells, CD2+ cells, CD8+ cells, CD4+ cells, a/b T cells and/or g/d T cells).
According to one embodiment, the lymphocytopenia comprises B lymphocytopenia (i.e. abnormally low level of B lymphocytes).
According to one embodiment, the lymphocytopenia comprises NK lymphocytopenia (i.e. abnormally low level of natural killer cells).
The methods of the present invention can also be used to increase lymphocyte counts in a subject having low lymphocyte counts and not having lymphocytopenia (e.g. adults having lymphocyte counts higher than 1,000 lymphocytes per microliter of blood but lower than 2000 lymphocytes per microliter of blood).
According to one embodiment, as a result of the treatment described herein, the lymphocyte counts in the subject increase by at least about 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 100 % or more, as compared to the lymphocyte counts prior to treatment.
Lymphocyte counts can be determined by any method known in the art, such as by standard blood test (e.g. complete blood counts (CBC)).
Lymphocytopenia may be caused by disease progression (e.g. cancer, infection, autoimmune disease) or by disease treatment (e.g. ultraviolet A irradiation, steroid treatment, chemotherapy, or hematopoietic stem cell transplantation) as discussed below.
According to one embodiment, lymphocytopenia is associated with various diseases and conditions. For example, lymphocytopenia may be associated with a malignant disease, with a non- malignant tumor, with an infection, with an autoimmune disease, with aplastic anemia, with an immune deficiency and/or with a nuclear or radiation exposure.
According to one embodiment, lymphocytopenia is associated with a non-malignant tumor or lesion (benign tumor that is not a malignant).
According to some embodiments of the invention, the non-malignant tumor includes, but is not limited to, an adenoma, a lipoma, a fibroma and a myoma. According to one embodiment, lymphocytopenia is associated with a malignant disease (also termed cancer).
According to some embodiments of the invention, the malignant disease is a solid tumor or tumor metastasis.
According to some embodiments of the invention, the malignant disease is a carcinoma, a lymphoma, a blastoma, a sarcoma, and a leukemia.
According to some embodiments of the invention, the malignant disease is a hematological malignancy.
According to some embodiments of the invention, the hematological malignancy comprises a leukemia or a lymphoma.
Exemplary hematological malignancies include, but are not limited to, leukemia [e.g., acute lymphatic, acute lymphoblastic, acute lymphoblastic pre-B cell, acute lymphoblastic T cell leukemia, acute - megakaryoblastic, monocytic, acute myelogenous, acute myeloid, acute myeloid with eosinophilia, B cell, basophilic, chronic myeloid, chronic, B cell, eosinophilic, Friend, granulocytic or myelocytic, hairy cell, lymphocytic, megakaryoblastic, monocytic, monocytic- macrophage, myeloblastic, myeloid, myelomonocytic, plasma cell, pre-B cell, promyelocytic, subacute, T cell, lymphoid neoplasm, predisposition to myeloid malignancy, acute nonlymphocytic leukemia, T-cell acute lymphocytic leukemia (T-ALL) and B-cell chronic lymphocytic leukemia (B- CLL)] and lymphoma [e.g., Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt, cutaneous T cell, histiocytic, lymphoblastic, T cell, thymic, B cell, including low grade/follicular; small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia] .
According to some embodiments of the invention, the malignant disease is a myeloproliferative disease, such as but not limited to, Solid tumors, Benign Meningioma, Mixed tumors of salivary gland, Colonic adenomas; Adenocarcinomas, such as Small cell lung cancer, Kidney, Uterus, Prostate, Bladder, Ovary, Colon, Sarcomas, Liposarcoma, myxoid, Synovial sarcoma, Rhabdomyosarcoma (alveolar), Extraskeletel myxoid chonodro sarcoma, Ewing's tumor; Other cancer types include Testicular and ovarian dysgerminoma, Retinoblastoma, Wilms' tumor, Neuroblastoma, Malignant melanoma, Mesothelioma, Myeloma, Colorectal cancer, Breast cancer, Skin cancer, Pancreatic cancer, Prostate cancer, Esophageal cancer and Ovarian cancer. According to one embodiment, lymphocytopenia is associated with an aplastic anemia, including but not limited to, acquired aplastic anaemia and inherited bone marrow failure syndromes (e.g. Fanconi anaemia).
According to one embodiment, lymphocytopenia is associated with an immune deficiency, including but not limited to, severe combined immunodeficiency syndromes (SCID), X-linked agammaglobulinemia (XLA), common variable immunodeficiency (CVID), and AIDS.
According to one embodiment, lymphocytopenia is associated with a viral, a bacterial, a parasitic, or a fungal infection. Exemplary infections include, but are not limited to, AIDS, viral hepatitis, tuberculosis, typhoid fever, histoplasmosis, influenza or malaria.
According to one embodiment, lymphocytopenia is associated with an autoimmune disease, such as but not limited to, psoriasis or lupus.
According to one embodiment, lymphocytopenia is associated with a nuclear or radiation exposure e.g. ionizing radiation including, but not limited to, cosmic, alpha, beta, gamma or X- rays, or neutrons. Nuclear or radiation exposure typically refers to large doses of ionizing radiation (e.g. 0.25 - 4 Gy) which can lead to Acute Radiation Syndrome (ARS) i.e. radiation poisoning.
According to one embodiment, lymphocytopenia is associated with treatment of the disease or disorder. Accordingly, lymphocytopenia may be instigated directly by the disease or condition (e.g. immune deficiency, hematopoietic malignancy, infection, radiation exposure). Additionally or alternatively, lymphocytopenia may be instigated by a treatment modality associated with the disease or condition (e.g. chemotherapy, immunotherapy, radiation therapy, immunosuppression, transplantation of a cell or tissue graft), as discussed below.
According to a specific embodiment, lymphocytopenia is associated with a hematopoietic stem cell transplantation employed for treatment of a malignancy (e.g. hematological malignancy, e.g. leukemia or lymphoma), aplastic anemia, immune deficiency (e.g. SCID), infection (e.g. viral infection) or autoimmune disease.
According to one embodiment, lymphocytopenia is associated with an anti-cancer drug. Anti-cancer drugs include, but are not limited to Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflomithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-nl; Interferon Alfa-n3; Interferon Beta- 1 a; Interferon Gamma- 1 b; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimu stine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Taxol; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofuirin; Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride. Additional antineoplastic agents include those disclosed in Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner), and the introduction thereto, 1202-1263, of Goodman and Gilman's "The Pharmacological Basis of Therapeutics", Eighth Edition, 1990, McGraw-Hill, Inc. (Health Professions Division).
According to one embodiment, lymphocytopenia is associated with chemotherapy, immunotherapy or radiation therapy. Exemplary chemotherapy which may cause lymphocytopenia include, but are not limited to, Abarelix, Actinomycin D, Aldesleukin, Aldesleukin, Alemtuzumab, Alitretinoin, Allopurinol, Altima (Pemetrexed), Altretamine, Amifostine, Anastrozole, Arsenic trioxide, Asparaginase, Azacitidine, Bevacuzimab, Bexarotene, Bleomycin, Bortezomib, Busulfan, Calusterone, Capecitabine, Carboplatin, Carmustine, Celecoxib, Cetuximab, Cisplatin, Cladribine, Clofarabine, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin, Darbepoetin alfa, Darbepoetin alfa, Daunorubicin liposomal, Daunorubicin, Decitabine, Denileukin diftitox, Dexrazoxane, Dexrazoxane, Docetaxel, Doxorubicin, Dromostanolone propionate, Elliott's B Solution, Epirubicin, Epoetin alfa, Erlotinib, Estramustine, Etoposide, Exemestane, Filgrastim, Floxuridine, Fludarabine, Fluorouracil 5-FU, Fulvestrant, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Goserelin acetate, Histrelin acetate, Hydroxyurea, Ibritumomab Tiuxetan, Idarubicin, Ifosfamide, Imatinib mesylate, Interferon alfa-2a, Interferon alfa- 2b, Irinotecan, Lenalidomide, Letrozole, Leucovorin, Leuprolide Acetate, Levamisole, Lomustine, CCNET, Meclorethamine, Nitrogen mustard, Megestrol acetate, Melphalan, L-PAM, Mercaptopurine 6-MP, Mesna, Methotrexate, Mitomycin C, Mitotane, Mitoxantrone, Nandrolone phenpropionate, Nelarabine, Nofetumomab, Oprelvekin, Oprelvekin, Oxaliplatin, Paclitaxel, Palifermin, Pamidronate, Pegademase, Pegaspargase, Pegfilgrastim, Pemetrexed disodium, Pentostatin, Pipobroman, Plicamycin mithramycin, Porfimer sodium, Procarbazine, Quinacrine, Rasburicase, Rituximab, Sargramostim, Sorafenib, Streptozocin, Sunitinib maleate, Tamoxifen, Temozolomide, Teniposide VM-26, Testolactone, Thioguanine 6-TG, Thiotepa, Topotecan, Toremifene, Tositumomab, Treosulfan, Trastuzumab, Tretinoin ATRA, Uracil Mustard, Valrubicin, Vinblastine, Vinorelbine, Zoledronate and Zoledronic acid.
Exemplary immunotherapy which may cause lymphocytopenia include, but are not limited to, monoclonal antibodies (e.g. Alemtuzumab, Trastuzumab, Ibritumomab tiuxetan, Brentuximab vedotin, Ado-trastuzumab emtansine, Denileukin diftitox, Blinatumomab, Bevacizumab and Cetuximab), Immune checkpoint inhibitors (e.g. Pembrolizumab, Nivolumab, Cemiplimab, Atezolizumab, Avelumab, Durvalumab, Ipilimumab), interleukins (e.g. IL-2), interferons (e.g. IFN- alfa, IFN-beta, IFN-gamma), immunomodulating drugs (e.g. Thalidomide, Lenalidomide, and Pomalidomide).
According to one embodiment, lymphocytopenia is associated with irradiation therapy.
According to one embodiment, lymphocytopenia is associated with total body irradiation
(TBI).
According to one embodiment, the TBI comprises a single or fractionated irradiation dose within the range of 0.25-0.5 Gy, 0.25-1 Gy, 0.25-2.5 Gy, 0.25-5 Gy, 0.25-10 Gy, 0.5-1 Gy, 0.5- 1.5 Gy, 0.5-2.5 Gy, 0.5-5 Gy, 0.5-7.5 Gy, 0.5-10 Gy, 0.5-15 Gy, 1-1.5 Gy, 1-2 Gy, 1-2.5 Gy, 1-3 Gy, 1- 3.5 Gy, 1-4 Gy, 1-4.5 Gy, 1-1.5 Gy, 1-7.5 Gy, 1-10 Gy, 2-3 Gy, 2-4 Gy, 2-5 Gy, 2-6 Gy, 2-7 Gy, 2- 8 Gy, 2-9 Gy, 2-10 Gy, 3-4 Gy, 3-5 Gy, 3-6 Gy, 3-7 Gy, 3-8 Gy, 3-9 Gy, 3-10 Gy, 4-5 Gy, 4-6 Gy, 4-7 Gy, 4-8 Gy, 4-9 Gy, 4-10 Gy, 5-6 Gy, 5-7 Gy, 5-8 Gy, 5-9 Gy, 5-10 Gy, 6-7 Gy, 6-8 Gy, 6-9 Gy, 6-10 Gy, 7-8 Gy, 7-9 Gy, 7-10 Gy, 8-9 Gy, 8-10 Gy, 10-12 Gy or 10-15 Gy.
According to one embodiment, lymphocytopenia is associated with total lymphoid irradiation (TLI, i.e. exposure of all lymph nodes, the thymus, and spleen).
According to a specific embodiment, the TLI comprises an irradiation dose within the range of 0.25-0.5 Gy, 0.25-1 Gy, 0.25-2.5 Gy, 0.25-5 Gy, 0.25-10 Gy, 0.5-1 Gy, 0.5-1.5 Gy, 0.5-2.5 Gy, 0.5-5 Gy, 0.5-7.5 Gy, 0.5-10 Gy, 0.5-15 Gy, 1-1.5 Gy, 1-2 Gy, 1-2.5 Gy, 1-3 Gy, 1-3.5 Gy, 1-4 Gy,
1-4.5 Gy, 1-1.5 Gy, 1-7.5 Gy, 1-10 Gy, 2-3 Gy, 2-4 Gy, 2-5 Gy, 2-6 Gy, 2-7 Gy, 2-8 Gy, 2-9 Gy, 2- 10 Gy, 3-4 Gy, 3-5 Gy, 3-6 Gy, 3-7 Gy, 3-8 Gy, 3-9 Gy, 3-10 Gy, 4-5 Gy, 4-6 Gy, 4-7 Gy, 4-8 Gy, 4-9 Gy, 4-10 Gy, 5-6 Gy, 5-7 Gy, 5-8 Gy, 5-9 Gy, 5-10 Gy, 6-7 Gy, 6-8 Gy, 6-9 Gy, 6-10 Gy, 7-8 Gy, 7-9 Gy, 7-10 Gy, 8-9 Gy, 8-10 Gy, 10-12 Gy, 10-15 Gy, 10-20 Gy, 10-30 Gy, 10-40 Gy, 10-50 Gy, 0.5-20 Gy, 0.5-30 Gy, 0.5-40 Gy or 0.5-50 Gy.
According to one embodiment, lymphocytopenia is associated with partial body irradiation (e.g. specific exposure of the lungs, kidney, brain etc.).
According to a specific embodiment, the partial body irradiation comprises an irradiation dose within the range of 0.25-0.5 Gy, 0.25-1 Gy, 0.25-2.5 Gy, 0.25-5 Gy, 0.25-10 Gy, 0.5-1 Gy, 0.5- 1.5 Gy, 0.5-2.5 Gy, 0.5-5 Gy, 0.5-7.5 Gy, 0.5-10 Gy, 0.5-15 Gy, 1-1.5 Gy, 1-2 Gy, 1-2.5 Gy, 1-3
Gy, 1-3.5 Gy, 1-4 Gy, 1-4.5 Gy, 1-1.5 Gy, 1-7.5 Gy, 1-10 Gy, 2-3 Gy, 2-4 Gy, 2-5 Gy, 2-6 Gy, 2-7 Gy, 2-8 Gy, 2-9 Gy, 2-10 Gy, 3-4 Gy, 3-5 Gy, 3-6 Gy, 3-7 Gy, 3-8 Gy, 3-9 Gy, 3-10 Gy, 4-5 Gy, 4- 6 Gy, 4-7 Gy, 4-8 Gy, 4-9 Gy, 4-10 Gy, 5-6 Gy, 5-7 Gy, 5-8 Gy, 5-9 Gy, 5-10 Gy, 6-7 Gy, 6-8 Gy, 6-9 Gy, 6-10 Gy, 7-8 Gy, 7-9 Gy, 7-10 Gy, 8-9 Gy, 8-10 Gy, 10-12 Gy, 10-15 Gy, 10-20 Gy, 10-30 Gy, 10-40 Gy, 10-50 Gy, 0.5-20 Gy, 0.5-30 Gy, 0.5-40 Gy or 0.5-50 Gy.
According to one embodiment, lymphocytopenia is associated with administration of an immunosuppressive drug. Immunosuppressive drugs include, but are not limited to, Calcineurin Inhibitors, Antiproliferative agents, mTOR inhibitors and Steroids.
Examples of immunosuppressive drugs include, but are not limited to, Tacrolimus (also referred to as FK-506 or fujimycin, trade names: Prograf, Advagraf, Protopic), Mycophenolate Mofetil, Mycophenolate Sodium, Prednisone, methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine ( sulphas alazopyrine), gold salts, D- penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE), etanercept, TNF.alpha. blockers, a biological agent that targets an inflammatory cytokine, and Non-Steroidal Anti-Inflammatory Drug (NSAIDs). Examples of NSAIDs include, but are not limited to acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, tramadol, rapamycin (sirolimus) and rapamycin analogs (such as CCI-779, RAD001, AP23573). These agents may be administered individually or in combination.
According to one embodiment, lymphocytopenia is associated with a combination of any of the above described therapeutic protocols (e.g. chemotherapeutic agent and TBI, chemotherapeutic agent and TLI, antibody immunotherapy and chemotherapeutic agent, etc.).
According to one embodiment, the lymphocytopenia is not associated with transplantation of a cell or tissue graft.
According to one embodiment, the lymphocytopenia is not associated with transplantation of hematopoietic stem cells.
According to one embodiment, lymphocytopenia is associated with transplantation of a cell or tissue graft.
As used herein, the phrase“cell or tissue graft” refers to a bodily cell (e.g. a single cell or a group of cells) or tissue (e.g. solid tissues or soft tissues, which may be transplanted in full or in part). Exemplary tissues which may be transplanted according to the present teachings include, but are not limited to, liver, pancreas, spleen, kidney, heart, lung, skin, intestine and lymphoid/hematopoietic tissues (e.g. lymph node, Peyer’s patches, thymus or bone marrow). Exemplary cells which may be transplanted according to the present teachings include, but are not limited to, immature hematopoietic cells including stem cells. The present invention also contemplates transplantation of whole organs (e.g. solid organs), such as for example, kidney, heart, lung, liver, pancreas, intestine or spleen.
According to one embodiment, the cell or tissue graft comprises co-transplantation of more than one cell or tissue type (e.g. co-transplantation of hematopoietic stem cells and a solid organ or tissue e.g. kidney, liver, lung etc. or co-transplantation of a number of solid organs or tissues e.g. liver and intestine, lung and heart, etc.).
According to one embodiment, the hematopoietic stem cells and the solid organ or obtained from the same donor.
According to one embodiment, the cell or tissue graft does not comprise hematopoietic stem cells.
According to one embodiment, the cell or tissue graft comprises hematopoietic stem cells. According to one embodiment, the cell or tissue graft comprises hematopoietic progenitor cells.
According to one embodiment, the hematopoietic stem cell transplantation is used to treat a disease or condition selected from the group consisting of a hematologic malignancy, an aplastic anemia, a viral infection, or an immune deficiency.
As used herein the phrase“hematopoietic stem cells” or“HSCs” refers to a hematopoietic tissue or cell preparation comprising precursor hematopoietic cells (e.g. immature hematopoietic cells). Such tissue/cell preparation includes or is derived from a biological sample, for example, bone marrow, mobilized peripheral blood (e.g. mobilization of CD34 cells to enhance their concentration), cord blood (e.g. umbilical cord), fetal liver, yolk sac and/or placenta. Additionally, purified CD34+ cells or other hematopoietic stem cells such as CD131+ cells can be used in accordance with the present teachings, either with or without ex-vivo expansion.
Hematopoietic stem cells typically have the capacity to self-renew (i.e. expand) and to differentiate into progenitor cells, which are more developmentally committed to a cell line than are stem cells, but which are nevertheless undifferentiated or immature in comparison to those cells that have differentiated and matured into specialized cells. Progenitor cells may be identified by expression of a cell marker including, but not limited to, CD45, CD34, CD38, SCA1, CD59, CD90/Thyl and/or IL-3 R alpha.
Accordingly, the term hematopoietic stem and progenitor cells may relate to hematopoietic stem cells, to progenitor cells or to a combination of these cells.
Accordingly, hematopoietic stem cells can be committed to a particular line of differentiation, e.g. common myeloid progenitors (CMP), common lymphoid progenitors (CLP), Colony-Forming Unit-Granulocyte/Erythrocyte/Macrophage/Megakaryocyte (CFU-GEMM). These then give rise to hematopoietic lineage committed progenitor cells including, but are not limited to, CFU-GM (colony forming unit-granulocyte-monocyte), CFU-E (colony forming unit-erythrocyte), BFU-E (burst forming unit-erythrocyte), CFU-G (colony forming unit- granulocyte), CFU-eo (colony forming unit-eosinophil), and CFU-Meg (colony forming unit-megakaryocyte).
Hematopoietic stem cells are capable of differentiating into any of granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages, dendritic cells) and lymphocytes (common lymphoid progenitors, pre-B, pro-B, mature B, pre-T, pro-B, mature T and NKT lymphocytes and NK cells).
According to one embodiment, the hematopoietic stem cells are human hematopoietic stem cells. According to one embodiment, the progenitor cells are human progenitor cells.
According to one embodiment, the hematopoietic stem cells comprise T cell depleted hematopoietic stem cells, i.e. a population of hematopoietic cells which are depleted of T lymphocytes. The T cell depleted hematopoietic stem cells, may include e.g. CD34+, CD33+ and/or CD56+ cells. The T cell depleted hematopoietic stem cells may be depleted of CD3+ cells, CD2+ cells, CD8+ cells, CD4+ cells, a/b T cells and/or g/d T cells.
According to one embodiment, the hematopoietic stem cells comprise T cell depleted G-CSF mobilized blood cells enriched for CD34+ immature hematopoietic cells.
Depending on the application, the method may be effected using a cell or tissue graft which is syngeneic or non-syngeneic (e.g. allogeneic or xenogeneic) with the subject.
As used herein, the term“syngeneic” cells refer to cells which are essentially genetically identical with the subject or essentially all lymphocytes of the subject. Examples of syngeneic cells include cells derived from the subject (also referred to in the art as“autologous”), from a clone of the subject, or from an identical twin of the subject.
As used herein, the term“non-syngeneic” cells refer to cells which are not essentially genetically identical with the subject or essentially all lymphocytes of the subject, such as allogeneic cells or xenogeneic cells.
As used herein, the term“allogeneic” refers to cells which are derived from a donor who is of the same species as the subject, but which is substantially non-clonal with the subject. Typically, outbred, non-zygotic twin mammals of the same species are allogeneic with each other. It will be appreciated that an allogeneic cell may be HLA identical, partially HLA identical or HLA non identical (i.e. displaying one or more disparate HLA determinant) with respect to the subject.
As used herein, the term“xenogeneic” refers to a cell which substantially expresses antigens of a different species relative to the species of a substantial proportion of the lymphocytes of the subject. Typically, outbred mammals of different species are xenogeneic with each other.
According to an embodiment of the present invention, the subject is a human being and the cells are from a human origin (e.g. autologous or non-autologous).
According to one embodiment, the subject is a human being and the cells are from a xenogeneic origin (e.g. porcine origin).
Depending on the application and available sources, the cell or tissue graft of the present invention may be obtained from a prenatal organism, postnatal organism, an adult or a cadaver donor. Moreover, depending on the application needed, the cell or tissue graft may be naive or genetically modified. Determination of the type of cell or tissue graft to be used is well within the ability of one of ordinary skill in the art. Furthermore, any method known in the art may be employed to obtain a cell or tissue graft (e.g. for transplantation).
Thus, for example, hematopoietic stem cells or progenitor cells may be obtained by collection of bone marrow from the rear pelvic bones. Alternatively, hematopoietic stem cells or progenitor cells may be obtained by collecting peripheral blood (e.g. peripheral blood stem cells (PBSCs)) from a donor following a cell immobilization protocol (e.g. following administration of granulocyte colony stimulating factor (G-CSF)). Methods of collecting peripheral blood are well known in the art and include, but are not limited to, drawing of up to 500-1000 ml whole blood from a donor and collection in a container containing an anti-coagulant (e.g. heparin or citrate) or by apheresis, a procedure in which the peripheral blood of an individual is passed through an apparatus, yielding a predominant constituent (e.g. mononuclear cells), and returning the other constituents to the subject's circulation. Alternatively, cells may be obtained by in-vitro or ex-vivo culture of cells. It will be appreciated that the cells of the invention may be of fresh or frozen (e.g., cryo-preserved) preparations.
According to one embodiment, hematopoietic stem cells or progenitor cells may be collected from cord blood.
Transplanting the cell or tissue into the subject may be effected in numerous ways, depending on various parameters, such as, for example, the cell or tissue type; the type, stage or severity of the recipient's disease (e.g. organ failure); the physical or physiological parameters specific to the subject; and/or the desired therapeutic outcome.
Following transplantation of the cell or tissue transplant into the subject according to the present teachings, it is advisable, according to standard medical practice, to monitor the growth functionality and immunocompatability of the organ according to any one of various standard art techniques. For example, the functionality of an organ or tissue transplant may be monitored following transplantation by standard functional tests. Structural development of the cells or tissues may be monitored via computerized tomography, or ultrasound imaging. In cases of hematopoietic stem cell transplantation it is advisable to monitor the occurrence and severity of graft rejection and graft versus host disease (GVHD).
Depending on the transplantation context, in order to facilitate engraftment of the cells, the method typically comprises conditioning the subject prior to transplantation under conditioning protocols, e.g. reduced intensity conditioning (RIC), such pre-transplant conditioning protocol may lead to lymphocytopenia.
According to some embodiments of the invention, the conditioning protocol comprises a total body irradiation (TBI), total lymphoid irradiation (TLI), partial body irradiation (e.g. specific exposure of the lungs, kidney, brain etc.), myeloablative conditioning and/or non-myeloablative conditioning, e.g. with different combinations including, but not limited to, co-stimulatory blockade, chemotherapeutic agent and/or antibody immunotherapy. According to some embodiments of the invention, the conditioning comprises a combination of any of the above described conditioning protocols (e.g. chemotherapeutic agent and TBI, co- stimulatory blockade and chemotherapeutic agent, antibody immunotherapy and chemotherapeutic agent, etc.).
Examples of conditioning agents which may be used to condition the subject and which can cause lymphocytopenia include, without limitation, irradiation and pharmacological agents.
Examples of pharmacological agents which can cause lymphocytopenia include myelotoxic drugs (e.g. busulfan, dimethyl mileran, melphalan and thiotepa), lymphocytotoxic drugs (e.g. lymphocytotoxic antibodies) and immunosuppressant drugs (discussed in detail above).
Additionally or alternatively, the method may further comprise conditioning the subject with an immunosuppressive regimen prior to, concomitantly with, or following transplantation of the cell or tissue transplant. It will be appreciated that the immunosuppressive regime may lead to lymphocytopenia.
Examples of suitable types of immunosuppressive regimens include administration of immunosuppressive drugs and/or immunosuppressive irradiation (both of which are discussed above).
Ample guidance for selecting and administering suitable immunosuppressive regimens for transplantation is provided in the literature of the art (for example, refer to: Kirkpatrick CH. and Rowlands DT Jr., 1992. JAMA. 268, 2952; Higgins RM. et ah, 1996. Lancet 348, 1208; Suthanthiran M. and Strom TB., 1996. New Engl. J. Med. 331, 365; Midthun DE. et ah, 1997. Mayo Clin Proc. 72, 175; Morrison VA. et ah, 1994. Am J Med. 97, 14; Hanto DW., 1995. Annu Rev Med. 46, 381; Senderowicz AM. et ah, 1997. Ann Intern Med. 126, 882; Vincenti F. et ah, 1998. New Engl. J. Med. 338, 161; Dantal J. et al. 1998. Lancet 351, 623).
Regardless of the cause of lymphocytopenia and in order to increase lymphocyte counts, the method is effected by administering to the subject a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity.
The term“inhibitor of a cannabinoid receptor CB2 expression and/or activity” as used herein refers to a natural or synthetic compound which acts as an inhibitor of CB2 gene expression (at the DNA or RNA level) and/or as an inhibitor of CB2 receptor activity (at the protein level).
The term "CB2 receptor" as used herein refers to the cannabinoid receptor type 2. According to a specific embodiment, the CB2 receptor is human CB2 receptor: an exemplary CB2 amino acid sequence is provided in GenBank accession number NP_00l832, and an exemplary CB2 nucleotide sequence is provided in GenBank accession number NM_00l84l.
A variety of molecules which interfere with transcription and/or translation (e.g., RNA silencing agents) or on the protein level (e.g., aptamers, small molecules and inhibitory peptides, antagonists, enzymes that cleave the polypeptide, antibodies and the like) can be used according to the present teachings.
Down regulation of expression and/or activity may be either transient or permanent.
According to a specific embodiment, the downregulation is transient.
According to specific embodiments, down regulating expression refers to the absence of mRNA and/or protein, as detected by RT-PCR or Western blot, respectively.
According to other specific embodiments down regulating expression refers to a decrease in the level of mRNA and/or protein, as detected by RT-PCR or Western blot, respectively. The reduction may be by at least a 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % or at least 99 % reduction.
According to a specific embodiment, down regulating activity refers to the absence of CB2 activity (e.g. lack of activation of factors in the CB2 cell signaling pathway, such as inhibition of adenylyl cyclase activity and reduced cAMP levels, or phosphorylation of the inhibitory Tyr-505 of the leukocyte-specific protein tyrosine kinase (Lck)), as detected e.g. by ELISA or Western Blot.
According to other specific embodiments, down regulating activity refers to a decrease in the level of CB2 activity (e.g. reduction in the activation of factors in the CB2 cell signaling pathway, such as inhibition of adenylyl cyclase activity and reduced cAMP levels, or phosphorylation of the inhibitory Tyr-505 of the leukocyte- specific protein tyrosine kinase (Lck)), as detected e.g. by ELISA or Western Blot. The reduction may be by at least a 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % or at least 99 % reduction.
Non-limiting examples of compounds capable of inhibiting a cannabinoid receptor CB2 expression are described hereinbelow.
Down-regulation at the nucleic acid level
Down-regulation at the nucleic acid level is typically effected using a nucleic acid agent, having a nucleic acid backbone, DNA, RNA, mimetics thereof or a combination of same (also referred to herein as a polynucleotide agent). The nucleic acid agent may be encoded from a DNA molecule or provided to the cell per se. According to one embodiment, for downregulation of cannabinoid receptor CB2, the agent is typically administered to hematopoietic cells (e.g. immune cells) expressing the cannabinoid receptor CB2.
Thus, downregulation of cannabinoid receptor CB2 can be achieved by RNA silencing. As used herein, the phrase "RNA silencing" refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression] mediated by RNA molecules which result in the inhibition or "silencing" of the expression of a corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.
As used herein, the term "RNA silencing agent" refers to an RNA which is capable of specifically inhibiting or "silencing" the expression of a target gene. In certain embodiments, the RNA silencing agent is capable of preventing complete processing (e.g., the full translation and/or expression) of an mRNA molecule through a post-transcriptional silencing mechanism. RNA silencing agents include non-coding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated. Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs.
In one embodiment, the RNA silencing agent is capable of inducing RNA interference.
In another embodiment, the RNA silencing agent is capable of mediating translational repression.
According to an embodiment of the invention, the RNA silencing agent is specific to the target RNA (e.g., cannabinoid receptor CB2) and does not cross inhibit or silence other targets or a splice variant which exhibits 99% or less global homology to the target gene, e.g., less than 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% global homology to the target gene; as determined by PCR, Western blot, Immunohistochemistry and/or flow cytometry.
RNA interference refers to the process of sequence- specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs).
Following is a detailed description on RNA silencing agents that can be used according to specific embodiments of the present invention.
DsRNA, siRNA and shRNA - The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes. The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single- stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex.
Accordingly, some embodiments of the invention contemplate use of dsRNA to downregulate protein expression from mRNA.
According to one embodiment dsRNA longer than 30 bp are used. Various studies demonstrate that long dsRNAs can be used to silence gene expression without inducing the stress response or causing significant off-target effects - see for example [Strat et al., Nucleic Acids Research, 2006, Vol. 34, No. 13 3803-3810; Bhargava A et al. Brain Res. Protoc. 2004;13:115-125; Diallo M., et al., Oligonucleotides. 2003;13:381-392; Paddison P.J., et al., Proc. Natl Acad. Sci. USA. 2002;99:1443-1448; Tran N., et al., FEBS Lett. 2004;573:127-134]
Accordingly, some embodiments of the invention contemplate use of siRNA to downregulate protein expression from mRNA.
The term "siRNA" refers to small inhibitory RNA duplexes (generally between 18-30 base pairs) that induce the RNA interference (RNAi) pathway. Typically, siRNAs are chemically synthesized as 21 mers with a central 19 bp duplex region and symmetric 2-base 3 '-overhangs on the termini, although it has been recently described that chemically synthesized RNA duplexes of 25-30 base length can have as much as a lOO-fold increase in potency compared with 21 mers at the same location. The observed increased potency obtained using longer RNAs in triggering RNAi is suggested to result from providing Dicer with a substrate (27 mer) instead of a product (21 mer) and that this improves the rate or efficiency of entry of the siRNA duplex into RISC.
The strands of a double-stranded interfering RNA (e.g., a siRNA) may be connected to form a hairpin or stem-loop structure (e.g., a shRNA). Thus, as mentioned, the RNA silencing agent of some embodiments of the invention may also be a short hairpin RNA (shRNA).
The term "shRNA", as used herein, refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The number of nucleotides in the loop is a number between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11. Some of the nucleotides in the loop can be involved in base-pair interactions with other nucleotides in the loop. Examples of oligonucleotide sequences that can be used to form the loop include 5'- CAAGAGA-3' and 5’-UUACAA-3’ (International Patent Application Nos. WO2013126963 and WO2014107763). It will be recognized by one of skill in the art that the resulting single chain oligonucleotide forms a stem-loop or hairpin structure comprising a double-stranded region capable of interacting with the RNAi machinery.
Synthesis of RNA silencing agents suitable for use with some embodiments of the invention can be effected using any method known in the art such as disclosed in PCT publication no. W02010137020, incorporated herein by reference in its entirety.
Potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (www(dot)ncbi(dot)nlm(dot)nih(dot)gov/BLAST/). Putative target sites which exhibit significant homology to other coding sequences are filtered out.
Qualifying target sequences are selected as template for siRNA synthesis. Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55 %. Several target sites are preferably selected along the length of the target gene for evaluation. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction. Negative control siRNA preferably includes the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.
For example, suitable siRNAs directed against cannabinoid receptor CB2 can be the siRNA commercially available from Origene or from Santa Cruz Biotechnology (SCBT).
It will be appreciated that, and as mentioned hereinabove, the RNA silencing agent of some embodiments of the invention need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides.
miRNA and miRNA mimics - According to another embodiment the RNA silencing agent may be a miRNA.
The term "microRNA", "miRNA", and "miR" are synonymous and refer to a collection of non-coding single- stranded RNA molecules of about 19-28 nucleotides in length, which regulate gene expression. miRNAs are found in a wide range of organisms (viruses. fwdarw .humans) and have been shown to play a role in development, homeostasis, and disease etiology.
A number of studies have looked at the base-pairing requirement between miRNA and its mRNA target for achieving efficient inhibition of translation (reviewed by Bartel 2004, Cell 1 lb- 281). In mammalian cells, the first 8 nucleotides of the miRNA may be important (Doench & Sharp 2004 GenesDev 2004-504). However, other parts of the microRNA may also participate in mRNA binding. Moreover, sufficient base pairing at the 3’ can compensate for insufficient pairing at the 5’ (Brennecke et al, 2005 PLoS 3-e85). Computation studies, analyzing miRNA binding on whole genomes have suggested a specific role for bases 2-7 at the 5’ of the miRNA in target binding but the role of the first nucleotide, found usually to be“A” was also recognized (Lewis et at 2005 Cell 120-15). Similarly, nucleotides 1-7 or 2-8 were used to identify and validate targets by Krek et al. (2005, Nat Genet 37-495).
The target sites in the mRNA may be in the 5' UTR, the 3' UTR or in the coding region. Interestingly, multiple miRNAs may regulate the same mRNA target by recognizing the same or multiple sites. The presence of multiple miRNA binding sites in most genetically identified targets may indicate that the cooperative action of multiple RISCs provides the most efficient translational inhibition.
miRNAs may direct the RNA induced silencing complex (RISC) to downregulate gene expression by either of two mechanisms: mRNA cleavage or translational repression. The miRNA may specify cleavage of the mRNA if the mRNA has a certain degree of complementarity to the miRNA. When a miRNA guides cleavage, the cut is typically between the nucleotides pairing to residues 10 and 11 of the miRNA. Alternatively, the miRNA may repress translation if the miRNA does not have the requisite degree of complementarity to the miRNA. Translational repression may be more prevalent in animals since animals may have a lower degree of complementarity between the miRNA and binding site.
The term "microRNA mimic" or“miRNA mimic” refers to synthetic non-coding RNAs that are capable of entering the RNAi pathway and regulating gene expression. miRNA mimics imitate the function of endogenous miRNAs and can be designed as mature, double stranded molecules or mimic precursors (e.g., or pre-miRNAs). miRNA mimics can be comprised of modified or unmodified RNA, DNA, RNA-DNA hybrids, or alternative nucleic acid chemistries (e.g., LNAs or 2'-0,4'-C-ethylene-bridged nucleic acids (ENA)). For mature, double stranded miRNA mimics, the length of the duplex region can vary between 13-33, 18-24 or 21-23 nucleotides. The miRNA may also comprise a total of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides. The sequence of the miRNA may be the first 13-33 nucleotides of the pre-miRNA. The sequence of the miRNA may also be the last 13-33 nucleotides of the pre-miRNA.
Preparation of miRNAs mimics can be effected by any method known in the art such as chemical synthesis or recombinant methods.
It will be appreciated from the description provided herein above that contacting cells with a miRNA may be effected by transfecting the cells with e.g. the mature double stranded miRNA, the pre-miRNA or the pri-miRNA. The pre-miRNA sequence may comprise from 45-90, 60-80 or 60-70 nucleotides.
The pri-miRNA sequence may comprise from 45-30,000, 50-25,000, 100-20,000, 1,000- 1,500 or 80-100 nucleotides.
For example, suitable miRNAs directed against cannabinoid receptor CB2 can be the miRNA available from Origene.
Antisense - Antisense is a single stranded RNA designed to prevent or inhibit expression of a gene by specifically hybridizing to its mRNA. Downregulation of a cannabinoid receptor CB2 can be effected using an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding cannabinoid receptor CB2.
Design of antisense molecules which can be used to efficiently downregulate a cannabinoid receptor CB2 must be effected while considering two aspects important to the antisense approach. The first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells, while the second aspect is design of an oligonucleotide which specifically binds the designated mRNA within cells in a way which inhibits translation thereof.
The prior art teaches of a number of delivery strategies which can be used to efficiently deliver oligonucleotides into a wide variety of cell types [see, for example, Jaaskelainen et al. Cell Mol Biol Lett. (2002) 7(2):236-7; Gait, Cell Mol Life Sci. (2003) 60(5):844-53; Martino et al. J Biomed Biotechnol. (2009) 2009:410260; Grijalvo et al. Expert Opin Ther Pat. (2014) 24(7):80l- 19; Falzarano et al, Nucleic Acid Ther. (2014) 24(l):87-l00; Shilakari et al. Biomed Res Int. (2014) 2014: 526391; Prakash et al. Nucleic Acids Res. (2014) 42(l3):8796-807 and Asseline et al. J Gene Med. (2014) 16(7-8): 157-65] .
In addition, algorithms for identifying those sequences with the highest predicted binding affinity for their target mRNA based on a thermodynamic cycle that accounts for the energetics of structural alterations in both the target mRNA and the oligonucleotide are also available [see, for example, Walton et al. Biotechnol Bioeng 65: 1-9 (1999)]. Such algorithms have been successfully used to implement an antisense approach in cells.
In addition, several approaches for designing and predicting efficiency of specific oligonucleotides using an in vitro system were also published (Matveeva et al., Nature Biotechnology 16: 1374 - 1375 (1998)].
Thus, the generation of highly accurate antisense design algorithms and a wide variety of oligonucleotide delivery systems, enable an ordinarily skilled artisan to design and implement antisense approaches suitable for downregulating expression of known sequences without having to resort to undue trial and error experimentation.
Nucleic acid agents can also operate at the DNA level. Downregulation of cannabinoid receptor CB2 can also be achieved by inactivating the gene (e.g., cannabinoid receptor CB2) via introducing targeted mutations involving loss-of function alterations (e.g. point mutations, deletions and insertions) in the gene structure, typically referred to as genome editing. Exemplary methods used to introduce nucleic acid alterations to a gene of interest and agents for implementing same include engineered endonucleases, meganucleases, ZFNs and TALENs, CRISPR-Cas system and Site-Specific Recombinases.
As used herein, the phrase“loss-of-function alterations” refers to any mutation in the DNA sequence of a gene (e.g., cannabinoid receptor CB2) which results in downregulation of the expression level and/or activity of the expressed product, i.e., the mRNA transcript and/or the translated protein. Non-limiting examples of such loss-of-function alterations include a missense mutation, i.e., a mutation which changes an amino acid residue in the protein with another amino acid residue and thereby abolishes the enzymatic activity of the protein; a nonsense mutation, i.e., a mutation which introduces a stop codon in a protein, e.g., an early stop codon which results in a shorter protein devoid of the enzymatic activity; a frame-shift mutation, i.e., a mutation, usually, deletion or insertion of nucleic acid(s) which changes the reading frame of the protein, and may result in an early termination by introducing a stop codon into a reading frame (e.g., a truncated protein, devoid of the enzymatic activity), or in a longer amino acid sequence (e.g., a readthrough protein) which affects the secondary or tertiary structure of the protein and results in a non functional protein, devoid of the enzymatic activity of the non-mutated polypeptide; a readthrough mutation due to a frame- shift mutation or a modified stop codon mutation {i.e., when the stop codon is mutated into an amino acid codon), with an abolished enzymatic activity; a promoter mutation, i.e., a mutation in a promoter sequence, usually 5' to the transcription start site of a gene, which results in down-regulation of a specific gene product; a regulatory mutation, i.e., a mutation in a region upstream or downstream, or within a gene, which affects the expression of the gene product; a deletion mutation, i.e., a mutation which deletes coding nucleic acids in a gene sequence and which may result in a frame- shift mutation or an in-frame mutation (within the coding sequence, deletion of one or more amino acid codons); an insertion mutation, i.e., a mutation which inserts coding or non-coding nucleic acids into a gene sequence, and which may result in a frame-shift mutation or an in-frame insertion of one or more amino acid codons; an inversion, i.e., a mutation which results in an inverted coding or non-coding sequence; a splice mutation i.e., a mutation which results in abnormal splicing or poor splicing; and a duplication mutation, i.e., a mutation which results in a duplicated coding or non-coding sequence, which can be in-frame or can cause a frame- shift. According to specific embodiments loss-of-function alteration of a gene may comprise at least one allele of the gene.
According to other specific embodiments loss-of-function alteration of a gene comprises both alleles of the gene.
Methods of introducing nucleic acid alterations to a gene of interest are well known in the art [see for example Menke D. Genesis (2013) 51: - 618; Capecchi, Science (1989) 244:1288-1292; Santiago et al. Proc Natl Acad Sci USA (2008) 105:5809-5814; International Patent Application Nos. WO 2014085593, WO 2009071334 and WO 2011146121; US Patent Nos. 8771945, 8586526, 6774279 and UP Patent Application Publication Nos. 20030232410, 20050026157,
US20060014264; the contents of which are incorporated by reference in their entireties] and include targeted homologous recombination, site specific recombinases, PB transposases and genome editing by engineered nucleases. Agents for introducing nucleic acid alterations to a gene of interest can be designed publically available sources or obtained commercially from Transposagen, Addgene and Sangamo Biosciences.
Down-regulation at the polypeptide level
According to specific embodiments the agent capable of downregulating a cannabinoid receptor CB2 is an antibody or antibody fragment capable of specifically binding cannabinoid receptor CB2. Preferably, the antibody specifically binds at least one epitope of a cannabinoid receptor CB2.
As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof (such as Fab, F(ab')2, Fv, scFv, dsFv, or single domain molecules such as VH and VF) that are capable of binding to an epitope of an antigen.
Suitable antibody fragments for practicing some embodiments of the invention include a complementarity-determining region (CDR) of an immunoglobulin light chain (referred to herein as “light chain”), a complementarity-determining region of an immunoglobulin heavy chain (referred to herein as“heavy chain”), a variable region of a light chain, a variable region of a heavy chain, a light chain, a heavy chain, an Fd fragment, and antibody fragments comprising essentially whole variable regions of both light and heavy chains such as an Fv, a single chain Fv (scFv), a disulfide- stabilized Fv (dsFv), an Fab, an Fab’, and an F(ab’)2.
As used herein, the terms "complementarity-determining region" or "CDR" are used interchangeably to refer to the antigen binding regions found within the variable region of the heavy and light chain polypeptides. Generally, antibodies comprise three CDRs in each of the VH (CDR HI or HI; CDR H2 or H2; and CDR H3 or H3) and three in each of the VL (CDR LI or LI; CDR L2 or L2; and CDR L3 or L3).
The identity of the amino acid residues in a particular antibody that make up a variable region or a CDR can be determined using methods well known in the art and include methods such as sequence variability as defined by Rabat et al. (See, e.g., Rabat et ah, 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C.), location of the structural loop regions as defined by Chothia et al. (see, e.g., Chothia et al., Nature 342:877- 883, 1989.), a compromise between Rabat and Chothia using Oxford Molecular's AbM antibody modeling software (now Accelrys®, see, Martin et al., 1989, Proc. Natl Acad Sci USA. 86:9268; and world wide web site www(dot)bioinf-org(dot)uk/abs), available complex crystal structures as defined by the contact definition (see MacCallum et al., J. Mol. Biol. 262:732-745, 1996) and the "conformational definition" (see, e.g., Makabe et al., Journal of Biological Chemistry, 283:1156- 1166, 2008).
As used herein, the“variable regions” and "CDRs" may refer to variable regions and CDRs defined by any approach known in the art, including combinations of approaches.
Functional antibody fragments comprising whole or essentially whole variable regions of both light and heavy chains are defined as follows:
(i) Fv, defined as a genetically engineered fragment consisting of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) expressed as two chains;
(ii) single chain Fv (“scFv”), a genetically engineered single chain molecule including the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
(iii) disulfide-stabilized Fv (“dsFv”), a genetically engineered antibody including the variable region of the light chain and the variable region of the heavy chain, linked by a genetically engineered disulfide bond.
(iv) Fab, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme papain to yield the intact light chain and the Fd fragment of the heavy chain which consists of the variable and CH1 domains thereof; (v) Fab’, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin, followed by reduction (two Fab’ fragments are obtained per antibody molecule);
(vi) F(ab’)2, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin (i.e., a dimer of Fab’ fragments held together by two disulfide bonds); and
(vii) Single domain antibodies or nanobodies are composed of a single VH or VL domains which exhibit sufficient affinity to the antigen.
Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).
Antibody fragments according to some embodiments of the invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)].
Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These Methods for producing these single-chain antigen binding proteins (sFv) are described, for example, by [Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
Another form of an antibody fragment is a peptide coding for a single complementarity determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et ah, Nature, 321:522-525 (1986); Riechmann et ah, Nature 332:323-327 (1988); Verhoeyen et ah, Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boemer et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10,: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).
According to one embodiment, the antibody is a humanized antibody.
According to one embodiment, the antibody is a monoclonal antibody.
According to one embodiment, the antibody is a humanized monoclonal antibody.
According to a specific embodiment, the cannabinoid receptor CB2 targeting antibodies are commercially available from e.g. Abcacm, OriGene, Santa Cruz Biotechnology (SCBT), Antibodies -online .
According to a specific embodiment, the cannabinoid receptor CB2 targeting antibody is H60 (discussed in detail in Savonenko et al., PLoS One. 2015; 10(6): e0l296l8).
Another agent which can be used along with some embodiments of the invention to downregulate cannabinoid receptor CB2 is an aptamer. As used herein, the term“aptamer” refers to double stranded or single stranded RNA molecule that binds to specific molecular target, such as a protein. Various methods are known in the art which can be used to design protein specific aptamers. The skilled artisan can employ SELEX (Systematic Evolution of Ligands by Exponential Enrichment) for efficient selection as described in Stoltenburg R, Reinemann C, and Strehlitz B (Biomolecular engineering (2007) 24(4):381-403).
Another agent capable of downregulating cannabinoid receptor CB2 would be any molecule which binds to and/or cleaves cannabinoid receptor CB2. Such molecules can be a small molecule, cannabinoid receptor CB2 antagonists, or cannabinoid receptor CB2 inhibitory peptide.
According to one embodiment, the agent capable of downregulating cannabinoid receptor CB2 binds to at least one residue in transmembrane domain 3, 4 and/or 5 of cannabinoid receptor CB2.
According to one embodiment, the agent capable of downregulating cannabinoid receptor CB2 binds to at least one residue in extracellular loop 2 of cannabinoid receptor CB2.
According to one embodiment, the agent capable of downregulating cannabinoid receptor CB2 binds to at least one residue in transmembrane domain 4-extracellular loop 2-transmembrane domain 5 region of cannabinoid receptor CB2.
According to a specific embodiment, the agent capable of downregulating cannabinoid receptor CB2 binds to at least one residue in the transmembrane domain of cannabinoid receptor CB2 through hydrogen bonds and/or aromatic and hydrophobic interactions.
According to a specific embodiment, the agent capable of downregulating cannabinoid receptor CB2 binds to residue Ser161.
According to a specific embodiment, the agent capable of downregulating cannabinoid receptor CB2 binds to residue Ser165.
According to a specific embodiment, the agent capable of downregulating cannabinoid receptor CB2 binds to residue Cys175.
According to a specific embodiment, the agent capable of downregulating cannabinoid receptor CB2 binds to any or all of residues Ser180-Phe183.
According to one embodiment, the inhibitor of cannabinoid receptor CB2 activity is a CB2 receptor antagonist. The CB2 receptor antagonist may be a true antagonist or inverse agonist, and includes any chemical entity that, upon administration to a subject, results in inhibition or down- regulation of a biological activity associated with activation of the CB2 receptor in the subject. Such CB2 receptor antagonist includes any agent that can block CB2 receptor activation or any of the downstream biological effects of CB2 receptor activation. For example, such a CB2 receptor antagonist can act by occupying the ligand binding site or a portion thereof of the CB2 receptor, thereby making the receptor inaccessible to its natural ligand so that its normal biological activity is prevented or reduced.
Exemplary inverse agonists which may be used according to the methods of the invention include, but are not limited to, SR144528, AM630, JTE-907, COR170, GPla, SCH336, RO6957022, AM1241 and Sch.4l43l9.
According to a specific embodiment, the inhibitor of cannabinoid receptor CB2 activity is SR144528.
According to a specific embodiment, the inhibitor of cannabinoid receptor binds to CB2 with a higher affinity compared to cannabinoid CB1 receptor (e.g. at least a 500-fold higher affinity).
According to a specific embodiment, the inhibitor of CB2 is a small molecule having an IC50 for CB2 < 8 mM, e.g. having an IC50 for CB2 in a range of 0.1 - 8 mM, e.g. 1 - 8 pM).
According to one embodiment, the agent capable of decreasing an activity or expression of cannabinoid receptor CB2 downregulates an activity or expression of cannabinoid receptor CB2 in hematopoietic cells (e.g. immune cells).
It will be appreciated that the methods of some embodiments of the invention (e.g. enhancing lymphocyte count or recovery) can be performed within a subject {i.e., in vivo), within cells derived from a subject {i.e., ex vivo or in vitro) or within a hematopoietic stem cell line {i.e., in vitro).
According to one aspect, there is provided an ex vivo method of enhancing lymphocyte recovery, the method comprising ex vivo contacting hematopoietic stem and/or progenitor cells with an effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby enhancing lymphocyte recovery. According to some embodiments, the method of ex vivo enhancing lymphocyte recovery is effected by contacting hematopoietic stem and/or progenitor cells with an inhibitor of a cannabinoid receptor CB2 expression and/or activity. Contacting may be effected for several minutes (e.g. about 5, 10, 15, 20, 30, 40, 50, 60, 70, 80 or 90 minutes) or for several hours (e.g. about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24hours).
The cell culture may further comprise tissue culture medium, supplemented with appropriate supplements e.g. L-glutamine, antibiotics, etc. according to known procedures (e.g., as described in the ATCC protocols).
According to one embodiment, after culture the hematopoietic stem and/or progenitor cells may be stored (e.g. for later use), expanded in culture or administered to a subject in need thereof (as discussed in detail herein). The inhibitor of a cannabinoid receptor CB2 expression and/or activity of some embodiments of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
Herein the term "active ingredient" refers to the inhibitor of a cannabinoid receptor CB2 expression and/or activity accountable for the biological effect.
Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.
Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
Techniques for formulation and administration of drugs may be found in“Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.
Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.
Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.
Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (inhibitor of a cannabinoid receptor CB2 expression and/or activity) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., lymphocytopenia) or prolong the survival of the subject being treated.
Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
Animal models such as immunocompetent mice, immunodeficient mice (e.g. SCID mice) or animal models for hematopoietic cancer (e.g. a B-CLL animal model such as the NOD-SCID mouse chimera as described previously by Shimoni A et al. A model for human B-chronic lymphocytic leukemia in human/mouse radiation chimera: evidence for tumor-mediated suppression of antibody production in low-stage disease. Blood. 1997; 89:2210-2218) can be used to determine therapeutic efficacy of the agents of the present invention in vivo.
Dosage amount and interval may be adjusted individually to provide the active ingredient at a sufficient amount to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
The above mentioned agents may be administered to a subject in a single administration or in plurality of administrations (e.g. 2, 3, 4, 5 or more). If plurality of administrations is employed, the agent may be administered over the course of one day, several days, several weeks, several months or several years. One of ordinary skill in the art is capable of determining the dosage amount and the course of treatment based on the subject being treated and the level of mobilization required. Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
According to one embodiment, in order to increase lymphocyte counts the method further comprises administering to the subject lymphocytes such as donor lymphocyte infusion (DLI). According to one embodiment, the lymphocyte infusion includes T cells (e.g. CD8+ T cells, CD4+ T cells, B cells, monocytes, macrophages, and/or NK cells).
It is expected that during the life of a patent maturing from this application many relevant inhibitors of cannabinoid receptor CB2 expression and/or activity will be developed and the scope of the term inhibitor of a cannabinoid receptor CB2 expression and/or activity is intended to include all such new technologies a priori.
As used herein the term“about” refers to ± 10 %.
The terms "comprises", "comprising", "includes", "including",“having” and their conjugates mean "including but not limited to".
The term“consisting of’ means“including and limited to”.
The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases“ranging/ranges between” a first indicate number and a second indicate number and“ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
As used herein, the term“treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
EXAMPLES
Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non-limiting fashion. Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et ah, (1989); "Current Protocols in Molecular Biology" Volumes I- III Ausubel, R. M., ed. (1994); Ausubel et ah, "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et ah, "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I- III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984);“Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
GENERAL MATERIALS AND EXPERIMENTAL PROCEDURES
Cannabis extracts and cannabinoids
Cannabis Sativa and Indika extracts with high content in THC or CBD (i.e. THC-BDS/CBD BDS, respectively) were supplied by Cannabliss (Cannabliss, Israel). The THC, CBN and CBD contents of the extracts were quantified against a commercial THC, CBN and CBD standards (Izun Pharma, Israel). Inhibitors
SR144528 - a CB2 receptor antagonist was purchased from Abeam. A967079 - a TRPA1 Receptor antagonist and BCTC - a TRPV1 Receptor antagonist were purchased from Alomone Labs, Israel. GSK2193874 - a TRPV4 antagonist was purchased from SIGMA- ALDRICH, Israel. GW9662 - a PPARy antagonist was purchased from Enzo Life Sciences, New York, USA.
Mice
Female 8- to l l-week-old C57BL/6 and BALB/c mice were purchased from Envigo, Jerusalem, Israel and CB2 knockout mice (CNR /_) were bred in the specific pathogen-free (SPF) facility of the Authority of Biological and Biomedical Models at the Hebrew University of Jerusalem. The study was approved by the Institutional Animal Care and Use Committee of the Hebrew University of Jerusalem in accordance with national laws and regulations for the protection of animals. Mice were housed under specific pathogen-free (SPF) conditions.
Syngeneic BMT model
C57BL/6 or CB2 knockout mice underwent lethal whole-body irradiation by single exposure to 10 Gy and were reconstituted with 8 x 106 donor C57BL/6 or CB2 knockout BM cells the following day. Cannabis extracts/cannabinoids (5 mg/kg) were administered intraperitoneally (IP), from the day of transplantation, every other day, for two weeks. Mice were monitored for weight loss. Once a week, blood was collected from the mice tail into Ethylenediaminetetraacetic acid (EDTA) coated capillary tubes. A complete blood count (CBC) with differentials was performed using a validated BC-2800Vet Auto Hematology Analyzer (Mindray).
Allogeneic BMT model
BALB/c mice underwent lethal whole-body irradiation by single exposure to 8 Gy and were reconstituted with 8 x 106 donor C57BL/6 BM cells and 2 x 106 spleen cells the following day. Cannabis extracts/cannabinoids (5 mg/kg) were administered intraperitoneally (IP), from the day of transplantation, every other day, for two weeks. For GVHD evaluation, mice were monitored daily for weight loss, diarrhea, ruffled skin, and survival. GVHD score, based on all of the aforementioned factors (rated on a scale of 0-4), was calculated as previously described (32).
Treatment-induced lymphocytopenia without BMT model
C57BL/6 and CB2 KO mice underwent non-lethal whole -body irradiation by single exposure to 6 Gy. Once a week, blood was collected from the mice tail into EDTA coated capillary tubes. CBC with differentials was performed using a validated BC-2800Vet Auto Hematology Analyzer (Mindray). Lymphocyte activation assays
A total of 1 x 106 carboxyfluorescin diacetate succinimidyl ester (CFSE) labeled C57BL/6 splenocytes cells/well were plated in 96- well flat bottom plates with RPMI 1640 medium supplemented with 10 % FCS, 1 % penicillin/streptomycin, and 1 % L-glutamine (Biological Industries, Beit Haemek, Israel). Splenocytes were activated with anti-CD3 antibodies (Biolegend, USA) at the presence of indicated concentrations of cannabis extracts/cannabinoids for 4 days. For proliferation test, CFSE levels on the cells were determined using FACS analysis. Cytokine concentration in the culture media was quantified using the ELISA Ready SET Go kits (eBioscience, San Diego, CA, USA), according to the manufacturer’s instructions. All determinations were made in triplicates.
Statistical analysis
Data from the BMT studies are described as mean values on dot plot showing individual values (lymphocyte and platelet count) in the indicated time point. Data from in vitro studies are represented as mean ± SD. Single comparisons to control were made using two-tailed Student’s t- test, with P value < 0.05 considered statistically significant.
EXAMPLE 1
CBD is a stronger inhibitor of in vitro lymphocyte activation, as compared to THC
First, in vitro methods were utilized in order to learn about the influence of pure CBD/THC and cannabis extracts on lymphocyte function. Cannabis extracts with high content of CBD or THC were named CBD BDS/ THC BDS (Botanical Drug Substance). These extracts were used, in addition to the pure cannabinoids, for two reasons: first, most of the patients are currently treated with cannabis based treatment and not with pure cannabinoids. Second, it was suggested that the combination of cannabinoids with other active molecules in the plant may have better results in medical use (known as the entourage effect) (15).
The effect of cannabis/cannabinoids on the proliferation of activated lymphocytes was analyzed. Succinimidyl ester (CFSE) labeled C57B1/6 mouse splenocytes were activated with anti- CD3 antibodies in the presence of pure cannabinoids, CBD BDS or THC BDS at different concentrations. Cell proliferation was assessed using CFSE FACS analysis. Interestingly, in vitro the inhibitory effect of pure cannabinoids on lymphocyte activation was stronger as compared to cannabis extract. Whether in the form of pure cannabinoids or cannabis extract, CBD inhibited proliferation significantly better than THC (Figure 1A). Similar results were obtained using Balb/C splenocytes (Figure 5A) or human Peripheral Blood Mononuclear Cells (PBMC) (Figure 5B). Next, the supernatant from the same experiments was used to test the effect of cannabinoids treatment on cytokine secretion upon lymphocyte activation. Four different cytokines were tested: IL-17, secreted in Thl7 reaction; IL-10, secreted from Treg; TNF-a, secreted in Thl reaction; and IL-5, secreted in Th2 reaction. The levels of secreted cytokines were examined using ELISA assay. Shown in Figures 1B-E are the results of 3 pg/ml treatment with pure cannabinoids and 10 pg/ml treatment with the cannabis extracts, containing approximately 30 % of the designated cannabinoid. The results for IL-17 and IL-10 after treatment with various other concentrations are illustrated in Figure 6A-B.
As evident from the results, all treatments significantly reduced IL-17 secretion (Figure 1B and Figure 6A). CBD BDS had the strongest effect with only 0.25 % in the supernatant as compared to untreated activated lymphocytes (control). IL-10 secretion was significantly increased by all treatments (Figure 1C and Figure 6B). Pure CBD had the strongest effect, with 1806 % IL-10 in the supernatant (as compared to control). Notably, pure CBD had a stronger effect then pure THC, but CBD BDS had less effect then THC BDS. All treatments led to a small elevation in TNF-a secretion (Figure 1D), which was significant in all treatments except THC BDS. The levels of IL-5 secretion were affected by THC BDS and pure CBD treatments (Figure 1E).
Overall these results show that the cannabinoids CBD and THC have an inhibitory effect on lymphocyte activation, associated with reduction in the secretion of the inflammatory IL-17 cytokine and an elevation in the secretion of the regulatory cytokine IL-10.
EXAMPLE 2
THC and CBD affect lymphocyte activation by different mechanisms
The cannabinoid receptor CB2 is highly expressed in immune cells (16, 17). To elucidate whether CB2 is involved in the effects of THC and CBD on lymphocytes, CB2 knock-out mice (CB2 KO) were used. First, splenocytes extracted from CB2 KO mice were used (Figure 7A) in a CFSE lymphocyte proliferation assay, similar to the assay in Figure 1A. The inhibitory effect of pure THC, but not pure CBD, was abolished in CB2 KO derived splenocytes (Figure 1F). Interestingly, the inhibitory effect of THC BDS was maintained.
These results indicate that the CB2 receptor is the main mediator for THC’s effect on lymphocytes. However, the effect CBD clearly does not involve CB2 signaling. Several mediators were suggested for CBD effects on mammalian cells (6, 7). To search the molecules which are involved in CBD’s effect on lymphocyte activation, several inhibitors were used together with CBD in a CFSE lymphocyte proliferation assay. A967079, BCTC and GSK2193874 are antagonist to TRP channels TRPA1, TRPV1 and TRPV4 respectively, which have been demonstrated to mediate CBD signaling. However, the present results demonstrate that none of these antagonists interfered with CBD’s inhibitory effect on lymphocyte activation (Figures 8A-C). Another potential mediator of CBD signaling is the nuclear receptor PPAR-g (18). The present inventors found that GW9662, the antagonist for PPAR-g, partially reversed the effect of CBD on lymphocyte proliferation (Figure 1G).
EXAMPLE 3
Cannabinoid treatment alters hematologic rehabilitation after bone marrow transplantation To test the influence of THC and CBD and cannabis extracts on hematopoiesis after BMT, a syngeneic transplantation model was utilized. C57BL/6 mice underwent lethal whole-body irradiation and were reconstituted with 8 x 106 donor C57BL/6 BM cells the following day (Figure 2A). 5 mg/kg of Cannabis extracts/pure cannabinoids/vehicle were administered intraperitoneally (IP), from the day of transplantation, every other day, for two weeks. Once a week, starting one week after transplantation, blood was collected from the mice tail and CBC with differentials was performed. Both pure cannabinoids and cannabis extracts had a significant inhibitory effect on lymphocyte recovery (Figures 2B and 2C). Among the tested compounds, pure THC had the strongest effect with a mean of 39 % inhibition as compared to vehicle treated mice (control), 3 weeks after transplantation (Figure 2B, right). The inhibitory effect of CBD treatment was significantly lower. Interestingly there was no significant difference between CBD BDS and THC BDS treatment (Figure 2C, right). The counts of monocytes and granulocytes were not affected by the treatment (data not shown). Platelets recovery was significantly improved only in the group that received THC BDS treatment, with a mean of 10 % improvement as compared to control, 2 weeks after transplantation (Figures 2D-E).
These results demonstrate that cannabis/cannabinoids treatments affect hematological reconstitution after bone marrow transplantation and that different cannabinoid drugs have different effects.
EXAMPLE 4
CB2 receptor has an inhibitory effect on lymphocytes recovery
Since THC had the strongest inhibitory effect on lymphocyte recovery, the present inventors wanted to examine the involvement of CB2 in this process. First, syngeneic BMT mice were administered with CB2 antagonist SR144528 once a day for one week from the day of transplantation. Once a week, starting one week after transplantation, blood was collected from the mice tail and CBC with differentials was performed. The present results demonstrate significantly improved recovery of lymphocytes in the treated group (Figure 3A). The average percent of initial weight during the experiment was similar in treated and non-treated groups. No signs of toxicity were evident.
To clarify whether this improvement is due to an effect on the grafted cells or on the accepting environment, CB2 KO mice were used as donors/acceptors in BMT experiments. The normal blood counts of CB2 KO female mice are similar to the WT C57BL/6 counts (Figure 7B). C57BL/6 mice underwent lethal whole-body irradiation and were reconstituted with 8 x 106 donor CB2 KO or C57BL/6 BM cells the following day. A significantly higher lymphocyte count was found in the group that received CB2 KO transplant as compared to control, starting from the second week after transplantation (Figure 3B). When C57BL/6 BM cells were transplanted to CB2 KO or C57BL/6 recipient mice lymphocyte counts were not significantly different (Figure 3C). Thus, these results illustrate that CB2 affects lymphocyte recovery in the engrafted cells rather than the host environment.
Altogether, these experiments demonstrate the inhibitory role of CB2 in rehabilitation of blood lymphocytes after bone marrow transplantation.
EXAMPLE 5
Cannabis/ Cannabinoids administration for GVHD prophylaxis
Several studies, as well as the in vitro assays described above (Figures 1A-E), indicate that cannabinoids have anti-inflammatory function (9). Yeshurun, et.al demonstrated the beneficial effect of the cannabinoid CBD as GVHD prophylaxis in patients (14). The present inventors therefore decided to compare the immunosuppressive effect of CBD/THC and cannabis extracts on GVHD prophylaxis in a murine model.
Balb/C mice underwent whole-body irradiation followed by allogeneic BMT from C57BL/6 donor mice. 5 mg/kg of Cannabis extracts/pure cannabinoids/vehicle were administered IP, from the day of transplantation, every other day, for two weeks (Figure 4A). Mice chimerism was not affected by the treatment (Figures 9A-B). In this model, both CBD BDS and THC BDS significantly improved survival (Figure 4B, right), while pure cannabinoids had a significant but smaller effect (Figure 4B, left). Moreover, GVHD scores were significantly lower in mice receiving cannabis extracts (Figure 4C).
These results demonstrate that cannabis extracts are more potent modulators of allogeneic activation in-vivo than pure THC or CBD. EXAMPLE 6
CB2 has an inhibitory function in lymphocyte recovery in a model of treatment-induced lymphocytopenia without BMT
To investigate the role of CB2 in lymphocyte recovery in a model of treatment-induced lymphocytopenia without BMT, C57BL/6 and CB2 KO mice received non-lethal 6 Gy whole-body irradiation. A significantly higher lymphocyte count was found in CB2 KO group as compared to control, starting from the second week after transplantation (Figures 10A-B). In addition, faster normalization of lymphocytes/granulocyte ratio was accomplished in irradiated CB2 KO mice as compared to control (Figures 10C-D).
These results demonstrate that CB2 has an inhibitory role in lymphocyte recovery in treatment-induced lymphocytopenia which is not associated with BMT.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.
In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety.
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26. Patinkin D, Milman G, Breuer A, Fride E, Mechoulam R. Endocannabinoids as positive or negative factors in hematopoietic cell migration and differentiation. Eur J Pharmacol. 2008 Oct 24;595(l-3):l-6 27. Grambow E, Striider D, Klar E, Hinz B, Vollmar B. Differential effects of endogenous, phyto and synthetic cannabinoids on thrombogenesis and platelet activity. Biofactors. 2016 Nov l2;42(6):58l-590
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Claims

WHAT IS CLAIMED IS:
1. A method of treating a lymphocytopenia in a subject in need thereof, wherein said lymphocytopenia is not associated with hematopoietic stem cell transplantation, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby treating the lymphocytopenia.
2. A method of treating a lymphocytopenia associated with hematopoietic stem cell transplantation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby treating the lymphocytopenia associated with hematopoietic stem cell transplantation.
3. A method of increasing lymphocyte count in a human subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby increasing the lymphocyte count.
4. A therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity for use in treating a lymphocytopenia in a subject in need thereof, wherein said lymphocytopenia is not associated with hematopoietic stem cell transplantation.
5. A therapeutically effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity for use in treating a lymphocytopenia associated with hematopoietic stem cell transplantation in a subject in need thereof.
6. An ex vivo method of enhancing lymphocyte recovery, the method comprising ex vivo contacting hematopoietic stem and progenitor cells with an effective amount of an inhibitor of a cannabinoid receptor CB2 expression and/or activity, thereby enhancing lymphocyte recovery.
7. The method of claim 6, further comprising administering said hematopoietic stem and progenitor cells to a subject in need thereof.
8. The method of claim 1 or 2, or inhibitor of a cannabinoid receptor CB2 expression and/or activity for use of claim 4 or 5, wherein said lymphocytopenia is not associated with transplantation of a solid organ or tissue.
9. The method of claim 1 or 2, or inhibitor of a cannabinoid receptor CB2 expression and/or activity for use of claim 4 or 5, wherein said lymphocytopenia is associated with transplantation of a solid organ or tissue.
10. The method of any one of claims 1-2 or 8-9, or inhibitor of a cannabinoid receptor CB2 expression and/or activity for use of any one of claims 4, 5 or 8-9, wherein the lymphocytopenia is associated with administration of an anti-cancer therapy.
11. The method of any one of claims 1-2 or 8-10, or inhibitor of a cannabinoid receptor CB2 expression and/or activity for use of any one of claims 4, 5 or 8-10, wherein the lymphocytopenia is associated with administration of a chemotherapeutic agent, a radiation therapy or an immunotherapy.
12. The method of any one of claims 1-2 or 8-11, or inhibitor of a cannabinoid receptor CB2 expression and/or activity for use of any one of claims 4-5 or 8-11, wherein said subject has a disease or condition selected from the group consisting of a malignant disease, a non-malignant tumor, an infection, an autoimmune disease, an aplastic anemia and a nuclear or radiation exposure.
13. The method of any one of claims 2 or 10-12, or inhibitor of a cannabinoid receptor CB2 expression and/or activity for use of any one of claims 5 or 10-12, wherein said hematopoietic stem cell transplantation is used to treat a disease or condition selected from the group consisting of a hematologic malignancy, an aplastic anemia or an immune deficiency.
14. The method of any one of claims 2, 10-12 or 13, or inhibitor of a cannabinoid receptor CB2 expression and/or activity for use of any one of claims 5, 10-12 or 13, wherein said hematopoietic stem cell transplantation is obtained from a source selected from the group consisting of bone marrow stem cells, peripheral blood stem cells and cord blood stem cells.
15. The method of any one of claims 2, 10-12 or 13-14, or inhibitor of a cannabinoid receptor CB2 expression and/or activity for use of any one of claims 5, 10-12 or 13-14, wherein said hematopoietic stem cell transplantation comprises T cell depleted hematopoietic stem cells.
16. The method of any one of claims 2, 10-11 or 13-15, wherein said inhibitor of said CB2 is administered to the subject prior to, concomitantly with and/or subsequent to said hematopoietic stem cell transplantation.
17. The method of any one of claims 2, 10-11 or 13-16, or inhibitor of a cannabinoid receptor CB2 expression and/or activity for use of any one of claims 5, 10-11 or 13-14, wherein said hematopoietic stem cell transplantation is autologous with the subject.
18. The method of any one of claims 2, 10-11 or 13-16, or inhibitor of a cannabinoid receptor CB2 expression and/or activity for use of any one of claims 5, 10-11 or 13-14, wherein said hematopoietic stem cell transplantation is non-autologous with the subject.
19. The method or inhibitor of a cannabinoid receptor CB2 expression and/or activity for use of claim 18, wherein the subject is monitored for graft versus host disease.
20. The method of any one of claims 1-3 or 6-19, or inhibitor of a cannabinoid receptor CB2 expression and/or activity for use of any one of claims 4-5, 8-14 or 17-19, wherein said inhibitor of said CB2 is a genome editing agent or an RNA silencing agent.
21. The method of any one of claims 1-3 or 6-19, or inhibitor of a cannabinoid receptor CB2 expression and/or activity for use of any one of claims 4-5, 8-14 or 17-19, wherein said inhibitor of said CB2 is an antagonist or an inverse agonist.
22. The method or inhibitor of a cannabinoid receptor CB2 expression and/or activity for use of any one of claims 1-20, wherein said inhibitor is selected from the group consisting of a small molecule, an antibody and an aptamer.
23. The method or inhibitor of a cannabinoid receptor CB2 expression and/or activity for use of claim 21 or 22, wherein said inhibitor is selected from the group consisting of SR144528, AM630, JTE-907, COR170, GPla, SCH336, RO6957022 and AM1241.
24. The method of any one of claims 1-2 or 8-23, or inhibitor of a cannabinoid receptor CB2 expression and/or activity for use of any one of claims 4-5, 8-14 or 17-23, wherein said lymphocytopenia comprises T lymphocytopenia, B lymphocytopenia and/or NK lymphocytopenia.
25. The method of any one of claims 1-2 or 8-24, or inhibitor of a cannabinoid receptor CB2 expression and/or activity for use of any one of claims 4-5, 8-14 or 17-24, wherein said lymphocytopenia is determined when a complete blood count of the subject shows a lymphocyte count lower than the age- appropriate reference.
26. The method of any one of claims 1-3 or 7-25, or inhibitor of a cannabinoid receptor CB2 expression and/or activity for use of any one of claims 4-5, 8-14 or 17-25, wherein said subject is a human subject.
PCT/IL2019/050458 2018-04-23 2019-04-22 Use of cb2 inhibitors for lymphocyte recovery WO2019207582A1 (en)

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