WO2003077832A2 - Dexanabinol et analogues de dexanabinol regulant des genes associes a l'inflammation - Google Patents

Dexanabinol et analogues de dexanabinol regulant des genes associes a l'inflammation Download PDF

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Publication number
WO2003077832A2
WO2003077832A2 PCT/IL2003/000223 IL0300223W WO03077832A2 WO 2003077832 A2 WO2003077832 A2 WO 2003077832A2 IL 0300223 W IL0300223 W IL 0300223W WO 03077832 A2 WO03077832 A2 WO 03077832A2
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Prior art keywords
saturated
cyclic
unsaturated
inflammatory
branched
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PCT/IL2003/000223
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WO2003077832A3 (fr
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Aaron Garzon
Ayelet Avraham
George Fink
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Pharmos Corporation
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Priority to AU2003214608A priority Critical patent/AU2003214608A1/en
Priority to EP03710188A priority patent/EP1485083A4/fr
Priority to CA002479676A priority patent/CA2479676A1/fr
Priority to IL16387703A priority patent/IL163877A0/xx
Priority to JP2003575886A priority patent/JP2005526768A/ja
Publication of WO2003077832A2 publication Critical patent/WO2003077832A2/fr
Publication of WO2003077832A3 publication Critical patent/WO2003077832A3/fr
Priority to US10/942,504 priority patent/US20050137251A1/en

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Definitions

  • the present invention relates to pharmaceutical compositions comprising as an active ingredient non-psychotropic cannabinoid derivatives that modulate the expression of genes involved in inflammatory and immune processes.
  • Regulating the transcription of pro and anti-inflammatory mediators has useful therapeutic application for prevention and treatment of acute and chronic inflammation, autoimmune diseases and related disorders, pain, infections, liver diseases, cardiovascular disorders, gastrointestinal disorders, disorders of the central and peripheral nervous system including neurodegenerative diseases, respiratory diseases, renal diseases, post-operative complications, tissue rejection and certain types of cancer.
  • Cannabinoids Cannabis sativa preparations have long been known as therapeutic agents to treat various diseases.
  • THC tetrahydrocannabinol
  • Cannabis sativa preparations have long been known as therapeutic agents to treat various diseases.
  • THC tetrahydrocannabinol
  • medicinal chemists to develop numerous cannabinoid analogs.
  • novel compounds were designed to exhibit the therapeutically beneficial properties of THC without the clinically undesirable psychotropic effects.
  • Potential therapeutic applications have classically included known attributes of marijuana itself such as anti- emesis, analgesia, anti-glaucoma and appetite stimulation.
  • More recently recognized roles for non-psychotropic cannabinoids are as neuroprotective and anti-inflammatory agents.
  • the diverse cannabinoid effects are generally attributed to the activation or inhibition of various types of receptors. Nevertheless, the mechanisms underlying some therapeutic effects of cannabinoid derivatives remain unclear.
  • COX-2 is one of the cyclooxygenase iso forms involved in the metabolism of arachidonic acid (AA) toward prostaglandins (PG) and other eicosanoids, a family of compounds known to exhibit inflammatory properties and known to be involved in inflammation.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • COX activity by modifying the enzyme active site thereby preventing the transformation of the AA substrate to PGE (Hinz B. et al., J. Pharm. Exp. Ther. 300: 367- 375, 2002).
  • TNF- ⁇ converting enzyme TACE
  • Inflammation is one of the most important processes involved in the defense of an organism; however, when it occurs in some organs, such as brain, in response to an insult, or if it is inappropriate, such as in autoimmune diseases, inflammation can be harmful and therefore requires pharmacological treatment.
  • the inflammatory response involves many effector mechanisms that produce a multiplicity of vascular and cellular reactions. Vasodilatation, increased microvascular permeability, chemotaxis, cellular activation, pain and finally repair are mediated by the local production and release of several specific mediators. Cytokines, chemokines, arachidonic acid derivatives (prostaglandins, thromboxanes and leukotrienes), oxygen and possibly nitrogen radicals, play a regulatory role in this complex and highly balanced process.
  • pro-inflammatory and anti-inflammatory mediators have pleiotropic effects, including the property of regulating one another. Therefore it is not surprising that the previously described cytokines, cytokine regulators, chemokines and "pro-inflammatory" enzymes are involved in numerous diseases where they can be either deleterious or beneficial. When appropriately regulated and balanced, these agents protect the host by activating defense mechanisms and therefore their complete inhibition is not desirable. However, if the inflammation is inappropriate and the expression of those mediators is highly dysregulated, then tissue damage may result. Compounds that would selectively and simultaneously down regulate pro-inflammatory mediators and up-regulate anti-inflammatory ones without totally blocking their physiological beneficial effects would have a clear therapeutic benefit for a wide range of disease states.
  • the present invention relates to pharmaceutical compositions comprising as an active ingredient non-psychotropic cannabinoids and their derivatives that are now disclosed unexpectedly to act as direct or indirect regulators of genes involved in inflammatory mechanisms.
  • the present invention encompasses any synthetic or natural cannabinoid which is essentially devoid of appreciable psychomimetic activity.
  • synthetic non-psychotropic derivatives of dexanabinol also known as HU-211.
  • the PGE 2 inhibitory activity displayed by the preferred compounds does not occur at the level of the COX-2 enzymatic activity, but rather at the level of gene regulation.
  • novel non-psychotropic cannabinoids are useful for the treatment of acute and chronic inflammation, autoimmune diseases and related disorders, pain, infections, liver diseases, cardiovascular disorders, gastrointestinal disorders, disorders of the central and peripheral nervous system including neurodegenerative diseases, respiratory diseases, renal diseases, post-operative complications, tissue rejection and certain types of cancer through means not previously envisioned.
  • compounds of the invention act at the level of gene transcription by down-regulating pro-inflammatory mediators or by up-regulating anti-inflammatory ones or by having both activities simultaneously serve as a basis for treating a wide range of conditions with said compounds.
  • the present invention encompasses pharmaceutical compositions for decreasing the transcription of at least one of the pro-inflammatory mediators COX-2, IL-l ⁇ , IL-2, iNOS, TNF- ⁇ and MCP-1, comprising as an active ingredient a compound of general formula (I): Formula I
  • R- t is selected from the group consisting of a) R' where R' is selected from the group consisting of
  • R 2 is selected from the group consisting of a) a halogen, b) a linear, branched or cyclic, saturated or unsaturated C ⁇ -C 6 alkyl, and c) -OR wherein R is selected from the group consisting of
  • R is hydrogen or a linear, branched or cyclic, saturated or unsaturated C ⁇ -C 6 alkyl optionally containing a terminal -OR" or -OC(O)R'" moiety wherein R'" is hydrogen or a linear, branched or cyclic, saturated or unsaturated Ci-C ⁇ alkyl, and
  • R 3 is selected from the group consisting of a) a linear, branched or cyclic, saturated or unsaturated C ⁇ -C ⁇ 2 alkyl, b) -OR a , in which R a is a linear, branched or cyclic, saturated or unsaturated C 2 -C 9 alkyl which may be substituted at the terminal carbon atom by a phenyl group, and c) a linear, branched or cyclic, saturated or unsaturated C ⁇ -C 7 alkyl-OR" wherein R" is as previously defined; and pharmaceutically acceptable salts, esters or solvates thereof.
  • the present invention also encompasses pharmaceutical composition for increasing the transcription of at least one of the anti-inflammatory cytokine IL-10, the protective cytokine IL-6 and of the suppressors of cytokine signaling SOCS-1 and SOCS-3, comprising as an active ingredient a compound of general formula (I) as previously defined.
  • R ⁇ is a heterocyclic moiety selected from the group consisting of an imidazolyl, an imidazolinyl, a morpholino, a piperidyl, a piperazinyl, a pyrazolyl, a pyrrolyl, a pyrrolidinyl, a triazolyl, and a tetrazolyl, wherein each cyclic moiety may optionally be further substituted with at least one substituent selected from the group consisting of C ⁇ - 6 alkyl, C ⁇ - 6 alkyloxy, C ⁇ _ 6 alkylthio, keto, carboxy, nitro, saturated or unsaturated cyclic moieties or aromatic or heterocyclic moieties wherein each ring comprises 3-8 carbon
  • Ri is selected from the group consisting of hydroxyl, imidazole, pyrazole, oxazole, isoxazole, tetrahydropyridine, pyrazoline, oxazoline, pyrrolidine, imidazoline, 2-thio-imidazole, 2- methylthio-imidazoline, 4-methyl-2-imidazoline, 4,4-dimethyl-2-imidazoline, methyl sulf ⁇ de, methylsulfoxide, acetamido, benzamide, cyano, 1,2,4-triazole, 1,3,4-triazole, 1,2,3,4-tetrazole, 1,2,3,5-tetrazole, thiophene, phenyl, morpholine, thiomorpholine, thiazolidine, glycerol, piperazine, 4-piperidinopiperidine, 4-methylpiperidine and tetrahydropyran.
  • Ri is selected from the group consisting of mono or di-substituted amines wherein the substituent is selected from the group consisting of an d-6 alkyl, C ⁇ - 6 alkyloxy, C ⁇ - 6 alkylthio, imidazolyl, an imidazolinyl, a morpholino, a piperidyl, a piperazinyl, a pyrazolyl, a pyrrolyl, a pyrrolidinyl, a triazolyl, and a tetrazolyl, wherein each cyclic moiety may optionally be further substituted with at least one substituent selected from the group consisting of C ⁇ .
  • each ring comprises 3-8 carbons optionally interrupted by 1-4 heteroatoms, said heteroatoms each independently selected from the group consisting of N, O, and S, wherein each ring optionally is further substituted with one or more groups selected from the group consisting of C ⁇ _ 6 alkyl, C ⁇ - 6 alkyloxy, d- 6 alkylthio, keto, carboxy, or nitro, wherein d. 6 alkyl, C ⁇ - 6 alkoxy and C ⁇ - 6 alkylthio are intended to include saturated and unsaturated linear, branched and cyclic structures.
  • a pharmaceutical composition which down-regulates gene expression of at least one the pro-inflammatory mediators COX-2, IL-l ⁇ , IL-2, iNOS, TNF- ⁇ and MCP-1, and up-regulates gene expression of at least one of the anti-inflammatory cytokine IL-10, the protective cytokine IL-6 and of the suppressors of cytokine signaling SOCS-1 and SOCS-3, comprising as an active ingredient a compound of the general formula (I) wherein R 2 is OH R 3 is 1,1- dimethylheptyl, there is a double bond between C6 and Cl, and Ri is selected from the group consisting of hydroxyl, 2-mercaptoimidazole, imidazole, pyrazole, 4-methylpiperidine, and 4-piperidino-piperidine.
  • compositions may contain in addition to the active ingredient conventional pharmaceutically acceptable carriers, diluents and excipients necessary to produce a physiologically acceptable and stable formulation.
  • compositions can be administered by any conventional and appropriate route including oral, parenteral, intravenous, intramuscular, intralesional, subcutaneous, transdermal, intrathecal, rectal or intranasal.
  • the pharmaceutical compositions Prior to their use as medicaments for preventing, alleviating or treating an individual in need thereof, the pharmaceutical compositions will be formulated in unit dosage.
  • the selected dosage of active ingredient depends upon the desired therapeutic effect, the route of administration and the duration of treatment desired.
  • a further aspect of the present invention provides a method of preventing, alleviating or treating a patient by regulating pro- and anti-inflammatory mediators selected from COX-2, IL-l ⁇ , IL-2, iNOS, TNF- ⁇ , MCP-1, IL-10, IL-6, SOCS-1 and SOCS-3, by administering to said patient a therapeutically effective amount of pharmaceutical composition containing as an active ingredient a compound of general formula (I) as previously defined.
  • a further aspect of the present invention relates to the use for the manufacture of a medicament for preventing, alleviating or treating a disease by regulating pro- and anti- inflammatory mediators selected from COX-2, IL-l ⁇ , IL-2, iNOS, TNF- ⁇ , MCP-1, IL-10, IL-6, SOCS-1 and SOCS-3, of a compound of general formula (I) substantially as shown in the specification.
  • Figure 1 shows the effect of various doses of dexanabinol and its analogs on IL-2 in Jurkat cells activated with PMA and Calcium ionophore.
  • panel A the down-regulatory effect is measured at the level of IL-2 gene expression by real-time RT-PCR.
  • panel B the down- regulatory effect is measured at the level of IL-2 secretion.
  • Figure 2 shows the down-regulatory effect of 10 ⁇ M of dexanabinol and its analogs on COX-2 gene expression in Jurkat cells activated with PMA and Calcium ionophore, as measured by real-time RT-PCR.
  • Figure 3 shows the effect of dexanabinol and its analogs on gene expression in the brains of mice submitted to MCAo 18 hrs before the measurements.
  • panel A the down- regulatory effect is measured on the pro-inflammatory mediators COX-2, MCP-1 and IL-2.
  • panel B the up-regulatory impact is measured on anti-inflammatory IL-10.
  • Figure 4 shows the effect of PRS-211,092 (A) as compared to vehicle ( ⁇ ) on expression of various inflammatory related genes as a function of time from ConA induction of liver injury.
  • A IL-2; B: MCP-1; C: TNF- ⁇ ; D: IL-l ⁇ ; E: IL-6; F: SOCS-1; and G: SOCS-3.
  • Figure 5 shows the effect of various doses of PRS-211,092 and PRS-211,220 on NF-AT driven expression of luciferase in activated T cells.
  • Figure 6 shows the effect of various doses of dexanabinol, PRS-211,092 and PRS-211,220, as well as Celecoxib and Dexamethasone (DXM), as compared to vehicle, on paw thickness in carrageenan induced paw edema.
  • DXM Dexamethasone
  • Figure 7 shows the effect of various doses of dexanabinol on tumor growth of in vivo implanted LoNo colorectal cancer cells.
  • Figure 8 shows the effect of dexanabinol and PRS-211,220 in vivo in the MPTP model of neurodegeneration.
  • the neurological outcome is measured in a short-term study.
  • the functional outcome is measured using the rotarod test in a long-term study.
  • compositions of the present invention cause an increase in gene expression of at least one of the anti-inflammatory cytokine IL-10, the protective cytokine IL-6 and of the suppressors of cytokine signaling SOCS-1 and SOCS-3.
  • the mechanism of action can either be through direct regulation of gene expression or through indirect feedback mechanisms. It will be noted that the compounds of the present invention have been tested for their impact on gene expression on a limited set of genes selected for their known involvement in the immunomodulatory and/or anti-inflammatory signaling cascades. Assaying the effect of those non-psychotropic cannabinoid derivatives on a larger set of genes, such as found in microarrays, may reveal additional genes that are involved in the new gene regulatory action herein disclosed.
  • the present invention relates to THC-type compounds which are characterized by an absolute stereochemistry at the positions 3 and 4 of the molecule (3S,4S), which is opposite to the (3R,4R) configuration in the natural series.
  • the natural compounds of the (3R,4R) configuration produce undesirable psychotropic "cannabis" type effects, which preclude their use for other therapeutically interesting effects.
  • the compounds of the invention being of the (3S,4S) configuration are substantially devoid of the undesired psychotropic effect and thus can be used for the treatment of various diseases and disorders.
  • the term "essentially free” qualitatively refer to (3S,4S) compounds of high optical purity substantially devoid of the undesired psychotropic effect lying with the (3R,4R) enantiomer.
  • the quantitative criterion of the minimum acceptable degree of optical purity of an intended therapeutic enantiomer is dictated by the pharmacological potency of the opposite enantiomer.
  • the enantiomeric pair HU-210 and HU-211, respectively of (3R,4R) and (3S,4S) configuration is an extreme example of such a situation, HU-210 being hundred times more psychoactive than natural ⁇ 9 -THC, the major active constituent in marijuana.
  • the very highly undesirable psychotropic effects of HU- 210 require that HU-211 should be of very high enantiomeric purity of at least 99.8% (Mechoulam R. et al., Tetrahedron Asymmetry 1(5): 315-8, 1990).
  • prodrug represents compounds which are rapidly transformed in vivo to the parent compounds of formula (I), for example by hydrolysis in blood.
  • Some of the compounds of formula (I) are capable of further forming pharmaceutically acceptable salts and esters.
  • “Pharmaceutically acceptable salts and esters” means any salt and ester that is pharmaceutically acceptable and has the desired pharmacological properties. Such salts include salts that may be derived from an inorganic or organic acid, or an inorganic or organic base, including amino acids, which is not toxic or undesirable in any way.
  • the present invention also includes within its scope solvates of compounds of formula (I) and salts thereof, for example, hydrates. All of these pharmaceutical forms are intended to be included within the scope of the present invention.
  • “inhibiting, reducing, or decreasing effect” is the ability to reduce the activity under discussion by at least 20%, preferably 40%, more preferably 60% and most preferably 80% or greater.
  • enhancing or increasing effect is the ability to increase the activity under discussion by at least 2 folds, preferably 3 folds, more preferably 4 folds and most preferably 5 folds or more.
  • prophylactically effective is intended to qualify the amount of compound which will achieve the goal of prevention, reduction or eradication of the risk of occurrence of the disorder, while avoiding adverse side effects.
  • therapeutically effective is intended to qualify the amount of compound that will achieve, with no adverse effects, alleviation, diminished progression or treatment of the disorder, once the disorder cannot be further delayed and the patients are no longer asymptomatic.
  • the compositions of the present invention are prophylactic as well as therapeutic.
  • the "individual" or “patient” for purposes of treatment includes any human or mammalian subject affected by any of the diseases where the treatment has beneficial therapeutic impact.
  • the alkyl substituents can be saturated or unsaturated, linear branched or cyclic, the latter only when the number of carbon atoms in the alkyl chain is equal or superior to 3.
  • genes affected by compounds of the invention are listed below and the diseases wherein abnormal regulation of said genes is implicated in the pathological progression are briefly reviewed.
  • prostaglandins have been known for some time to play a major role in the inflammatory process. They are involved as mediators of pain, edema and vascular permeability in arthritic diseases and they have been postulated to be involved in the pathophysiology of colorectal cancer.
  • the biosynthesis of prostaglandins depends upon the action of cyclooxygenase (COX), recently found to exist in the human as cyclooxygenase type 1 (COX-1) and cyclooxygenase type 2 (COX-2).
  • Both enzymes are involved in the synthesis of prostaglandins, COX-1 constitutively and COX-2 following induction by a number of agents including mitogens, endotoxins, hormones, cytokines, stress conditions and growth-factors.
  • prostaglandins have both physiological and pathological roles, it has been assumed that the constitutive COX-1 was responsible for the important physiological functions for example in the GI tract, while the inducible COX-2 was mainly responsible for the pathological effects of prostaglandins in inflamed tissues.
  • recent pharmacological studies have shown that COX-2 is not exclusively expressed in inflamed tissues, but is constitutively present in several organs where it synergizes with COX-1 in maintaining homeostasis.
  • COX-2 inhibitors already exist on the market and have a wide range of therapeutic benefits. COX-2 inhibitors were already found effective in the treatment of osteoarthritis, rheumatoid arthritis, ocular inflammation, acute and chronic menstrual pain, gastritis caused by bacterium helicobacter pylori.
  • a selective inhibitor would have potential anti-cancer effects, such as with breast and colorectal cancer, would be useful in the treatment of polyps and angiogenesis and be an attractive candidate for the treatment of neurological damage either resulting from spinal cord injury, cerebral ischemia or neurodegenerative disorders such as Alzheimer disease and Parkinson's disease or AIDS associated dementia.
  • a positive role for COX-2 inhibition has also been suggested in chronic liver diseases such as cirrhosis. Recent finding suggest that COX-2 is a major source of systemic prostacyclin synthesis, and its increased production is observed in patients with signs of platelet activation such as unstable angina, severe atherosclerosis and during angioplasty.
  • Interleukin- l ⁇ is a potent pro-inflammatory cytokine that has been implicated in a broad spectrum of diseases.
  • Cells known to express IL-l ⁇ include astrocytes, adrenal cortical cells, NK cells, macrophages and monocytes, endothelial cells, keratinocytes, megakaryocytes and platelets, neurons, neutrophils, oligodendroglia, osteoblasts, Schwann cells, trophoblasts, and T cells plus fibroblasts.
  • IL-l ⁇ is a key factor in several inflammatory disorders that accompany for example septic shock, IBD, pancreatitis, ulcerative colitis, pulmonary inflammation and wound healing.
  • IL-l ⁇ central nervous system
  • Exacerbation may be a result of either the direct cytotoxic action of IL-l ⁇ on resident cells in the CNS or it may be a result of secondary bystander damage by the leukocytes recruited to the brain in response to IL- l ⁇ production.
  • IL-l ⁇ Inappropriate production of IL-l ⁇ was also observed in immune disorders such as allergy, systemic lupus erythematosus (SLE), psoriasis, graft versus host disease and MS.
  • IL-l ⁇ is up-regulated in patients suffering from Gaucher's disease (GD), and it is speculated that this over-expression may relate to the pathophysiology of some of the clinical manifestations of GD.
  • GD Gaucher's disease
  • pancreatic beta-cell destruction leading to insulin dependent diabetes mellitus shows dependence on IL-1.
  • IL-l ⁇ which might affect the production of other various cytokines as well as the regulation of other cellular factors, has been implicated in the progression of benign oncologic conditions to severe and often fatal malignancies, in atherosclerosis and other cardiovascular disorders, in infectious diseases, in renal and liver dysfunction, acute respiratory distress syndrome (ARDS), in ischemic and reperfusion injury and multiple organ failure.
  • ARDS acute respiratory distress syndrome
  • the long list of diseases wherein IL-l ⁇ was found implicated supports its central role as a pivotal pro-inflammatory mediator.
  • the induction of the IL-2 gene is a key event in T cell activation that is required for the resting cells to become effector cells.
  • a cascade of cytoplasmic signaling molecules leads to the assembly of several transcription factors at their corresponding sites on the IL-2 promoter region in the nucleus.
  • Each one of these transcription factors including AP-1, NF- ⁇ B and NFAT, is regulated by different signaling pathways, which act in concert to elicit full activation of the IL-2 gene.
  • Similar mode of IL-2 gene regulation is obtained by T cell activation with phorbol myristate acetate (PMA) and calcium ionophore.
  • PMA phorbol myristate acetate
  • Immunosuppressive drugs such as cyclosporin A strongly inhibit the transcription of the IL-2 gene and their contribution to the therapeutic arsenal is well known.
  • Compounds able to inhibit the production of the cytokine IL-2 are potential immunosuppressive drugs important in the treatment of disorders where T cell activation plays a pivotal role, in particular inflammatory conditions with an etiology including an autoimmune component such as arthritis, rheumatic diseases, systemic lupus erythematosus, myasthenia gravis, inflammatory bowel disease, chronic liver disease, heart failure, multiple sclerosis, inflammatory demyelinating neuropathies, psoriasis, diabetes type 1, parasitic infections, uveitis and other ocular inflammatory conditions, Sj ⁇ gren's syndrome, glomerulonephritis and transplant rejection.
  • an autoimmune component such as arthritis, rheumatic diseases, systemic lupus erythematosus, myasthenia gravis, inflammatory bowel disease, chronic
  • Interleukin-6 is critical to the regulation of the immune and haematopoietic systems.
  • the pleiotropic nature of this cytokine family has resulted in a variety of pseudonames based on its multiple biological functions.
  • IL-6 elicits B cells to undergo proliferation and differentiation into antibody-forming cells; assists in IL-4 dependant IgE synthesis and T cell activation, growth and differentiation.
  • IL-6 also acts in conjunction with IL-3 to induce the proliferation of pluripotent haematopoietic progenitors.
  • this cytokine induces the expression of acute phase proteins.
  • IL-6 is secreted by T cells, B cells, mast cells, monocytes, macrophages, hepatocytes, fibroblasts, endothelial cells, keratinocytes and many tumor cell lines.
  • Adipocytes, bone marrow stroma cells, mesangial cells and some cell types of the central nervous system also produce this cytokine.
  • IL-6 production is generally correlated with cell activation.
  • IL-6 has been described as both a pro-inflammatory and anti-inflammatory mediator, and its levels are altered in a number of diseases: in inflammatory and autoimmune diseases, such as RA and other forms of arthritis, SLE, ulcerative colitis, Crohn's disease, pancreatitis, diabetes, MS, psoriasis; in infectious diseases, such as HIV, bacterial infections and sepsis, viral and bacterial meningitis; in oncologic disorders such as metastatic melanoma, cervical cancer, myeloid leukemia, multiple myeloma, Hodgkin's disease, metastatic renal cell carcinoma, prostate tumors; in renal insufficiency and dialysis, such as glomerulonephritis, nephropaties and hemodialysis; in liver diseases, such as chronic liver diseases, alcoholic liver cirrhosis, hepatitis and hepatectomies; in kidney, bone marrow or liver transplantation; in AD, bums victims and patients suffering from myocardi
  • IL-6 is considered to be an early marker of injury severity following trauma. The nature of IL-6 action depends upon time and site of expression, which ultimately influence if this cytokine will act as pro- or anti- inflammatory. However, recent studies suggest that IL-6 should be considered as a protective cytokine in the overall balance of cytokine regulation. Proper regulation of this important cytokine will have clear beneficial therapeutic impact.
  • Interleukin- 10 down-regulates the production of pro-inflammatory cytokines and chemokines by activated macrophages, monocytes, polymorphonuclear leukocytes and eosinophils. It has been recently suggested that part of this down-regulation is achieved by elevation in the levels of SOCS molecules (Suppressors Of Cytokine Signaling). Therefore IL-10 is an anti-inflammatory cytokine that plays a role in suppressing immune and inflammatory responses. There is evidence that IL-10 can control both T helper 1 (Thl) type of responses and also Th2 mediated inflammatory processes.
  • Thl T helper 1
  • IL-10 has a beneficial effect on a variety of acute and chronic inflammatory and autoimmune events including but not limited to rheumatoid arthritis, ischemia-reperfusion injury, atherosclerosis, psoriasis, pemphigus, allergic contact sensitivity reactions, uveitis, organ transplantation, injury, infection and sepsis, inflammatory bowel disease, acute pancreatitis, asthma, nephro toxic nephritis and certain malignancies.
  • iNOS Nitric oxide (NO) is a short lived molecule required for many physiological functions in host defence, inflammation and immunity. NO is synthesized by the enzyme nitric oxide synthase (NOS) and overproduced during various pathological inflammatory states.
  • nNOS neuronal
  • eNOS entothelial
  • iNOS inducible
  • nNOS neuronal
  • eNOS entothelial
  • iNOS inducible
  • nNOS and iNOS Overproduction of NO by nNOS and iNOS have been reported in a number of clinical disorders including acute and chronic neurodegenerative disorders, convulsions, pain, septic shock, asthma, tissue damage following inflammation, Crohn's disease, SLE, osteoarthritis, rheumatoid arthritis, allograft rejection and in certain cancers.
  • iNOS is induced by endotoxin or pro-inflammatory cytokines and in turn modulate expression of the latter.
  • Selective inhibition of nNOS or iNOS is expected to provide a novel therapeutic approach to various diseases.
  • Monocyte chemoattractant protein-1 (MCP-1) is a pro-inflammatory chemokine of the C-C family, responsible for the recruitment and activation of mainly monocytes, macrophages, basophils, mast cells, T cells, and natural killer cells.
  • the activated monocytes which are recruited to the site of injury, secrete in turn inflammatory agents such as TNF- ⁇ , IL-l ⁇ , nitric oxide and prostaglandins.
  • MCP-1 can be involved in beneficial processes such as wound healing, but when expressed in excess it becomes involved in the pathophysiology of a large number of inflammatory and autoimmune diseases. MCP-1 has been implicated in a large number of diseases that affect various organs by means of acute or chronic inflammation.
  • Pathological sites of action of MCP-1 include the skeleton with disorders such as rheumatoid arthritis and various types of osseous inflammation, the kidneys with nephrites, nephritic syndromes and nephrosis characterized by glomerular nephritides, the eyes with uveitis, vitreoretinal disorders, proliferative diabetic retinopathy, and other ocular inflammatory conditions, the cardiovascular system with MCP-1 involvement in the early stages of atherosclerosis, in restenosis and in the inflammatory response following myocardial infarction, the respiratory system with alveolitis, asthma and lung fibrosis or allergic inflammation, the digestive tract with inflammatory bowel diseases (IBD) including both ulcerative colitis and Crohn's disease.
  • IBD inflammatory bowel diseases
  • the nervous system is also an important target where MCP-1 up- regulation has been observed in various types of pathology.
  • MCP-1 increases in MCP-1 level have been observed following both head trauma and brain ischemia as in stroke, and in immune mediated inflammation as seen in experimental autoimmune encephalomyelitis (EAE) or multiple sclerosis (MS), and in the inflammatory phenomenon associated with neurodegenerative disorders such as Alzheimer's disease (AD).
  • EAE experimental autoimmune encephalomyelitis
  • MS multiple sclerosis
  • AD Alzheimer's disease
  • MCP-1 is also involved in the development of peripheral nervous system (PNS) disorders characterized by mononuclear cell infiltration and related demyelinating disorders.
  • PNS peripheral nervous system
  • MCP-1 is involved in vasculitis, angiogenesis, tumor growth and metastasis, graft rejection, certain types of bacterial, parasitic or viral infections, psoriasis, pemphigus and related disorders, delayed type hypersensitivity reactions of the skin, Hodgkin's disease and a number of chronic diseases characterized by a significant infiltrate of monocytes, including sarcoidosis, Wegener's granulomatosis and tuberculosis. MCP-1 is also very important in cases where complications occur following surgical interventions such as, for example, angioplasty, atherectomy, circulatory recovery techniques, transplants, organ replacement, tissue replacement and prosthetic implants. Thus, blocking MCP-1 production and therefore leukocyte recruitment to target tissues in inflammatory and autoimmune disease would be a highly effective intervention.
  • Cytokines exert their biological activity through binding to specific cell surface receptors, that initiate the appropriate intracellular signal transduction cascade which in turn lead to the physiological outcome.
  • many common themes have emerged in cytokine signal transduction.
  • the Janus kinases Upon receptor multimerisation, the Janus kinases (JAKs) are activated and phosphorylate, among other proteins, the signal transducers and activators of transcription (STATs). Dimers of phosphorylated STATs then move into the nucleus where they bind to recognition sequence in target genes to increase transcription.
  • STATs signal transducers and activators of transcription
  • Dimers of phosphorylated STATs then move into the nucleus where they bind to recognition sequence in target genes to increase transcription.
  • Such signaling pathway needs to be also negatively regulated to ensure the timely switch off of the biological response.
  • SOCS proteins are an important element in a classic negative feedback loop that regulates JAK/STAT signal transduction initiated by many cytokines. Following cytokine activation the cell will not only display increased transcription of the genes important in mediating the biological effects of the cytokine but also genes encoding the SOCS protein which limit the biological effect of the cytokine. At least seven SOCS proteins were identified till now and their activity is presently being unraveled.
  • SOCS-1 and SOCS-3 are induced by about the same set of cytokines, including IL-6, Growth Hormone (GH), IL-10 and GM-CSF, though with different efficiency.
  • SOCS-1 inhibits IL-6, GH signaling, IL-2, IL-4, Interferons (IFN) ⁇ , ⁇ and ⁇ , LIF, oncostatin M and trombopoeitin.
  • SOCS-3 down-regulates leptin, GH, IFN- ⁇ , IFN- ⁇ and IFN- ⁇ .
  • Tumor necrosis factor has been attributed a central role in inflammatory processes. In all disorders with an inflammatory component the common process is that the initial injury, whether initiated by an infectious, chemical, or environmental agent, produces focal tissue necrosis in the target organ. As a result of this damage, tissue-fixed macrophages and circulating monocytes migrate to the damaged site, become activated and secrete products that cause additional cell damage or induction of inflammatory products thus amplifying the response. TNF- ⁇ is a cytokine produced primarily by monocytes and macrophages.
  • TNF- ⁇ is cytotoxic and regulates inflammatory processes through induction for instance of IL-1, IL-6, IL-8, macrophage inflammatory protein (MIP)-2, granulocyte-macrophage colony stimulating factor (GM- CSF) and adhesion molecules.
  • MIP macrophage inflammatory protein
  • GM- CSF granulocyte-macrophage colony stimulating factor
  • TNF- ⁇ is involved in septic shock syndrome, autoimmune and inflammatory processes including Crohn's disease, brain injury, venous thromboses, arteriosclerosis, vasculitis, IBD, MS, EAE, SLE, AD, PD, AIDS dementia, contact dermatitis, mixed connective tissue disease, arthritis, organ specific toxic response, hepatic injury during sepsis and reperfusion, chronic inflammatory lung diseases, muscle wasting and cachexia.
  • the therapeutic importance of TNF- ⁇ blockade is tremendous, nevertheless it should be kept in mind that TNF- ⁇ is also involved in normal physiological and repair processes and totally eliminating this cytokine would be detrimental.
  • compositions according to the present invention will be useful for treating indications having an inflammatory or autoimmune mechanism involved in their etiology or pathogenesis.
  • Such diseases or disorders are exemplified by multiple sclerosis, amyotrophic lateral sclerosis, systemic lupus erythematosis, myasthenia gravis, Sjogren's syndrome, diabetes mellitus type I, late onset diabetes type 2, sarcoidosis; skeletal and connective tissue disorders including arthritis, rheumatoid arthritis, osteoarthritis and rheumatoid diseases; ocular inflammation related disorders including uveitis, vitreoretinal disorders, proliferative diabetic retinopathy, allergic conjunctivitis; metabolic diseases that involve abnormalities in lipid peroxisomes and lipid peroxidation and/or oxidative stress; skin related disorders including psoriasis, pemphigus and related syndromes, delayed-type hypersensitivity and contact dermatitis; respiratory diseases including cystic fibrosis, chronic bronchitis, emphysema, chronic obstructive pulmonary disease, asthma, allergic r
  • the pharmaceutical compositions comprising as an active ingredient a compound regulating the gene expression of the pro- and anti-inflammatory mediators selected from COX-2, IL-l ⁇ , IL-2, iNOS, TNF- ⁇ , MCP-1, IL-10, IL-6, SOCS-1 and SOCS-3, act as neuroprotectors.
  • a compound regulating the gene expression of the pro- and anti-inflammatory mediators selected from COX-2, IL-l ⁇ , IL-2, iNOS, TNF- ⁇ , MCP-1, IL-10, IL-6, SOCS-1 and SOCS-3.
  • composition of the present invention may also be effective in treating demyelinating disorders and certain chronic degenerative diseases that are characterized by gradual selective neuronal loss such as Parkinson's disease, Alzheimer's disease, AIDS dementia, Huntington's chorea, amyotrophic lateral sclerosis, Kennedy's syndrome, motor neuron disease and prion-associated neurodegeneration.
  • compositions according to the present invention will be useful in treating pain including peripheral, visceral, neuropathic, inflammatory and referred pain.
  • compositions of the present invention may also be effective in cardiovascular protection and/or treatment of atheroma, atherosclerosis, and consequences thereof, restenosis, angioplasty, myocardial ischemia and myocardial infarction.
  • Another feature of the present invention is the ability of the disclosed compounds in chemoprevention in oncological processes including polyps, tumor growth, angiogenesis and metastasis of certain types of cancer, including breast and colon cancer.
  • compositions of the present invention which down-regulate gene expression of at least one the pro-inflammatory mediators COX-2, IL-l ⁇ , IL-2, iNOS, TNF- ⁇ , and MCP-1, comprise as an active ingredient a compound of the general formula (I): Formula I
  • Ri is selected from the group consisting of a) R' where R' is selected from the group consisting of
  • R 2 is selected from the group consisting of a) a halogen, b) a linear, branched or cyclic, saturated or unsaturated C ⁇ -C 6 alkyl, and c) -OR wherein R is selected from the group consisting of
  • R" is hydrogen or a linear, branched or cyclic, saturated or unsaturated C ⁇ -C 6 alkyl optionally containing a terminal -OR" or -OC(O)R'" moiety wherein R'" is hydrogen or a linear, branched or cyclic, saturated or unsaturated C ⁇ -C 6 alkyl, and
  • R 3 is selected from the group consisting of a) a linear, branched or cyclic, saturated or unsaturated C ⁇ -C ⁇ 2 alkyl, b) -OR a , in which R a is a linear, branched or cyclic, saturated or unsaturated C 2 -C 9 alkyl which may be substituted at the terminal carbon atom by a phenyl group, and c) a linear, branched or cyclic, saturated or unsaturated C ⁇ -C 7 alkyl-OR'" wherein R'" is as previously defined; and pharmaceutically acceptable salts, esters or solvates thereof.
  • compositions of the present invention which up-regulate gene expression of at least one of the anti-inflammatory cytokine IL-10, the protective cytokine IL-6 and of the suppressors of cytokine signaling SOCS-1 and SOCS-3, comprise as an active ingredient a compound of the general formula (I):
  • Ri is selected from the group consisting of a) R' where R' is selected from the group consisting of
  • R 2 is selected from the group consisting of a) a halogen, b) a linear, branched or cyclic, saturated or unsaturated C ⁇ -C 6 alkyl, and c) -OR wherein R is selected from the group consisting of
  • R" is hydrogen or a linear, branched or cyclic, saturated or unsaturated C ⁇ -C 6 alkyl optionally containing a terminal -OR" or -OC(O)R'" moiety wherein R'" is hydrogen or a linear, branched or cyclic, saturated or unsaturated C ⁇ -C 6 alkyl, and
  • R 3 is selected from the group consisting of a) a linear, branched or cyclic, saturated or unsaturated C ⁇ -C ⁇ alkyl, b) -OR a , in which R a is a linear, branched or cyclic, saturated or unsaturated C 2 -C 9 alkyl which may be substituted at the terminal carbon atom by a phenyl group, and c) a linear, branched or cyclic, saturated or unsaturated C ⁇ -C 7 alkyl-OR" wherein R" is as previously defined; and pharmaceutically acceptable salts, esters or solvates thereof.
  • R 2 is hydroxy or lower acyloxy and wherein R 3 is dimethylheptyl or a dimethylalkyl radical with a total of at least 7 carbon atoms.
  • Ri is a heterocyclic moiety selected from the group consisting of an imidazolyl, an imidazolinyl, a morpholino, a piperidyl, a piperazinyl, a pyrazolyl, a pyrrolyl, a pyrrolidinyl, a triazolyl, and a tetrazolyl, wherein each cyclic moiety may optionally be further substituted with at least one substituent selected from the group consisting of C ⁇ _ 6 alkyl, C ⁇ 6 alkyloxy, C _ 6 alkylthio, keto, carboxy, nitro, saturated or unsaturated cyclic moieties or aromatic or heterocyclic moieties wherein each ring comprises 3-8 carbons optionally interrupted by 1-4 heteroatoms, said heteroatoms each independently selected from the group consisting of N, O, and S, wherein each ring optionally is further substituted with one or more groups selected from the group consisting of C ⁇
  • Ri is selected from the group consisting of hydroxyl, imidazole, pyrazole, oxazole, isoxazole, tetrahydropyridine, pyrazoline, oxazoline, pyrrolidine, imidazoline, 2-thio-imidazole, 2- methylthio-imidazoline, 4-methyl-2-imidazoline, 4,4-dimethyl-2-imidazoline, methyl sulfide, methylsulfoxide, acetamido, benzamide, cyano, 1,2,4-triazole, 1,3,4-triazole, 1,2,3,4-tetrazole, 1,2,3, 5-tetrazole, thiophene, phenyl, morpholine, thiomorpholine, thiazolidine, glycerol, piperazine, 4-piperidinopiperidine, 4-methylpiperidine and tetrahydropyran.
  • Ri is selected from the group consisting of mono or di-substituted amines wherein the substituent is selected from the group consisting of an C ⁇ - 6 alkyl, d- 6 alkyloxy, d- 6 alkylthio, imidazolyl, an imidazolinyl, a morpholino, a piperidyl, a piperazinyl, a pyrazolyl, a pyrrolyl, a pyrrolidinyl, a triazolyl, and a tetrazolyl, wherein each cyclic moiety may optionally be further substituted with at least one substituent selected from the group consisting of d.
  • each ring comprises 3-8 carbons optionally interrupted by 1-4 heteroatoms, said heteroatoms each independently selected from the group consisting of N, O, and S, wherein each ring optionally is further substituted with one or more groups selected from the group consisting of C ⁇ - 6 alkyl, d- 6 alkyloxy, C ⁇ . 6 alkylthio, keto, carboxy, or nitro, wherein C ⁇ - 6 alkyl, - 6 alkoxy and C ⁇ - 6 alkylthio are intended to include saturated and unsaturated linear, branched and cyclic structures.
  • a pharmaceutical composition which down-regulates gene expression of at least one the pro-inflammatory mediators COX-2, IL-l ⁇ , IL-2, iNOS, TNF- ⁇ and MCP-1, and up-regulates gene expression of at least one of the anti-inflammatory cytokine IL-10, the protective cytokine IL-6 and of the suppressors of cytokine signaling SOCS-1 and SOCS-3, comprising as an active ingredient a compound of the general formula (I) wherein R 2 is OH, R 3 is 1,1- dimethylheptyl, there is a double bond between C6 and Cl, and Ri is selected from the group consisting of hydroxyl, 2-mercaptoimidazole, imidazole, pyrazole, 4-methylpiperidine, and 4-piperidino-piperidine.
  • compositions of particular interest comprise as an active ingredient compounds within formula (I) previously disclosed as HU-211, also known as dexanabinol, PRS-211,092, PRS-211,095, PRS-211,220, PRS-211,251 and PRS-211,257 in International Patent application WO 01/98289.
  • novel compositions contain in addition to the active ingredient conventional pharmaceutically acceptable carriers, diluents and excipients necessary to produce a physiologically acceptable and stable formulation.
  • Some compounds of the present invention are characteristically hydrophobic and practically insoluble in water with high lipophilicity, as expressed by their high octanol/water partition coefficient expressed as log P values, and formulation strategies to prepare acceptable dosage forms will be applied. Enabling therapeutically effective and convenient administration of the compounds of the present invention is an integral part of this invention. For water soluble compounds standard formulations will be utilized.
  • Solid compositions for oral administration such as tablets, pills, capsules, softgels or the like may be prepared by mixing the active ingredient with conventional, pharmaceutically acceptable ingredients such as corn starch, lactose, sucrose, mannitol, sorbitol, talc, polyvinylpyrrolidone, polyethyleneglycol, cyclodextrins, dextrans, glycerol, polyglycolized glycerides, tocopheryl polyethyleneglycol succinate, sodium lauryl sulfate, polyethoxylated castor oils, non-ionic surfactants, stearic acid, magnesium stearate, dicalcium phosphate and gums as pharmaceutically acceptable diluents.
  • conventional, pharmaceutically acceptable ingredients such as corn starch, lactose, sucrose, mannitol, sorbitol, talc, polyvinylpyrrolidone, polyethyleneglycol, cyclodextrins, dextrans, gly
  • the tablets or pills can be coated or otherwise compounded with pharmaceutically acceptable materials known in the art, such as microcrystalline cellulose and cellulose derivatives such as hydroxypropylmethylcellulose (HPMC), to provide a dosage form affording prolonged action or sustained release.
  • pharmaceutically acceptable materials known in the art, such as microcrystalline cellulose and cellulose derivatives such as hydroxypropylmethylcellulose (HPMC), to provide a dosage form affording prolonged action or sustained release.
  • Other solid compositions can be prepared as suppositories, for rectal administration.
  • Liquid forms may be prepared for oral administration or for injection, the term including but not limited to subcutaneous, transdermal, intravenous, intrathecal, intralesional, adjacent to or into tumors, and other parenteral routes of administration.
  • the liquid compositions include aqueous solutions, with or without organic cosolvents, aqueous or oil suspensions including but not limited to cyclodextrins as suspending agent, flavored emulsions with edible oils, triglycerides and phospholipids, as well as elixirs and similar pharmaceutical vehicles, h addition, the compositions of the present invention may be formed as aerosols, for intranasal and like administration.
  • Topical pharmaceutical compositions of the present invention may be formulated as solution, lotion, gel, cream, ointment, emulsion or adhesive film with pharmaceutically acceptable excipients including but not limited to propylene glycol, phospholipids, monoglycerides, diglycerides, triglycerides, polysorbates, surfactants, hydrogels, petrolatum or other such excipients as are known in the art.
  • pharmaceutically acceptable excipients including but not limited to propylene glycol, phospholipids, monoglycerides, diglycerides, triglycerides, polysorbates, surfactants, hydrogels, petrolatum or other such excipients as are known in the art.
  • the pharmaceutical compositions Prior to their use as medicaments, the pharmaceutical compositions will generally be formulated in unit dosage.
  • the active dose for humans is generally in the range of from 0.05 mg to about 50 mg per kg body weight, in a regimen of 1-4 times a day.
  • the preferred range of dosage is from 0.1 mg to about 20 mg per kg body weight.
  • dosages would be determined by the attending physician, according to the disease to be treated, its severity, the method and frequency of administration, the patient's age, weight, gender and medical condition, contraindications and the like.
  • the dosage will generally be lower if the compounds are administered locally rather than systematically, and for prevention or chronic treatment rather than for acute therapy.
  • a further aspect of the present invention provides a method of preventing, alleviating or treating a patient by regulating pro- and anti-inflammatory mediators selected from COX-2, IL-l ⁇ , IL-2, iNOS, TNF- ⁇ , MCP-1, IL-10, IL-6, SOCS-1 and SOCS-3, by administering to said patient a therapeutically effective amount of pharmaceutical composition containing as an active ingredient a compound of general formula (I) as previously defined.
  • a further aspect of the present invention relates to the use for the manufacture of a medicament for preventing, alleviating or treating a disease by regulating pro- and anti- inflammatory mediators selected from COX-2, IL-l ⁇ , IL-2, iNOS, TNF- ⁇ , MCP-1, IL-10,
  • test compounds are prepared as follows: for in vitro assays the compounds are first dissolved in DMSO and then stepwise diluted in the assay buffer, generally tissue culture medium, down to a final concentration of 0.1% DMSO. For in vivo assays the test compounds are first diluted in cremopho ⁇ ethanol (70% and 30% w/w respectively) and further diluted 1:20 in physiological buffer, generally saline, to reach the appropriate dose. Alternatively, compounds can be first disolved in PEG:ethanol (1:1) and then diluted in Intralipid. Thus, the vehicle is the original "solvent" diluted in the appropriate buffer.
  • genes involved in inflammatory processes either directly, e.g. cytokines and chemokines, or indirectly, e.g. regulators of cytokine signaling pathways or transcription.
  • These genes are either encoding pro-inflammatory mediators, such as the cytokines IL-l ⁇ , IL-2, TNF- ⁇ , the chemokine MCP-1 and the enzyme COX-2, or anti-inflammatory mediators such as the anti- inflammatory cytokine IL-10, the protective cytokine IL-6 or upstream regulators of cytokine such as the suppressors of cytokine signaling SOCS-1 and SOCS-3.
  • Gene expression was assessed by RNA level quantitation and when possible by direct protein quantitation. Whatever the experimental system or the gene to be tested the following procedure were followed for quantitation.
  • RNA preparation and real-time RT-PCR Total RNA was prepared using SV total RNA isolation system (Promega). The cells or tissues were homogenized in lysis buffer. The lysates were transferred to an RNA isolation column, treated with DNAse, washed and eluted according to kit instructions. RNA concentrations were determined using GeneQuant II (Phamacia-Amersham). Complementary DNA (cDNA) was synthesized from total RNA using SUPERSCRIPT II reverse transcriptase (Life Technologies). 2 ⁇ g of total RNA were combined with an oligo
  • Quantitative real-time RT-PCR includes 1 ⁇ l of the cDNA, 300 nM of the appropriate forward and reverse primers (see below) and 7.5 ⁇ l of the reaction mix containing buffer, nucleotides, Taq polymerase and syber green (Syber Green master mix, Applied Biosystems), in a total reaction volume of 15 ⁇ l.
  • Gene amplification was obtained using the GeneAmp 5700 sequence detection system (Applied Biosystems).
  • Amplification included one stage of 10 minutes at 95°C followed by 40 cycles of a 2-steps loop: 20 seconds at 95°C, and 1 minute at 60°C.
  • the amount of the amplified product is measured by the fluorescence of the double strand DNA binding dye, syber Green.
  • the cycle of threshold (C T ) representing the PCR cycle at which an increase in fluorescence above a baseline signal can be first detected, is determined for each product.
  • a delay of one PCR cycle in the C T is translated into a two-fold decrease in starting template molecules and vice versa.
  • the changes in the C T of the specific gene product are normalized to the changes in the C T of a reference gene cyclophilin A or GAPDH. Results are expressed as fold increase of gene expression in the test system above the appropriate control, such as inactivated cell lines or vehicle "treated" animals, hi all cases, results are also normalized to either one of the reference house-keeping genes.
  • Human IL-2 forward 5'- GGGACTTAATCAGC AATATCAACGT-3 '
  • ELISA Enzyme Linked ImmunoSorbent Assay
  • the reaction is stopped and reading is carried out in a spectrophotometer at the appropriate wavelength.
  • Samples are tested at least in duplicate and the appropriate standard curve, consisting of serial dilutions of the recombinant target protein, is incorporated on each plate. Concentration of the protein in the sample is calculated from the standard curve.
  • dexanabinol and its analogs reduce the levels of secreted PGE 2 in LPS activated mouse macrophages (RAW 264.7) cells in vitro (WO 01/98289).
  • the IC 5 o for inhibition of PGE 2 secretion were determined and found to be 10 ⁇ M, 10 ⁇ M, 4 ⁇ M, and 8 ⁇ M, for dexanabinol, PRS-211,092, PRS-211,095 and PRS-211,220, respectively.
  • the IC 50 for inhibition of PGE secretion for the known anti- inflammatory drugs Celecoxib, Rofecoxib and NS-398 were respectively 5 nM, 100 nM and 100 nM in the same experimental setup.
  • Dulbecco's modified Eagle's medium (DMEM) with 4 mM L-glutamine adjusted to contain
  • RNA samples were extracted from the cells 2.5 hrs after activation and COX-2 gene expression levels were analyzed by realtime RT-PCR as previously described.
  • results of this experiment are expressed as fold activation of COX-2 over non- activated macrophages, after normalization to cyclophilin A expression.
  • cells are treated with vehicle only we observe a maximal 6-fold increase in COX-2 expression.
  • a decrease of 67% in RNA levels of COX-2 is observed when the activation is carried out in presence of 10 ⁇ M dexanabinol.
  • all three analogs PRS-211,092, PRS-211,095 and PRS-211,220 cause an inhibition of at least 50%.
  • mice brains intra cerebral ventricular (i.c.v.).
  • Each treatment group was composed of at least five C57/BL male mice (6-8 weeks old, 25 g average body weight,
  • mice were anesthetized with a mixture of 35 mg/kg pental and 8 mg/kg xylazine.
  • LPS was dissolved in saline at 20 ng/ ⁇ l and 5 ⁇ l were injected in each ventricule at a rate of 1 ⁇ l/min with the help of a syringe pump and a brain infusion canula. After each injection, the cannula is left in situ for one more minute to avoid reflux.
  • the various treatment groups, controls including the cremopho ⁇ ethanol vehicle, dexanabinol and its analogs (20 mg/kg), were injected i.p. (0.1 ml/10 g body weight) simultaneously with the i.c.v injection of LPS.
  • PRS-211,092 inhibited gene expression in a dose dependent manner with an IC 50 of 4 ⁇ M for iNOS and of 17.5 ⁇ M for IL-l ⁇ .
  • COX-2 gene expression was elevated 70-fold by activation and under these conditions the dose related effect of PRS-211,092 yielded an IC 50 of 21.5 ⁇ M.
  • the reduction in IL-l ⁇ gene expression correlates to a reduction in IL-l ⁇ secretion.
  • the IC 50 of dexanabinol, PRS-211,092 and PRS-211,220 for inhibition of IL-l ⁇ secretion are 6 ⁇ M, 3 ⁇ M and 4 ⁇ M, respectively.
  • PRS-211,095 was tested at the single concentration of 10 ⁇ M and found to inhibit IL-l ⁇ secretion by 70%, similarly to PRS-211,092 and PRS-211,220 that both yielded 76% inhibition at that same concentration.
  • the active ingredients of the present invention are effective not only in affecting COX-2 RNA level but also in specifically reducing RNA levels of additional inflammatory related genes such as IL-l ⁇ and iNOS.
  • the impact on gene expression correlates with a decrease in secretion of the relevant inflammatory mediators.
  • classical anti-inflammatory therapies which are designed to block one mediator at a time.
  • a compound that can reduce the expression and secretion of multiple inflammatory mediators simultaneously harbors in a single molecule the approach of combined therapy now used to replace the single target classical anti-inflammatory strategies.
  • the brains were cryosectioned (18 ⁇ m) at the level of the entire hippocampus, hnmunohistochemistry staining was carried out using polyclonal rat anti-mouse F4/80 (Serotec, USA) and goat anti-rat-IgG-peroxidase (Jackson, USA), for the detection of activated microglia cells.
  • the slides were stained using the 3,3- diaminobenzidine tetrahydrochloride (DAB) chromagen detection kit of the automated immunostaining system (Nantana, France). Quantitative analysis was carried out by counting the number of immunoreactive cells/mm 2 at the level of the hippocampus. Statistical analysis.
  • DAB 3,3- diaminobenzidine tetrahydrochloride
  • Results are expressed as mean ⁇ SD. Data were analyzed using analysis of variance (ANON A) followed by post-hoc Fisher test and t-test. A value of p ⁇ 0.05 is considered to be statistically significant. Twenty-four hours following LPS injection, we observed a rise in the level of IL-l ⁇ rnR ⁇ A, normalized to cyclophilin. LPS injected animals have 18 fold higher IL-l ⁇ mR ⁇ A levels than saline injected animals. Treatment with 20 mgkg of PRS-211,092, administered simultaneously with the LPS stimulation, reduced the amount of IL-l ⁇ mR ⁇ A fold activation by 56% as compared to its vehicle. Immunohistochemical analysis for the glial cell marker revealed that i.c.v.
  • the induction of the IL-2 gene is the hallmark event of T cell activation that is required for the resting cells to become effector cells. Similar mode of IL-2 gene regulation is obtained by T cell activation with PMA (phorbol-12-myristate- 13 -acetate) and calcium ionophore.
  • PMA phorbol-12-myristate- 13 -acetate
  • calcium ionophore The human acute lymphoma T cell line Jurkat (ATCC # TIB 152) was used to test the possible immunosuppressive effect of dexanabinol and its analogs on T cell activation.
  • the Jurkat cells were grown in RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, and 10% heat inactivated fetal bovine serum.
  • Cells were grown in tissue culture flasks and seeded at appropriate density into 24 wells tissue culture plates. 2 x 10 6 cells in one milliliter were stimulated using 10 ng/ml of PMA (Sigma) and 1 ⁇ M A23187 calcium ionophore (Sigma). Cyclosporin A (Sandoz), a known immunosuppressive drug, was used as positive control. The controls and test compounds were added at indicated concentrations one hour before stimulation. RNA samples were extracted from the cells 6 hrs after activation and IL-2 gene expression levels were analyzed by real-time RT-PCR as previously described.
  • Figure 1 A depicts the result of this experiment as fold activation of IL-2 over non- activated T cells. The results are plotted after normalization to cyclophilin A expression.
  • Dexanabinol and its analogs inhibited IL-2 gene expression in a dose related manner with maximal inhibitory activity at the highest dose tested, 10 ⁇ M.
  • dexanabinol also known as PRS-211,007
  • reduced IL-2 fold of activation by 41%, PRS- 211,092 by 69%, PRS-211,095 by 70% and PRS-211,220 by 84% (the latter not shown).
  • Cyclosporin A completely blocked IL-2 transcription at 10 nM and inhibited it to 50% at 1 nM.
  • IL-2 secretion In parallel, the supernatant from each well was collected 24 hours after the activation and analyzed for the presence of secreted IL-2 by ELISA. The principles of the assay are as previously described.
  • the secondary antibodies were conjugated with HRP, following substrate addition the peroxidase catalyzed color change is stopped by acidification.
  • the absorbance measured at 450 nm is proportional to the concentration of IL-2 in the sample or standard.
  • a standard curve is obtained by plotting the concentrations of recombinant IL- 2 standards versus their absorbances. The IL-2 concentrations in experimental samples are then determined using the standard curve.
  • Results are shown in Figure IB, where the amount of secreted IL-2 (ng/ml) is plotted for each treatment group.
  • the activated cells are treated with vehicle only the maximal level of LL-2 secretion is 8.73 ng/ml.
  • dexanabinol and its analogs PRS-211,092, PRS-211,095 and PRS-211,220 inhibited the secretion of IL-2 in a dose dependent manner at IC 50 of 8 ⁇ M, 0.4 ⁇ M, 1 ⁇ M and 2 ⁇ M respectively.
  • the positive control Cyclosporin A inhibited IL-2 concentrations in the growth medium with an IC 5 o of 0.06 nM in the same experimental setup.
  • Mast cells are multifunctional bone marrow derived cells that upon activation release many potent inflammatory mediators. Release is done either from preformed granules, trough the process of degranulation, or following stimulation-induced de novo synthesis.
  • the molecules released by Mast cells include biogenic amines such as histamine, chemokines, cytokines, enzymes, growth factors, peptides, arachidonic acid products and proteoglycans. It should be noted that mast cells are also known to play a key role in generating pain signal.
  • RBL-2H3 cells (ATCC # CRL-2256) are grown in EMEM medium with Earle's BSS, 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non essential amino acids, 1.0 mM sodium pyruvate, and 15% heat inactivated fetal calf serum.
  • Cells are grown in tissue culture flasks and seeded at appropriate density into 24 wells tissue culture plates. 2 x 10 5 cells in one milliliter are seeded. Following overnight incubation, the plated cells are preincubated for half an hour with test compounds and controls and then stimulated with 10 ng/ml of PMA (Sigma) and 1 ⁇ M A23187 calcium ionophore (Sigma).
  • the degranulation process is allowed to proceed at 37°C for various periods of time, depending on the level of analysis and the molecule monitored.
  • Cells are collected after half an hour for RNA preparation while supernatants are collected two and an half hours after stimulation, for the analysis of hexoaminidase and PGE 2 secretion, and IL-4 and TNF- ⁇ gene expression and secretion.
  • COX-2 gene expression RNA is collected one hour after stimulation.
  • the Src family inhibitor PP2 or the PKC inhibitor GF109203X both from Calbiochem
  • the controls and test compounds are added at indicated concentrations before stimulation.
  • the concentrations of the agent under study are measured in commercially available EIA, ELISA or enzymatic assays.
  • RNA samples are extracted from the cells at the appropriate predetermined time points after activation and gene expression levels are analyzed by real-time RT-PCR as previously described wherein the constitutively expressed COX-1 is used to normalize gene expression.
  • Example 8 Quantitation of gene expression in brain tissue following Middle Cerebral Artery Occlusion.
  • This model corresponds to cerebral ischemia as observed in stroke.
  • Mice C57/BL, male, 25 gr average body weight, Harlan, Israel
  • halothane in 30% oxygen and 70% nitrogen (4% for induction in an anesthesia chamber, and 1-2% in a facemask for maintenance).
  • a midline incision was made in the skin of the neck, and the tissue underneath was bluntly dissected.
  • the right common carotid artery (CCA) and its junction with the external carotid artery (EGA) and internal carotid artery (ICA) were explored by blunt dissection.
  • the branches of the ECA, the occipital and the superior thyroid artery, were then cauterized.
  • the CCA was then transiently closed by positioning around it a 5-0 silk suture material (Assut, Switzerland). Two cm pieces of the nylon suture material were cut and placed in a solution of 1% Poly-L-Lysine and then dried in an oven (60°C) for 60 minutes. The tip of each piece was rounded under a flame. The ECA is permanently occluded with the same type of suture material. A third closure, transient this time, was done in the ICA with 5-0 silk suture material. A small hole is cut in the ECA and the nylon thread is inserted into the ICA while avoiding entrance into the pterygopalatine artery. The thread is inserted 11 mm until a slight resistance is felt. Then a 5-0 silk suture knot secures the thread. One cm of the thread left outside are then cut. The skin wound is closed by 5-0 silk suture material.
  • Proprioceptive placing 0 2 * Scores are as follows: 0 no observable deficit, 1 limb flexion during hang test, 2 deficit on lateral push.
  • # Scores are as follows: 0 complete immediate placing, 1 incomplete or delayed placing (>2 seconds), 2 absence of placing. Only animals with total scores between 8 to 12 were included in the study.
  • the selected animals are resedated using the same method, the neck wound is then re-opened and the nylon thread is pulled out of the ICA. The skin wound is then closed with 5-0 silk suture material.
  • the controls and test compounds, (dexanabinol also known as PRS-211,007, PRS-211,092, PRS-211,095, and PRS-211,220) are administered 1 minute before the end of the insult. All treatments are delivered i.v. 5 mg/kg (except PRS-211,220 0.5 mg/kg).
  • Vehicle is administered 5 ml/kg. Each treatment group comprised 6 to 8 animals. The drugs were dissolved in PEG-Ethanol and diluted in Intralipid (Pharmacia Upjohn). Eighteen hours later, animals were sacrificed by i.p. injection of pentobarbitone sodium 100 mg/kg. Brains were then removed, and total RNA was prepared from the ipsilateral half of the brains. Gene expression levels were analyzed by real-time RT-PCR as previously described. Results are expressed as fold activation over sham operated animals. Gene expression was normalized to house-keeping gene cyclophilin.
  • COX-2 gene expression in MCAo brains Dexanabinol and its analogs were already shown to be effective in reducing brain damage after stroke and improving outcome in the middle artery cerebral occlusion (MCAo) model in rats and mice. The purpose of this experiment was to check if these functional improvements were achieved by the newly identified mechanism of action. Therefore, COX-2 RNA levels were assessed 18 hrs following MCAo. The results obtained with all genes tested are displayed in Figure 3. Vehicle only treated animals displayed a 5-fold activation of COX-2 gene expression versus sham operated animals. Treatment with dexanabinol and the PRS-211,092 clearly reduced this outcome by 38% and 48% respectively, in comparison with the vehicle treated group (Figure 3A).
  • MCP-1 gene expression in MCAo brains Chemokines are low molecular weight, secreted proteins that chemoattractant and activate specific subpopulations of leukocytes.
  • Monocyte chemoattractant protein- 1 MCP-1
  • MCP-1 RNA levels following MCAo were previously reported (Che et al, Brain Research 902: 171-7, 2001). hnmunohistochemistry studies showed that both ischemic neurons (after 12 hours of ischemic insult) and astrocytes (two days after insult) expressed MCP-1.
  • Dexanabinol and its analogs were already shown to be effective in reducing IL-2 both at the level of gene expression and at the level of secretion in activated T cells. Therefore, we wished to verify their impact in vivo and we assessed IL-2 RNA levels in mice brains 18 hrs following MCAo. Vehicle only treated animals displayed a 4- fold activation of IL-2 gene expression versus sham operated animals. Treatment with dexanabinol and the PRS-211,092 clearly reduced this outcome by 177% and 130% respectively, in comparison with the vehicle treated group (Figure 3A).
  • IL-10 is a potent anti-inflammatory cytokine strongly related to the previously described pro-inflammatory genes. Moreover, it has already been reported that IL-10 gene expression levels increase in rat brain following MCAo (Zhai et al, J. Neurol. Sci. 152:
  • the active ingredient of the present invention is effective in reducing in vivo the RNA level of 3 important pro-inflammatory mediators, COX-2, IL-2 and MCP-1, while it is increasing the RNA level of the anti- inflammatory cytokine IL-10. Consequently, the compounds may be therapeutically effective in the wide variety of iiximune/inflammatory related disorders. Moreover, it should be noted that compounds of the invention are specific both in terms of the inflammatory related genes they regulate and in terms of the cellular targets or tissues in which they act.
  • the concentration of cytokines was also determined in the Organs of interests.
  • the mice were killed by dislocation of the cervical vertebrae, at predetermined time points following ConA injection.
  • the spleen and the liver were removed. Part of the liver was fixed in 4% formaldehyde for histology and the other part was kept at -80°C for protein or RNA extraction.
  • the spleens were weighted and a small part of the spleen was fixed in 4% formaldehyde, while most of the organ is cultured according to the following procedure.
  • Each spleen is squeezed through a cell strainer with the rough end of a 5 ml syringe into 4 ml of RPMI medium.
  • ALT Alanine aminotransferase
  • ALT is an enzyme found mainly in the liver and it is measured to determine whether the liver is damaged or diseased in a variety of human conditions, especially hepatitis and cirrhosis. The level of its release into the bloodstream linearly correlates with the severity of the liver injury and thus it can be used both for diagnosis purposes and to monitor the efficiency of a treatment.
  • the time course of plasma ALT release after ConA injection is known from the literature to peak at eight hours. At this time point the ALT concentration measured was about 1700 units/1 and 5 mg/kg of PRS-211,092 when administered 30 minutes before ConA injection significantly reduced ALT concentrations by 57%.
  • the level of IL-2 gene expression was assessed in the spleen of the ConA injected mice, one and four hours following injury.
  • vehicle treated animals displayed at each time point respectively a 309-fold and a 997-fold increase in IL-2 RNA levels versus saline injected animals.
  • Treatment with 5 mg/kg i.v. of PRS-211,092 reduced this outcome by 46% when tested one hour after injury and by 32% when tested at four hours after injury.
  • the spleen was removed and splenocytes were cultured for 24 hours to allow the assessment of secretion levels.
  • TNF- ⁇ a cytokine produced mainly by activated macrophages
  • TNF- ⁇ has pleiotropic effects both beneficial, as in liver regeneration, and deleterious, when it has direct cytotoxic role in human hepatocytes.
  • TNF- ⁇ has been shown to be a crucial factor in immune mediated hepatitis and is also a mediator of hepatotoxicity in patients with alcoholic liver disease, fulminant hepatic failure and viral hepatitis. In most of these disorders, the concentration of TNF- ⁇ correlate inversely with patients survival.
  • TNF- ⁇ expression was assessed in the liver.
  • Increase in TNF- ⁇ gene expression is detected immediately, as early as 15 minutes following ConA injection (21- fold increase over saline injected animals), and already at this time point treatment with 5 mg/kg PRS-211,092 has an inhibitory effect of 32%.
  • the increase in TNF- ⁇ expression is correspondingly 162, 81 and 38-fold.
  • the test compound reduces these effects by 27% and 17%, and then maintains the same level of TNF- ⁇ expression (Figure 4C).
  • IL-l ⁇ is first up-regulated may have an initial positive effect, such as induction of SOCS-1, further sustained by its later inhibition, as suggested by the decrease in ALT at the end of the study that supports the overall hepatoprotective activity of the test compound.
  • PRS-211,092 reduces IL-6 ConA induced overexpression by 31%, and looses efficacy when the analysis is carried out eight hours after liver injury induction.
  • the effect of PRS-211,092 is statistically significant at all time points.
  • PRS-211,092 has a bimodal effect on IL-6 expression.
  • IL-10 gene expression in liver and spleen, and secretion, in the ConA model IL-10 gene expression in liver and spleen, and secretion, in the ConA model.
  • Treated animals displayed further increased IL-10 gene expression by 1.5 fold in the spleen as early as 15 minutes after injury followed by a 3.2 fold increase 1 hr after injury. A similar increase of about 1.4 fold is observed in the liver only 8 hours after injury, suggesting that IL-10 is first up-regulated in the spleen then in the liver.
  • the spleen was removed at one hour after injury and splenocytes were cultured for 24 hours to allow the assessment of secretion levels.
  • this in vivo model we show that the increase in IL-10 RNA levels is supported and correlated with an increase in protein secretion.
  • SOCS-3 as previously described. Both SOCS-1 and SOCS-3 have important recognized roles in the liver. SOCS-1 deficient mice suffer from three major abnormalities: lymphopoenia, macrophage infiltration of several organs including the liver, heart, lung and skin, and severe fatty degeneration of the liver. On the other hand, SOCS-3 is the main
  • cytokines and chemokine tested often act in concert and cross regulate one another to yield the final physiological outcome.
  • a biological marker of liver injury such as ALT, SAA-3 and haptoglobin
  • the acute phase response is an innate body defense seen during acute inflammation, infection and trauma, which involves the altered production of certain blood proteins termed acute phase proteins (APPs).
  • APPs acute phase proteins
  • activated macrophages and other leukocytes release pro-inflammatory cytokines such as TNF- ⁇ , IL-1 and IL-6.
  • cytokines in turn stimulate hepatocytes to synthesize and secrete acute phase proteins such as C-reactive protein (CRP), mannose-binding lectin (MBL), haptoglobin and serum amyloid A (SAA).
  • CRP C-reactive protein
  • MBL mannose-binding lectin
  • SAA serum amyloid A
  • PRS-211,092 significantly reduced the levels of liver gene expression of SAA-3 by 55% and 48%, and of haptoglobin by 56% and 66%, at the respective time points.
  • the positive control FK-506 yielded on average at both time points of four and eight hours after injury 97% and 78% reduction in gene expression of SAA-3 and haptoglobin respectively.
  • Hepatoprotection through inhibition of apoptosis in HepG2 cell lines The purpose of this study is to assess whether the hepatoprotective effect of the active compounds of the invention is achieved through inhibition of apoptotic events in cells of hepatic lineage.
  • the proteolytic cleavage of caspases and poly-ADP-ribose polymerase (PARP) is a known marker of apoptosis, and is investigated by immunoblotting or immunohistochemistry in HepG2 cells. Apoptosis is induced in these cells either by TNF- ⁇ , anti-CD95 or ethanol.
  • HepG2 (ATCC # HB-8065) are human hepatocellular carcinoma cells and are grown in Eagle's Minimum Essential medium, supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 1.5 g/1 sodium bicarbonate. A total of 1 x 10 cells/well are seeded in 6-well plates and are treated with either anti- CD95 (0.5 mg/ml) or with TNF- ⁇ (10 ng/ml), and cycloheximide (10 mg/ml). For ethanol induced apoptosis, the range of the inducer is 100 to 400 ⁇ M and cells must be cultured for 24 hours under those conditions.
  • Test compounds and controls are added to the cells one hour before the induction of apoptosis, unless otherwise stated. After 6 hours for TNF- ⁇ or anti-CD95 or 24 hours for ethanol induction, cells are washed in cold PBS and lysed in 1% Triton X-100, 50 mM Tris-HCl, pH 7.6, and 150 mM NaCl containing 3 mg/ml leupeptin, 3 mg/ml aprotinin, 3 mg/ml pepstatin A and 2 mM phenylmethylsulfonyl .uoride.
  • the cell lysates are separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene di.uoride membrane (Amersham, Germany).
  • Membranes are blocked with 5% milk powder in Tris-buffered saline and then incubated for 1 hour with 1 mg/ml of either anti- cleaved caspase-3, anti-cleaved caspase-7, or antibodies recognizing the full-length forms of the proteins.
  • Membranes are then washed 4 times with Tris-buffered saline/0.05 % Tween-20 and incubated with the respective peroxidase-conjugated secondary antibodies for 1 hour. After extensive washing, bound antibodies are detected by enhanced chemiluminescent staining.
  • Example 11 Inhibition of IL-2 related transcription factors in activated T cells.
  • IL-2 gene expression and secretion are inhibited by the active compounds of the invention both in vitro, in PMA/Calcium ionophore activated T cells, and in vivo in the MCAo model for determination of neuroprotection and in the ConA induced model of liver injury for determination of hepatoprotection.
  • IL-2 is tightly regulated at the level of transcription. The purpose of this study was to check if the test compounds have an effect on some of the transcription factors involved in the regulation of the IL-2 promoter.
  • the cells containing each reporter were plated in a 24 well plate at 10 6 cells/ml/well.
  • Test compounds were resuspended in DMSO, and added for one hour before cell activation, at predetermined doses wherein DMSO final concentration is 0.1 %. Cyclosporin A was used as positive control at the dose of 100 nM.
  • Cells were then stimulated with 10 ng/ml PMA and 2 ⁇ M calcium ionophore for 6 hours.
  • cells were collected, rinsed in PBS and lyzed for 15 minutes on ice in 50 ⁇ l luciferase lysis buffer
  • Results are shown in Figure 5, where we can see that activation of the cells yield a significant increase in NF-AT driven luciferase expression, from 60 luminescence units (LU) to 2449 LU.
  • Treatment of the cells with test compounds caused a dose dependent reduction in NF-AT driven luciferase expression.
  • PRS-211,092 has an IC 5 o of 1.5 ⁇ M and PRS-211,220 has an IC 50 of 3.9 ⁇ M.
  • 100 nM of cyclosporin A caused a total inhibition.
  • DNA-array based technologies are widely used in gene regulation research, most commonly to measure differential gene expression, that is comparing the relative level of RNA transcripts in different samples.
  • the purpose of this study is to allow a preliminary screen of the impact of dexanabinol and its analogs on the regulation of a large amount of genes.
  • the technology is based on hundreds (macro-arrays) to thousands (micro-arrays) of sequence-specific DNA fragments spotted on a solid matrix such as glass slides or membranes. RNA samples from the examined tissue or cells are reverse-transcribed into cDNA, labeled and hybridized with the array.
  • the number of labeled transcripts hybridized to a single spot is turned to a radioactive, fluorescence or chemiluminescence signal and detected by the appropriate instrument.
  • the quantification of the signal on each spot measures the level of expression of the specific gene.
  • membrane-based focused macroarrays each consisting of gene families representing a biological regulatory pathway such as cytokines or chemokine arrays, commercially made by SuperArray hie.
  • cDNA samples from mice brains after MCAo treated with either vehicle or test compound were labeled with biotin, hybridized to the array-membranes and detected using a chemiluminescence detector, according to SuperArray instructions.
  • the genes that are expressed differentially between treatment and control are subjected to confirmation analysis using real-time quantitative PCR, as previously described.
  • Example 13 Effect of the compounds in carrageenan induced paw edema.
  • mice Female Balb/c mice (20 gr average body weight, Harlan, Israel) are anesthetized with a combination of xylazine and pentobarbitone diluted in sterile saline, 15 and 6 mg/kg i.p. respectively. Anesthetized mice are injected subcutaneously, in the subplantar region of one (right) paw with 0.05 ml of 1% w/v Carrageenan in sterile water.
  • the contralateral (left) paw is not injected as data from the literature, confirmed by our own experience, showed that injection of 0.05 ml of normal saline did not affect later thickness or volume measurements.
  • the test compounds including known anti-inflammatory controls, are dissolved in cremophor: ethanol and further diluted 1:20 or 1:50 in sterile saline before i.p. injection that takes place immediately before the carrageenan injection. Three hours after injection the animals are resedated following the previously described procedure.
  • Paw thickness is measured using a dial thickness gauge (Spring-dial, constant low pressure gauge, Mitutoyo, TG/L-1, 0.01mm) and paw volume is measured using a plethysmometer (model #7150, Ugo Basile, Italy).
  • Paw Edema is expressed as the difference between the right treated and the left untreated paws of the same animal, either as ⁇ Paw Volume ( ⁇ PV) in millimeters cube or as ⁇ Paw Thickness ( ⁇ PT) in millimeters.
  • ⁇ PV Paw Volume
  • ⁇ PT Paw Thickness
  • Results are depicted in Figure 6 where the % inhibition of paw thickness, normalized to vehicle, is plotted against the dose of the test compound.
  • Dexanabinol yields a reduction of about 29%) in paw thickness at doses ranging from 0.2 to 0.5 mg/kg.
  • PRS-211,092 yields a reduction of about 22% in paw thickness at the very low dose of 0.25 mg/kg.
  • PRS- 211,220 reduces even more paw thickness by 31% at the even lower dose of 0.1 mg/kg.
  • Example 14 Effect of compounds in cancer chemoprotection.
  • Pancreatic tumor cell lines were obtained from ATCC.
  • Panc-1 ATCC # CRL-1469
  • DMEM fetal calf serum
  • Aspc-1 (ATCC # CRL-1682) were cultured in RPMI supplemented with 2 mM Glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L bicarbonate, antibiotics (penicillin, nystatin and streptomycin) and 20% heat inactivated fetal calf serum.
  • Cells were seeded in a 24 well plate (10 5 cells/ml/well) and grown overnight. The cells are incubated with the test compounds (1-100 ⁇ M) or vehicle (0.1% DMSO final concentration). Cell viability was determined 24 hours later using standard crystal violet staining.
  • the culture medium was removed from the wells and the cells were fixed by adding 1 ml/well of 2% formaldehyde in PBS for 10 minutes. Following fixation the cells are washed three times with PBS and 250 ⁇ l of 0.5% (w/v) crystal violet is added to each well and the plates were incubated for
  • the cells are stained for activated caspase 3 to determine whether they died through an apoptotic mechanism.
  • the medium from the wells is discarded and cells are fixed by adding 1 ml of 4% formaldehyde in PBS, for 10 min.
  • Cells are washed twice with PBS-0.1% Tween20 (PBS-T) and permeabihzed with cold methanol for 20 min.
  • the cells are washed twice with PBS-T and incubated with 1 ml blocking solution (3% BSA, PBS-T) for 30 min.
  • the primary antibody (rabbit anti- cleaved caspase 3 (aspl75) Cell Signaling Technology, diluted 1:50 with blocking solution) is added and the cells incubated for 60 min.
  • the tumor cells LoVo (originating from a colorectal tumor; ATCC # CCL-229) were grown in DMEM containing 4.5 g/L glucose, 2 mM Glutamine, 1% Pen/Strep and 10%) heat inactivated fetal calf serum. The cells were harvested using Trypsin-EDTA, the detached cells were rinsed in PBS, and counted. Predetermined amounts (lxlO 6 cells h constant volume of 0.12 ml/animal) were injected s.c.
  • mice average weight 20-25 gr, Harlan, Israel.
  • Each treatment group was composed of at least 7 animals. Each animal was clinically monitored daily. The growth of the tumor was also monitored during the daily visits but actual measurements were recorded once a week. When tumors reach the appropriate size, animals are treated with either vehicle, 5 ml/kg/day, or with our test compounds, in the range of 2.5 to 10 mg/kg/day.
  • mice Thirty-six out of the 40 implanted mice developed a visible tumor within 5 to 6 weeks from tumor implantation. All animals were treated first on the 36 th -42 nd day of tumor implantation and the treatment lasted 8 weeks. One animal died in each treatment group between the 5 th and the 7 th week of treatment. The results are expressed as percent of tumor growth at the various days of the treatment as compared to baseline day 1. The results are depicted in Figure 7 where we can observe that the efficacy of dexanabinol is inversely proportional to its dose in the range tested. 2.5 mg/kg ( ⁇ ) seems more efficient than 5 mg/kg (A), itself better than 10 mg/kg (x), which has very little effect, if at all, as compared to vehicle (— ⁇ --).
  • Example 15 Treatment of neurodegenerative disorders: the MPTP model
  • Parkinson's disease is a neurodegenerative disorder characterized by tremor, slowness of movements, stiffness and poor balance. Most, if not all, of these disabilities are due to a profound reduction in striatal dopamine content caused by loss of dopaminergic neurons in the Substantia Nigra pars compacta (SNpc) and of their projecting nerve fibers in the striatum.
  • SNpc Substantia Nigra pars compacta
  • MPTP l-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine
  • MPTP is a well known neurotoxin that can cause depletion of dopamine content in the striatum and a reduction in the number of nigrostriatal dopaminergic neurons in several species including humans (Turski L.
  • the aim of the present study is to examine the effect of compounds involved in gene regulation of pro- and anti-inflammatory mediators on the progression of MPTP- induced dopaminergic toxicity.
  • the study is carried out in two time windows, hi the short- term model, the neurological outcome is assessed, namely by measuring the effect of the test compounds on the number of immunoreactive cells.
  • the functional outcome is assessed in the rotarod system following various treatments.
  • mice C57/BL male mice, average weight 30 g, Harlan, Israel
  • mice were administered i.p. with 4 injections of MPTP (Sigma, USA) (20 mg/kg, 5 ml/kg) in saline (Teva Medical Israel) at 2 hours interval on day 1.
  • the test compounds and vehicle control (cremophor: ethanol diluted in saline) were injected i.p. once just before the first MPTP administration at a volume dose of 5 ml/kg.
  • Dexanabinol was tested at 10, 20, and 30 mgkg and PRS-211,220 was tested at 0.5, 1, and 5 mg/kg.
  • Each treatment group was composed of at least seven animals. Seven days following the MPTP treatment the animals are euthanized by i.p. administration of 100 mg/kg pentobarbitone sodium (CTS, Israel) and their brains are removed for tyrosine hydroxylase (TH) detection using immunohistochemistry.
  • CTS pentobarbitone sodium
  • TH immunoreactivity at the level of the SNpc Brains were fixed by immersion in 4% formaldehyde for at least 72 hours. The brains were then washed with PBS and transferred to 30% sucrose in PBS until they sank. After the brains sank in the sucrose they were frozen using the cryostat special fast-freezing technique (-60°C). The brains were cryosectioned (20 ⁇ m) at the level of the striatum and at the level of the substantia nigra (SN). Immunohistochemistry staining was carried out using Rabbit anti-tyrosine hydroxylase (1 :250, Calbiochem, USA).
  • the slides were stained using the 3,3-diaminobenzidine tetrahydrochloride (DAB) chromagen detection kit of the automated immunostaining system (Vantana, France). Quantitative analysis was carried out by counting the number of TH-in munoreactive cells/mm 2 at the widest area of the SNpc. Statistical analysis.
  • DAB 3,3-diaminobenzidine tetrahydrochloride
  • Results are expressed as mean ⁇ SD. Data were analyzed using analysis of variance (ANOVA) followed by post-hoc Fisher test. A value of p ⁇ 0.05 is considered to be statistically significant.
  • mice C57/BL male mice, average weight 30 g, Harlan, Israel
  • MPTP Sigma, USA
  • the test compounds and controls were injected i.p. once just before the first MPTP administration at a volume dose of 5 ml/kg.
  • Dexanabinol was tested at 20 mg/kg and PRS-211,220 was tested at 5 mg/kg.
  • Each treatment group was composed of at least fourteen animals.
  • Another parameter that was measured in this experiment is mortality, which was relatively high in this model. Animals that received only saline, and no MPTP, displayed, as expected, 0% mortality (0/14 animals). However, MPTP injected animals displayed 69% mortality (11/16 animals) over the period of the study, reflecting the severity of the model. Treatment with 20 mg/kg dexanabinol dramatically lowered this figure down to 11% mortality (1/9 animals), while treatment with 5 mg/kg PRS-211,220 was even more effective with 0% mortality (0/7 animals).
  • Pain mediated by the peripheral nervous system is tested in the 'formalin test' for cutaneous (peripheral) pain (Tjolson A. et al, Pain 51: 5-17, 1992).
  • test compounds are injected i.p.
  • formalin is injected s.c. in the plantar surface of the hind paw of a mouse 90 min after the test compound.
  • formalin administration is assessed (every 5 min for 1 hr) by the number of times the animal licks the formalin-injected paw.
  • the aim of this study is to assess the potential analgesic effects of our compounds in an animal model of neuropathic pain.
  • a peripheral monopathy was induced in the right hind limb of rats following a chronic constriction of the sciatic nerve (Bennet G.J. and Xie
  • Pre-surgery baseline values are ascertained as the mean of two pre-surgery values.
  • the animals are surgically prepared by constricting the right sciatic nerve with 4 chromic cat gut loose ligatures.
  • the animals that have developed mechanical allodyna are arbitrarily allocated to the various treatment groups based on the pre-surgery values.
  • the design is randomized, performed in a masked fashion as to whether drug or vehicle is being given.
  • the animals male Sprague-Dawley rats, are allowed to acclimatize to the behavioral testing equipment before testing. On the testing day, the animals are given a single dose of one of the test compounds in a volume of 2.5 ml/kg. Following 15 and 180 minutes a series of Von Frey filaments (pre-calibrated before testing) are applied to the plantar surface of the hind paw, from below. The filaments are applied in ascending order starting with the weakest force; 0.37 g or filament handle no. 3.61), and the withdrawal threshold for both the ipsilateral and contralateral hind paws is evaluated.
  • the withdrawal threshold is defined as being the lowest force of two or more consecutive Von Frey's filaments to elicit a reflex withdrawal response (i.e. a brief paw flick) and is measured in grams.
  • mice (average body weight 30 g, Harlan, Israel) are pre-treated with i.v. injection of either vehicle
  • mice are injected i.p. with 10 ml/kg of 0.6% acetic acid in water and the number of writhes are counted during a 5 minutes period, starting 5 minutes after the acetic acid administration.
  • a writhing is considered as contraction of the abdominal muscles accompanied by an elongation of the body and extension of the hind limb. Results are expressed as mean number of writhes ⁇ SEM. Data was analyzed using analysis of variance (ANOVA) followed by Tukey's post hoc test. A value of p ⁇ 0.05 was considered statistically significant.
  • ANOVA analysis of variance
  • Diabetes type I the NOD mice model.
  • the purpose of the present study is to establish a model in non-obese diabetic (NOD) mice to test the protective activity of dexanabinol and its analogs in an experimental setup relevant to human insulin-dependent diabetes mellitus.
  • NOD non-obese diabetic
  • NOD/It female mice 70-80 days old at study onset, Harlan, Israel are weighted at day 1. Their baseline glucose level is established using a drop of blood obtained by sectioning the tip of the tail and a glucometer with the appropriate glucosticks (Elite, Bayer). Mice are then injected i.p. with cyclophosphamide (Sigma) diluted in saline at a dose of 300 mg/kg. The appearance of glucose in the urine of the animals is monitored every two days using a urine multistick (Bayer). When this test indicates that the animals reach glucourea, then the level of glucose in the blood is reassessed during two consecutive days after overnight starvation.
  • Animals are defined as diabetic if their glucose blood levels are above 300 mg/dl. Three days following the diagnostic of diabetes, the animals are sacrificed by i.p. injection of 100 mg/kg pentobarbitone. Their spleen and pancreas are removed for further study including FACS analysis of the T cells subpopulations in the spleen and histo- and immuno-pathological evaluation of the pancreas.
  • the histopathological evaluation is carried out on ten Langerhans islands for each animal and the scoring is according to the following method (Sempe P. et al, Eur. J. Immunol. 21: 1163-9, 1991).
  • the severity of the damage is scored according to the level of mononuclear infiltrate: 0- no infiltration, 1- periductular infiltrate, 2- peri-islet infiltrate, 3- intra-islet infiltrate, 4- intra-islet infiltrate associated with ⁇ -cell destruction.
  • the mean score for the pancreas of each animal is calculated by dividing the total score by the number of islets examined. Example 20.
  • the purpose of the present study is to test the nephro-protective activity of dexanabinol and its analogs in an acute renal ischemia model in rats.
  • mice Male Sprague Dawley rats (250 gr average body weight, Harlan, Israel) are anesthetized with a combination of xylazine and pentobarbitone 8 and 35 mg/kg i.p. respectively. Then a 45-minutes ischemia is induced bilaterally on both kidneys. The sedated animals are positioned on their backs. The abdomen skin is shaved and cleaned with 70% ethanol. A midline skin incision is performed (2-3 cm long) and the abdomen is opened through an incision in the linea Alba. The kidneys are explored after gentle removal of the intestines to the opposite direction. While this is done, the intestines are covered with wet (warm saline 37°C) sterile sponges.
  • the renal arteries are isolated by blunt dissection from the surrounding fat, and occluded together with the renal veins in the kidney hilus by arterial micro clips (FST Canada). Kidneys that become pale immediately after artery occlusion are considered ischemic. Only animals showing that both kidneys are ischemic are included in the study. During the ischemic insult the intestines are returned into the abdominal cavity. The wound is covered with wet sponges (they were kept wet by rinsing warm saline). h addition, rectal temperature is monitored to remain between 37°C- 38°C. Rectal temperature is measured using a thermistor (YSI USA model 400) and a measuring unit (Cole Parmer model 8402-00).
  • Treatments are administered i.v. into the femoral vein at 5 ml/kg to 10 animals per group, immediately after the end of the ischemic insult. Results are compared to ischemic (vehicle treated) and sham (the same procedure, without renal artery occlusion).
  • Inflammatory bowel disease the acetic acid-induced model.
  • the purpose of this study is to evaluate the activity of test compounds in a masked study of acetic acid-induced inflammatory bowel disease in rats.
  • Table 1 Criteria for Scoring Disease Activity Index (DAr) of IBD.
  • DAI- combined score of weight loss, stool consistency, and bleeding/3. * Normal stool - well formed pellets; loose stools - pasty stool that does not stick to the anus; and diarrhea - liquid stools that sticks to the anus.
  • the clinical outcome is analyzed using analysis of variance (ANOVA) followed by Duncan's post-hoc test.
  • a non-parametric test (Wilcoxon Rank Sum Test) is used for evaluating the gross pathology findings.
  • EAE experimental allergic encephalomyelitis
  • DTH delayed type hypersensitivity
  • EAE is an autoimmune neurological disease elicited by sensitization of the animals to myelin basic protein from the central nervous system, which is also known as basic encephalitogenic protein.
  • Experimental autoimmune arthritis is induced in animals by immunization with collagen in complete Freund's adjuvant: the model is therefore named collagen induced arthritis (CIA).
  • Delayed type hypersensitivity is induced by the application of dinitrofluorobenzene according to a strict time-schedule, therefore the model generated correspond to allergic contact dermatitis in the human.
  • the purpose of the present study is to test the ability of our compounds to prevent or attenuate the clinical signs of these three autoimmune disease models.
  • autoimmune encephalomyelitis Various animal models of autoimmune encephalomyelitis are known in the art, depending on the method of induction, the strain of the animal and the antigen employed to induce the disease.
  • the impact of the test compounds is tested in EAE using Lewis rats in which the onset of disease is observed by the appearance of clinical symptoms about 10 days after induction. The disease progresses and the clinical score increases and peaks around day 15 and spontaneous recovery is observed around day 18 after induction of the disease.
  • the animals (at least 10 per test group at initiation of study) are maintained on a 12 hours light/12 hours dark regimen, at a constant temperature of 22°C, with food and water ad libitum.
  • EAE is induced in these animals by immunization with purified guinea pig myelin basic protein emulsified in Complete Freund's Adjuvant.
  • Guinea pig myelin basic protein (MBP) is prepared from spinal cord homogenates defatted with chloroform/ethanol and the isolated protein is purified using ion exchange chromatography. Each animal receives 50 ⁇ g of the purified protein.
  • a solution of MBP (0.5 mg/ml) is emulsified with an equal volume of Complete Freund's Adjuvant containing 4 mg/ml of mycobacterium tuberculosis, and each animal receives 100 ⁇ l (50 ⁇ l in each hind foot pad).
  • Animals are treated with test compounds or vehicle control, administered intravenously in a volume of 5 ml/kg, for three consecutive days starting from the onset of the disease ( ⁇ at day 10 following disease induction).
  • Methyl prednisolone is used as positive control and it is administered daily for 5 consecutive days i.v. at 20 mg/kg starting from day of disease induction by MBP injection.
  • the results are recorded as clinical score; score of 0 indicates a normal animal with no clinical signs, 1 indicates tail paralysis, 2 indicates paraplegia, 3 indicates quadriplegia, 4 indicates complete body paralysis and 5 indicates death.
  • mice (20 g average body weight, Harlan, Israel) were sensitized on day 0 and day 1 by application of 30 ⁇ l of 0.15% Dinitrofluorobenzene (DNFB) diluted in acetone on the shaved skin of the abdomen.
  • DNFB Dinitrofluorobenzene
  • Test compounds were administered at increasing doses from 0 to 15 mg/kg i.p. twice, the first injection was immediately after DNFB challenge (on day 6) and the second injection was 16 hours after challenge (on day 7).
  • Each treatment group comprised at least 7 animals.
  • Dexamethasone (DXM) was used as positive control. Ear thickness was determined (in 0.01 mm units) 24 hours after challenge (and 6 hours after second treatment on day 7) using a dial thickness gauge (Mitutoyo, Japan).
  • Results are analyzed as ear thickness of DNFB treated over DNFB untreated contralateral ear.
  • the impact of the test compound is further assessed by comparing its mean impact on the animals of the treatment group to the response generated by the appropriate vehicle only.

Abstract

L'invention concerne des compositions pharmaceutiques comprenant, en tant que principe actif, des dérivés cannabinoïdes, non psychotropes, qui modulent l'expression de gènes impliqués dans les processus immun et d'inflammation. La régulation de la transcription de médiateurs pro- et anti-inflammatoires est utile dans les applications thérapeutiques destinées à prévenir et à traiter l'inflammation aiguë et chronique, des maladies auto-immunes et des troubles associés, la douleur, des infections, des maladies hépatiques, des troubles cardio-vasculaires, gastro-intestinaux, des troubles des systèmes nerveux central et périphérique incluant des troubles neurodégénératifs, des maladies respiratoires, des maladies rénales, des complications post-opératoires, le rejet tissulaire et certains types de cancer.
PCT/IL2003/000223 2002-03-18 2003-03-16 Dexanabinol et analogues de dexanabinol regulant des genes associes a l'inflammation WO2003077832A2 (fr)

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AU2003214608A AU2003214608A1 (en) 2002-03-18 2003-03-16 Dexanabinol and dexanabinol analogs regulate inflammation related genes
EP03710188A EP1485083A4 (fr) 2002-03-18 2003-03-16 Dexanabinol et analogues de dexanabinol regulant des genes associes a l'inflammation
CA002479676A CA2479676A1 (fr) 2002-03-18 2003-03-16 Dexanabinol et analogues de dexanabinol regulant des genes associes a l'inflammation
IL16387703A IL163877A0 (en) 2002-03-18 2003-03-16 Dexanabinol and dexanabinol analogsregulate inflammation related genes
JP2003575886A JP2005526768A (ja) 2002-03-18 2003-03-16 炎症関連遺伝子を調節するデキサナビノール及びデキサナビノール類似体
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WO2005046714A2 (fr) * 2003-11-12 2005-05-26 Ares Trading S.A. Molecules antagonistes de cytokines
WO2006008183A1 (fr) * 2004-07-23 2006-01-26 Novartis Ag Biomarqueurs de la polyarthrite rhumatoide (pr)
WO2006024958A2 (fr) * 2004-08-09 2006-03-09 Novimmune S.A. Compositions cannabinoides et procedes d'utilisation correspondants
EP1752149A1 (fr) * 2005-07-29 2007-02-14 Laboratorios Del Dr. Esteve, S.A. CB1 Antagonists ou agonistes inverses comme agents thérapeutiques pour le traitement de l'inflammation impliquant l'expression de gène
EP1903866A1 (fr) * 2005-11-07 2008-04-02 Murty Pharmaceuticals, Inc. Administration amelioree de tetrahydrocannabinol
JP2008533107A (ja) * 2005-03-17 2008-08-21 プロイェクト、デ、ビオメディシナ、シーマ、ソシエダッド、リミターダ 自己免疫疾患および／または移植拒絶反応の予防および／または治療における５’−メチルチオアデノシン（ｍｔａ）の使用
WO2009007700A1 (fr) * 2007-07-06 2009-01-15 E-Therapeutics Plc Traitement de mélanome
WO2009047505A3 (fr) * 2007-10-10 2009-07-30 E Therapeutics Plc Traitement de mélanome
WO2011030106A1 (fr) 2009-09-10 2011-03-17 E-Therapeutics Plc Apoptose de cellules cancéreuses
WO2013160645A1 (fr) 2012-04-26 2013-10-31 E-Therapeutics Plc Dexanabinol ou un dérivé de celui-ci destiné à être utilisé dans le traitement du cancer dans des plages de doses de 2 à 30 mg/kg
WO2014021455A1 (fr) * 2012-08-03 2014-02-06 国立大学法人愛媛大学 Inhibiteur de l'activation d'une cellule immunitaire et son utilisation
CN102851295B (zh) * 2006-08-28 2015-01-07 长春华普生物技术有限公司 Toll 样受体调节性寡核苷酸及其用途
WO2017068349A1 (fr) 2015-10-23 2017-04-27 E-Therapeutics Plc Cannabinoïde pour utilisation en immunothérapie

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JP6470879B1 (ja) * 2017-03-08 2019-02-13 日清オイリオグループ株式会社 抗炎症剤、抗炎症用医薬組成物、抗炎症用食品組成物

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US7803383B2 (en) 2003-11-12 2010-09-28 Ares Trading S.A. Method of treatment comprising administration of a cytokine antagonist molecule
AU2004289105B2 (en) * 2003-11-12 2010-06-03 Ares Trading S.A. Cytokine antagonist molecules
WO2005046714A2 (fr) * 2003-11-12 2005-05-26 Ares Trading S.A. Molecules antagonistes de cytokines
JP2007515157A (ja) * 2003-11-12 2007-06-14 アレス トレイディング ソシエテ アノニム サイトカインアンタゴニスト分子
WO2005046714A3 (fr) * 2003-11-12 2005-11-17 Ares Trading Sa Molecules antagonistes de cytokines
EA010420B1 (ru) * 2003-11-12 2008-08-29 Арес Трейдинг С.А. Молекулы-антагонисты цитокинов
WO2006008183A1 (fr) * 2004-07-23 2006-01-26 Novartis Ag Biomarqueurs de la polyarthrite rhumatoide (pr)
WO2006024958A2 (fr) * 2004-08-09 2006-03-09 Novimmune S.A. Compositions cannabinoides et procedes d'utilisation correspondants
WO2006024958A3 (fr) * 2004-08-09 2006-12-28 Novimmune Sa Compositions cannabinoides et procedes d'utilisation correspondants
JP2008533107A (ja) * 2005-03-17 2008-08-21 プロイェクト、デ、ビオメディシナ、シーマ、ソシエダッド、リミターダ 自己免疫疾患および／または移植拒絶反応の予防および／または治療における５’−メチルチオアデノシン（ｍｔａ）の使用
EP1752149A1 (fr) * 2005-07-29 2007-02-14 Laboratorios Del Dr. Esteve, S.A. CB1 Antagonists ou agonistes inverses comme agents thérapeutiques pour le traitement de l'inflammation impliquant l'expression de gène
WO2007017125A1 (fr) * 2005-07-29 2007-02-15 Laboratorios Del Dr. Esteve, S.A. Antagonistes cb1 ou antagonistes inverses comme agents thérapeutiques pour le traitement de l'inflammation impliquant l’expression de gènes
EP1903866A1 (fr) * 2005-11-07 2008-04-02 Murty Pharmaceuticals, Inc. Administration amelioree de tetrahydrocannabinol
EP1903866A4 (fr) * 2005-11-07 2010-12-22 Murty Pharmaceuticals Inc Administration amelioree de tetrahydrocannabinol
CN102851295B (zh) * 2006-08-28 2015-01-07 长春华普生物技术有限公司 Toll 样受体调节性寡核苷酸及其用途
WO2009007700A1 (fr) * 2007-07-06 2009-01-15 E-Therapeutics Plc Traitement de mélanome
CN101808636B (zh) * 2007-07-06 2012-05-02 e-生物有限公司 黑色素瘤的治疗
KR101532242B1 (ko) * 2007-07-06 2015-06-29 이-테라퓨틱스 리미티드 흑색종의 치료
AU2008273927B2 (en) * 2007-07-06 2012-12-13 E-Therapeutics Plc Treatment of melanoma
US20110020217A1 (en) * 2007-10-10 2011-01-27 E-Therapeutics Plc Treatment of melanoma
WO2009047505A3 (fr) * 2007-10-10 2009-07-30 E Therapeutics Plc Traitement de mélanome
WO2011030106A1 (fr) 2009-09-10 2011-03-17 E-Therapeutics Plc Apoptose de cellules cancéreuses
CN102573833A (zh) * 2009-09-10 2012-07-11 e-生物有限公司 癌细胞凋亡
CN105935357A (zh) * 2009-09-10 2016-09-14 e-生物有限公司 癌细胞凋亡
WO2013160645A1 (fr) 2012-04-26 2013-10-31 E-Therapeutics Plc Dexanabinol ou un dérivé de celui-ci destiné à être utilisé dans le traitement du cancer dans des plages de doses de 2 à 30 mg/kg
CN104470509A (zh) * 2012-04-26 2015-03-25 e-生物有限公司 剂量范围在2至30mg/kg的地塞米诺或其衍生物在癌症治疗中的用途
WO2014021455A1 (fr) * 2012-08-03 2014-02-06 国立大学法人愛媛大学 Inhibiteur de l'activation d'une cellule immunitaire et son utilisation
US9517217B2 (en) 2012-08-03 2016-12-13 Ehime University Immune cell activation inhibitor and use thereof
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