[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

ZA200407182B - Dexanabinol and dexanabinol analogs regulate inflammation releated genes. - Google Patents

Dexanabinol and dexanabinol analogs regulate inflammation releated genes. Download PDF

Info

Publication number
ZA200407182B
ZA200407182B ZA200407182A ZA200407182A ZA200407182B ZA 200407182 B ZA200407182 B ZA 200407182B ZA 200407182 A ZA200407182 A ZA 200407182A ZA 200407182 A ZA200407182 A ZA 200407182A ZA 200407182 B ZA200407182 B ZA 200407182B
Authority
ZA
South Africa
Prior art keywords
saturated
cyclic
unsaturated
branched
inflammatory
Prior art date
Application number
ZA200407182A
Inventor
Aaron Garzon
Ayelet Avraham
George Fink
Original Assignee
Pharmos Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pharmos Corp filed Critical Pharmos Corp
Publication of ZA200407182B publication Critical patent/ZA200407182B/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4155—1,2-Diazoles non condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/453—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with oxygen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Landscapes

  • Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Neurosurgery (AREA)
  • Pulmonology (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • Diabetes (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Rheumatology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Endocrinology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Obesity (AREA)
  • Emergency Medicine (AREA)
  • Psychology (AREA)
  • Hospice & Palliative Care (AREA)
  • Psychiatry (AREA)
  • Pain & Pain Management (AREA)
  • Transplantation (AREA)
  • Vascular Medicine (AREA)

Description

MIDEXANABINOL AND DEXANABINOL ANALOGS
MREGULATE INFLAMMATION RELATED GENES
FIELD OF THE INVENTION
The present: invention relates to pharmaceutical compositions comprising as an active ingredient non-ps ychotropic cannabinoid derivatives that modulate the expression of genes involved in inflarnmatory and immune processes. Regulating the transcription of pro and anti-inflammatory’ mediators has useful therapeutic application for prevention and treatment of acutee and chronic inflammation, autoimmune diseases and related disorders, pain, infections, liver diseases, cardiovascular disorders, ga strointestinal disorders, disorders of the central and peripheral nervous system inclmding neurodegenerative diseases, respiratory diseases, renal diseases, post-operative comp ications, tissue rejection and certain types of cancer.
BACKGROUND OF THE INVENTION
Cannabinoids
Cannabis saliva preparations have long been known as therapeutic agents to treat various diseases. The identification of tetrahydrocannabinol (THC) as the active principle of marijuana prompted medicinal chemists to develop numerous cannabinoid analogs.
These novel compounds were designed to exhibit the therapeutically beneficial properties of THC without the clinically undesirable psychotropic effects. Potential therapeutic applications haves 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 arti-inflammatory agents.
The diverse cann_abinoid effects are generally attributed to the activation or inhibition of various types of receptors. Nevertheless, the mechanisms undexlying some therapeutic © 25 effects of cannabinoid derivatives remain unclear. . Several syn_thetic cannabinoid derivatives have been prepared since the discovery of the natural ligand with an emphasis toward therapeutic non-pesychoactive agents. An extended family of such compounds were disclosed for example in US Patent Nos. 4,179,517, 4,876,276, 5,538,993, 5,635,530, 6,096,740, and in international patent
WO ©03/077832 PCT/IL03/00223 application WO 01/98289. A pivotal member of this family of compounds is 1,1- dimethy-theptyl-(3S,4S)-7-hydroxy-A®-tetrahydrocannabinol, disclosed” in US 4,876,276 and den oted therein HU-211. HU-211 was subsequently assigned the trivial chemical name . dexanabinol. The neuroprotective action of dexanabinol, and later on its derivatives, was attributed in part to their ability to block the NMDA receptor. Moreover, the compounds ) seemed to share anti-oxidative and anti-inflammatory properties unrela-ted to their capacity to block the NMDA receptor. This anti-inflammatory activity was -associated with the ability of those compounds to reduce the secretion of PGE; produced by the enzyme cycloox ygenase-2 (COX-2). COX-2 is one of the cyclooxygenase isofosrms involved in the metabolism of arachidonic acid (AA) toward prostaglandins (PG) and eother eicosanoids, a family of compounds known to exhibit inflammatory properties and kimown to be involved in inflamnmation. Most conventional NSAIDs (non-steroidal anti-irmflammatory drugs) inhibit COX activity by modifying the enzyme active site theresby preventing the transformation of the AA substrate to PGE, (Hinz B. et al., J. Pharm. EZxp. Ther. 300: 367- 375,2002).
Moreover, dexanabinol and later on its analogs were found able to block the producti on or action of TNF-a, as disclosed in International Patent applications WO 97/1166 and WO 01/98289. It was postulated that the inhibition of the cytokine occurs at a post-tr anscriptional stage, since in a model of head injury dexanabinol did not affect the levels of TNF-a. mRNA (Shohami E. et al., J. Neuroimmuno. 72: 169 -77, 1997). Human
TNE-o is first translated into a 27 kd transmembrane precursor proteim, which is cleaved into the secreted 17 kd form by TNF-a converting enzyme (TACE). Based on RT-PCR experiments, Shoshany et al. reported that dexanabinol has no significant effect on TNF-a : mRNA whereas it significantly reduced the levels of TACE mRNJA, supporting the assumption that the drug acts at the level of secretion inhibition
Inflammation
Inflammation is one of the most important processes involved ira the defense of an : organism; however, when it occurs in some organs, such as brain, in ressponse to an insult, or if it is inappropriate, such as in autoimmune diseases, inflammation can be harmful and "30 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 nitrogera radicals, play a regulatory - role in this complex and highly balanced process. Following is a selection of genes whose protein product was shown to be involved in a variety” of pathways leading to ’ inflammation; COX-2, IL-2, MCP-1, IL-1B, iNOS, TNF-c., IL-6 and IL-10 and SOCS.
The involvement of these inflammatory mediators in various pathological conditions was extensively reviewed (Frangogianis N.G. et al., Cardio. Res. 53: 31-47, 2002; Gerard
C. et al., NJature Immuno. 2: 108-15, 2001; Nathan C., Nature: 420: 846-852, 2002; Quan N. et al., Histol. Histopathol. 17: 273-88, 2002). Blocking the effect of these inflammatory mediators by diverse strategies includes, in addition to the conventional small molecule drug appwoach, the preparation of antibodies, peptidiic mimetics or decoys, oligonucleotides, either antisense or triple-helix forming, directed either against the ligand or against dts receptor(s). Selective inhibition of COX-2 alone is disclosed for example in
US patent Nos. 5,783,597, 5,840,746 and 6,025,253 which provide small molecules for alleviating, preventing or treating inflammation, neurod-egenerative diseases and angiogenic disorders. US patent 6,344,323 discloses COX-2 antisense oligonucleotides and methods o f use thereof and US patent 5,776,502 discloses methods of transcriptional modulatiom of various genes including inflammatory mediators using molecules that bind to DNA, RNA or protein. The use of antibodies or peptides is exemplified in US patent 6,277,969 wwhich discloses antibodies to human TNF-o. and peptides thereof for diagnosis and treatment of pathologies involving this cytokine.
Despite the progress in this field, there remains an unmet: medical need for effective therapies for inflammatory diseases. The pro-inflammatoxy 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 mechamisms and therefore their _ 30 complete irhibition 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. . SUMMARY OF THE INVENTION 3 The present invention relates to pharmaceutical compositions conmprising as an active ingredient non-psychotropic cannabinoids and their derivatives that are now disclosed unexpectezdly to act as direct or indirect regulators of genes involvezd in inflammatory mechanis ms. The present invention encompasses any synthetic or meatural cannabinoid which is essentially devoid of appreciable psychomimetic activity. Curmrently preferred are synthetic non-psychotropic derivatives of dexanabinol, also known as H U-211.
Surpprisingly, it is now disclosed that the PGE, 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. Therefore, some novel non-psychotropic cannabinoids are useful for the treatment of acute and chronic inflammation, autoimmune diseases and related disorders, pain, infections, liver diseases, cardio~vascular disorders, gastrointestinal disorders, disorders of the central and peripheral nervous system including neurodegenerative diseases, respiratory diseases, renal disease=s, post-operative complications, tissue rejection and certain types of cancer through meaans not previously envisioned. The fact that 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, I'L-1p, IL-2, iNOS,
TNF-a. ancl MCP-1, comprising as an active ingredient a compound of ge=neral formula (I):
Formula I
R4 1 . 6 2 R,
Soo! . “yy (0) Rs; having the (3S,4S) configuration and being essentially free of the (3R,4R) enantiomer, wherein the dashed line indicates an optional C1-C2 or C6-C1 double bond, and wherein:
RR, is selected from the group consisting of a) RR’ where R’ is selected from the group consisting of
A) a linear or branched, saturated or unsaturated, carbon side chain comprising 1-8 carbon atoms optionally interrupted by 1-3 heteroatoms, and
B3) a saturated or unsaturated cyclic moiety, an aromatic moiety or a heterocyclic moiety; the cyclic moiety having from 5-2 0 atoms comprising one or two-ringed structures, wherein each ring comprises 3-8 carbons, optionally interrupted by 1-4 heteroatoms, and optionally further substituted with one or nore groups selected from i) a linear, branched or cyclic, saturated or uns-aturated C;-Ce alkyl, ii) a linear, branched or cyclic, saturated or uns-aturated C;-Cs alkoxy, iii) a linear, branched or cyclic, saturated or uns aturated C;-Cg alkylthio, iv) ahalogen, v) carboxyl, vi) —CO0,-C,-C, alkyl, wherein the alkyl can be linear, branched or cyclic, saturated or unsaturated, vii) keto, viii) nitro, ) ix) a saturated or unsaturated cyclic moiety, an zaromatic or a heterocyclic moiety; the cyclic moiety havimg from 5-20 atoms comprising one or two-ringed structures, wherein each ring comprises 3-8 carbons, optionally interrupte-d by 1-4 heteroatoms, s and optionally further substituted with one or more groups selected from 1)-viii) as defined above, b) an amine or an amide substituted with at least one substituent as defined in R’ . abosve, ¢) athiol, a sulfide, a sulfoxide, a sulfone, a thioester or a thicdamide optionally . substituted with one substituent as defined in R’ above, anA d) ahydroxyl or an ether -OR’ wherein R’ is as defined above;
R; is s elected from the group consisting of a) ahalogen, b) alinear, branched or cyclic, saturated or unsaturated C;-C¢ alkyl, and ¢) -OR wherein R is selected from the group consisting of
A) -R", wherein R" is hydrogen or a linear, branched or cyclic, saturated or unsaturated C;-Cg alkyl optionally containing a terminal -COR™ or -OC(O)R™ moiety wherein R" is hydrogen or a linear, branched or cyclic, saturated or unsaturated C,-Cg alkyl, and
B) -C(O)R™ wherein R" is as previously defined; and
Rj; is selected from the group consisting of a) alinear, branched or cyclic, saturated or unsaturated C;-C,= alkyl, b) -OR® in which Ris a linear, branched or cyclic, saturated or unsaturated C,-Co alkyl which may be substituted at the terminal carbon atom_ by a phenyl group, and c) alinear, branched or cyclic, saturated or unsaturated C,-C; alkyl-OR™ wherein R™ 1s as previously defined; and pharmaceutically acceptable salts, esters or solvates thereof.
T he present invention also encompasses pharmaceutical composition for increasing the tramascription 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, comprissing as an active ingredient a compound of general formula (I) as previously . defined...
Currently more preferred compounds are those wherein BR; is hydroxy or lower acyloxy and wherein Rj is dimethylheptyl or a dimethylalkyl ra_dical with a total of at least 7 carbon atoms.
According to currently preferred embodiments of the present invention Rj 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 C4 alkyl, Cy. ’ alkyloxy, Cs 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 mores groups selected from the group consisting of Ce alkyl, C,¢ alkyloxy, C,¢ alkylthio, keto, carboxy, or nitro, wherein C,.¢ alkyl, C,¢ alkoxy and C,.¢ alkylthio are intended to include saturated and unsaturated linear, branched and cyclic structuxes.
According to more preferred embodiments of tthe present invention R; is selected from the group consisting of hydroxyl, imidazosle, pyrazole, oxazole, isoxazole, tetrahydropyridine, pyrazoline, oxazoline, pyrrolidines, 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, phemyl, morpholine, thiomorpholine, thiazolidine, glycerol, piperazine, 4-piperidinopiperidine, 4-methylpiperidine and tetrahydropyran.
According to additional more preferred embodirments of the present invention R; is selected from the group consisting of mono or dli-substituted amines wherein the substituent is selected from the group consisting of” an Cig alkyl, Cis alkyloxy, Ci alkylthio, imidazolyl, an imidazolinyl, a morpholimo, 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 lezast one substituent selected from the ) group consisting of Cis alkyl, Cys alkyloxy, Cy. 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 Theteroatoms, said heteroatoms each independently selected from the group consisting of N, O, and §, wherein each ring optionally is further substituted with one or mores groups selected from the group consistimg of Cy alkyl, C,.s alkyloxy, C,.s alkylthio, keto, carboxy, or nitro, wherein Ci. alkyl, C6 alkoxy and C,; alkylthio are intended to include s-aturated and unsaturated linear, b-ranched and cyclic structures. ’ According to another currently preferred embodiment, we clisclose a pharmaceutical
S composition which down-regulates gene expression of at least One the pro-inflammatory mediatoxs COX-2, IL-1B, IL-2, iNOS, TNF-a. and MCP-1, and up-regulates gene expression of at least one of the anti-inflammatory cytokine IL-140, the protective cytokine
IL-6 and of the suppressors of cytokine signaling SOCS-1 and SCOCS-3, comprising as an active ingredient a compound of the general formula (I) where-in R; is OH, R; 1s 1,1- dimethyJdheptyl, there is a double bond between C6 and C1, and R, is selected from the group consisting of hydroxyl, 2-mercaptoimidazole, imidazomle, pyrazole, 4-methyl- piperidirae, and 4-piperidino-piperidine.
Thee pharmaceutical compositions may contain in addition to the active ingredient conventi onal pharmaceutically acceptable carriers, diluents and excipients necessary to produce a physiologically acceptable and stable formulation.
Th_e pharmaceutical compositions can be administered by any conventional and appropriate route including oral, parenteral, intravenous, intramuscular, intralesional, subcutan_eous, transdermal, intrathecal, rectal or intranasal.
Pri or 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 admin istration and the duration of treatment desired.
A further aspect of the present invention provides a method of preventing, alleviating or treatirig a patient by regulating pro- and anti-inflammatory mmediators selected from
COX-2, IL-1B, IL-2, iNOS, TNF-a, 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 medicam ent for preventing, alleviating or treating a disease by r-egulating pro- and anti- inflammatory mediators selected from COX-2, IL-18, IL-2, iNOS,. TNF-a, MCP-1, IL-10,
IL-6, SOCS-1 and SOCS-3, of a compound of genera? formula (I) substantially as shown in the specification.
BRIEF DESCRIPTION OF THE FIGURES
To assist in the understanding of the invention, and in particular of the data that are miven in the examples, the following drawing figures are presented herein:
Figure 1 shows the effect of various doses of dexanab-inol and its analogs on IL-2 in Jurkat cells activated with PMA and Calcium ionophore. In gpanel A the down-regulatory effect is rneasured at the level of IL-2 gene expression by real-time RT-PCR. In panel B the down- regulatory effect is measured at the level of IL-2 secretion. Eigure 2 shows the down-regulatory effect of 10 pM of dexanabinol and its analogs on
COX-2 gene expression in Jurkat cells activated wih PMA and Calcium ionophore, as measured by real-time RT-PCR.
Figure 3 shows the effect of dexanabinol and its analedgs on gene expression in the brains of mice submitted to MCAo 18 hrs before the me-asurements. In panel A the down- regulatory effect is measured on the pro-inflammatory mediators COX-2, MCP-1 and IL-2.
Im 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 (m) 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-qo; D: IL-1f; 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 dxiven expression of luciferase in activated T cells.
Figure 6 shows the effect of various doses of dexanabirol, 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. . 25 Fagure 7 shows the effect of various doses of dexanabinol on tumor growth of in vivo implanted LoVo colorectal cancer cells.
Figures 8 shows the effect of dexanabinol and PRS-211,2209 in vivo in the MPTP model of neurociegeneration. In panel A the neurological outcome is mneasured in a short-term study.
In pan el B the functional outcome is measured using the rotarod test in a long-term study.
DET AJLED DESCRIPTION OF THE INVENTION
“The present invention provides compositions effective to cause a reduction in gene expres sion of at least one of COX-2, IL-1B, IL-2, iNOS, T NF-o. and MCP-1. Moreover, compo-sitions of the present invention cause an increase in gene expression of at least one of the anti-inflammatory cytokine IL-10, the protectiv=e 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 mecharisms. It will be noted that the compounds of the pres ent invention have been tested for thedr impact on gene expression on a limited set of genes selected for their known involvement in the immunomodulatory and/or anti-inflammmatory signaling cascades.
Assaying the effect of those non-psychotropic cannabinoid derivatives on a larger set of 15S genes, such as found in microarrays, may reveal additional genes that are involved in the new gerne regulatory action herein disclosed.
Thhe 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 (BR4R» configuration produce undesirable psychotropic *“‘c annabis” type effects, which preclude their use for other therapeutically interesting effects. The compounds of the inventio-n being of the (35,45) configuration are substantially devoid of the undesired psychotropic effect and thus can be used for the treatment of various diseases and disorder s. Thus, in the present specification and claims which follow the term “essentially free” qualitatively refer to (3S,4S) compounds of high optical purity substantially devoid of the umdesired psychotropic effect lying with the (3R,4R) enantiomer. The quantitative . criterion of the minimum acceptable degree of optical purity of an intended therapeutic enantion-aer is dictated by the pharmacological potency of thie opposite enantiomer. The : higher thie psychotropic activity of the opposite enantiomer, the stricter is the requirement for optic al purity. Therefore, the quantitative meaning of “essentially free” depends upon the nature of each enantiomeric pair. The enantiomeric goair 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 A°-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 emantiomeric purity of at least 99.8% (Mechoulam R. et al., Tetrahedron Asymmetry 1(5): 315-8, 1990). ’ In the present specification the term “prodrug” represents compounds which are rapidly transformed in vivo to the parent compoumds of formula (I), for example by hydrolysis in blood. Some of the compounds of form ula (I) are capable of further forming pharmaceutically acceptable salts and esters. “Pharmaceutically acceptable salts and esters” means any salt and ester that is pharmaceuti cally 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, incltading 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.
In the present specification “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.
In the present specification “enhancing or increasing effect” is the ability to increase the activity under discussion by at least 2 folds, prefer ably 3 folds, more preferably 4 folds and most preferably 5 folds or more.
In the present specification and claims which follow “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. The term “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.
C30 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.
Theroughout this specification and the claims which fellow, the alkyl substituents can be satura ted 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. ' Thes genes affected by compounds of the invention are listed below and the diseases
S wherein aabnormal regulation of said genes is implicated in the pathological progression are ’ briefly re viewed.
COX-2
In addition to their specific physiological functions ir several organs, the kidney and gastrointestinal (GI) system in particular, prostaglandins h_ave been known for some time to play a major role in the inflammatory process. They arez involved as mediators of pain, edema armd vascular permeability in arthritic diseases and €hey have been postulated to be involved in the pathophysiology of colorectal cancer. The= biosynthesis of prostaglandins depends vapon 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 const-itutively and COX-2 following induction by a number of agents including mitogens, enclotoxins, hormones, cytokines, stress comditions and growth-factors. As 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 main ly responsible for the pathological effects of prosstaglandins in inflamed tissues.
However, recent pharmacological studies have shown that COX-2 is not exclusively expressed. in inflamed tissues, but is constitutively present in several organs where it synergizess with COX-1 in maintaining homeostasis. There fore, total inhibition of COX-2 might no® be desirable. Nevertheless, a selective inhibitor of COX-2 is expected to be 2S useful in treating the pathophysiological effects of prosta_glandins, by virtue of its anti- inflammatory, antipyretic and analgesic properties. Indeed COX-2 inhibitors already exist on the m arket and have a wide range of therapeutic benefits. COX-2 inhibitors were already found effective in the treatment of osteoarthritizs, rheumatoid arthritis, ocular ) inflammation, acute and chronic menstrual pain, gastritis ca_used by bacterium helicobacter pylon. In addition a selective inhibitor would have potenttial anti-cancer effects, such as with breast and colorectal cancer, would be useful in the treatment of polyps and angiogene=sis and be an attractive candidate for the treatmen t of neurological damage either resulting rom 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 im patients with signs of platelet activation such as unstable angina, severe atherosclerosis and during angioplasty.
IL-1B
Interleukin-1p (IL-1) is a potent pro-inflammatory cytokine that has been implicated in a broad spectrum of diseases. Cells known to express IL-1p 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-1B is a key factor in several inflammatory disorders that accompany for example septic shock, IBD, pancreatitis, ulcerative colitis, pulmonary inflammation and wound healing. It is also implicated in inflammation leading to conmective tissue destruction such as in rheumatoid arthritis, osteoarthritis, synovial inflammation and periodontal disease. Moreover, it is involved in central nervous system (CNS) pathologies, where it is thought to exacerbate neuronal loss. Its level is elevated in brain injury, ALS, AD, PD and anorexia. However, it remains unclear how the effects of IL-1B are mediated. Exacerbation may be a result of either the direct cytotoxic action of IL-1B 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- 1p production. Inappropriate production of [L-1B was also observed in immune disorders such as allergy, systemic lupus erythematosus (SLE), psoriasis, graft versus host disease and MS. IL-1p 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. The promotion of pancreatic beta-cell destruction leading to insulin dependent diabetes mellitus shows dependence on IL-1. The acquired expression of
IL-1B, 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 "30 cardiovascular disorders, in infectious diseases, in renal and liver dysfunction, acute respiratory distress syndrome (ARDS), im ischemic and reperfusion injury and multiple organ failure. The long list of diseases wherein IL-1p was found impli cated supports its central role as a pivotal pro-inflammatory mediator.
IL-2
T he 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. Following ligand binding to tThe T cell receptor together with the engagement of a co-stimulatory receptor such as CID28, 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 transcri ption factors, including AP-1, NF-kB and NFAT, is regulated by <lifferent signaling pathwaoys, 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 cal cium ionophore. Immunosuppressive drugs such as cyclosporin _A strongly inhibit the trarascription of the IL-2 gene and their contribution to the therapeu tic arsenal is well known. Compounds able to inhibit the production of the cytokine ICL-2 are potential immuneosuppressive drugs important in the treatment of disorders wheres T cell activation plays a pivotal role, in particular inflammatory conditions with an etio-logy including an autoimenune component such as arthritis, rheumatic diseases, systemic lupus erythematosus, myasthenia gravis, inflammatory bowel disease, chronic 1 iver disease, heart failure, multiple sclerosis, inflammatory demyelinating neuropathies, psoriasis, diabetes type 1, parasitic infections, uveitis and other ocular inflammatory comditions, Sjogren’s syndrorme, glomerulonephritis and transplant rejection.
IL-6
Iraterleukin-6 (IL-6) is critical to the regulation of the immune ard haematopoietic systemss. The pleiotropic nature of this cytokine family has resulted] in a variety of pseudomames based on its multiple biological functions. IL-6 elicits BB cells to undergo prolifer-ation and differentiation into antibody-forming cells; assists in II"_-4 dependant [gE synthessis and T cell activation, growth and differentiation. IL-6 also ascts in conjunction . with I1_-3 to induce the proliferation of pluripotent haematopoietic pro gemitors. In addition, this cytokine induces the expression of acute phase proteins. IL-6 is secreted by T cells, B +30 cells, rnast cells, monocytes, macrophages, hepatocytes, fibroblasts, endothelial cells, keratineocytes and many tumor cell lines. Adipocytes, bone marrow stroma cells, mesangial cells ard some cell types of the central nervous system also produce t'his cytokine. IL-6 production is generally correlated with cell activation. IL-6 has been described as both a pro-inflammatosry and anti-inflammatory mediator, and its levels zare altered in a number of diseases: in in-flammatory and autoimmune diseases, such as RA and other forms of arthritis, SLE, wilcerative colitis, Crohn’s disease, pancreatitis, diabetes, MS, psoriasis; in } infectious diseases, such as HIV, bacterial infections and se=psis, viral and bacterial meningitis; in Oncologic disorders such as metastatic melanoma, cervical cancer, myeloid : leukemia, multiiple myeloma, Hodgkin’s discase, metastatic rena 1 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 hespatectomies; in kidney, bone marrow or liver transplantation; in AD, burns victims and patients suffering from myocardial infarct. 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 cytokin-e will act as pro- or anti- inflammatory. However, recent studies suggest that 11-6 shosuld be considered as a protective cytokine in the overall balance of cytokine regulation. Proper regulation of this important cytok-ine will have clear beneficial therapeutic impact.
IL-10
Interleukim-10 (IL-10) down-regulates the production of pro-inflammatory cytokines and chemokines by activated macrophages, monocytes, polymorp_honuclear leukocytes and eosinophils. It kaas been recently suggested that part of this down—regulation is achieved by elevation in the levels of SOCS molecules (Suppressors Of Cytok-ine Signaling). Therefore
I-10 is an an ti-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 respons es and also Th2 mediated inflammatory processes. IL-10 has a beneficial effect on a variety of acute and chronic inflammatory and autoimmune events including but not limitecd to rheumatoid arthritis, ischemia-reperfusion injury, atherosclerosis, psoriasis, pemphigus, allergic contact sensitivity reactions, uveit is, organ transplantation, injury, infectiom and sepsis, inflammatory bowel disease, acuite pancreatitis, asthma, nephrotoxic nepwhritis and certain malignancies. iNOS
Nitric oxdde (NO) is a short lived molecule required for many physiological functions in hosst defence, inflammation and immunity. NO is symthesized by the enzyme nitric oxide sym thase (NOS) and overproduced during various pathological inflammatory states. Three distinct isoforms of NOS have been identified: neuronal (nNOS), entothelial
(eNOS) and inducible (iNOS). Overproduction of NO by nNOS and iNOS have been reportesd in a number of clinical disorders including acute and chromic neurodegenerative disorders, convulsions, pain, septic shock, asthma, tissue damage following inflammation, _ Crohn_’s disease, SLE, osteoarthritis, rheumatoid arthritis, allograft resjection and in certain cancers. iNOS is induced by endotoxin or pro-inflammatory cytokines and in tum : modulate expression of the latter. Selective inhibition of nNOS or iNOS is expected to providle a novel therapeutic approach to various diseases.
MCP -1
Monocyte chemoattractant protein-1 (MCP-1) is a pro-inflammmatory chemokine of the C—C family, responsible for the recruitment and activation o=f mainly monocytes, macro-phages, 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-o, IL-1, nitric oxide and prostaglandins. MCP-1 can be involved in beneficial processes such as wound healing, but when expressed En excess it becomes involved in the pathophysiology of a large number of inflammateory and autoimmune diseas es. MCP-1 has been implicated in a large number of diseases that affect various organs by means of acute or chronic inflammation. Pathological site=s of action of MCP-1 includ_e the skeleton with disorders such as rheumatoid arthritis &nd various types of osseowas inflammation, the kidneys with nephrites, nephritic syndmomes and nephrosis characterized by glomerular nephritides, the eyes with uveitis, vitreoretinal disorders, proliferative diabetic retinopathy, and other ocular inflammatory conditions, the cardio-vascular system with MCP-1 involvement in the early stages of atherosclerosis, in resteneosis and in the inflammatory response following myocardial infarction, the respiratory system with alveolitis, asthma and lung fibrosis or allergic inflammation, the digestdve tract with inflammatory bowel diseases (IBD) including both ulcerative colitis and Crohn’s disease. The nervous system is also an important targzet where MCP-1 up- regula tion has been observed in various types of pathology. In the ce=ntral nervous system, increases in MCP-1 level have been observed following both he-ad 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 pheno mmenon associated with neurodegenerative disorders such as Alzheimer’s disease (AD). Likewise, MCP-1 is also involved in the development of peripoheral nervous system (PNS» disorders characterized by mononuclear cell infiltration and melated demyelinating disorders. Moreover 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 monocwies, 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 tar_get tissues in inflammatory and autoimmune disease would be a highly effective intervention.
SOCS
Cytokines exert their biological activity through binding to specific cell surface receptors, that initiate the appropriate intracellular signal trarsduction cascade which in turn lead to the physiological outcome. Despite their dive-rse biological roles, many common themes have emerged in cytokine signal tramsduction. Upon receptor multimeri sation, the Janus kinases (JAKs) are activated and gohosphorylate, among other proteins, the signal transducers and activators of transcription (STATS). Dimers of phosphorylated STATs then move into the nucleus wheres 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. There are at least three families of proteins that inhibit JAK/STAT signaling and the suppressors of cytokine signaling (SOCS) belong to one of them. SOCS prote-ins 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 willl 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 theim activity is presently being unraveled. SOCS-1 and SOCS-3 are induced by about the samme set of cytokines, including : IL-6, Gro wth Hormone (GH), IL-10 and GM-CSF, though witl different efficiency. In turn
SOCS-1 inhibits IL-6, GH signaling, IL-2, IL-4, Interferorms (IFN) a, Bp and vy, LIF, oncostatinn M and trombopoeitin. SOCS-3 down-regulates leptin, GH, IFN-o, IFN-f and
IFN-y. Thus, induction of SOCS proteins by one cytokine alse attenuates the response for additional cytokines different from the original inducer, by a phenomenon of cross inhibitiosn. These proteins are potentially important regulators oef inflammatory and immune= responses of hematopoiesis and hormone response.
TNF-a
Tumor necrosis factor (TNF) has been attributed a central mole 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 eenvironmental agent, produces focal tissue necrosis in the target organ. As a result of thiss 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 iflammatory products thus amplifying the response. TNF-a is a cytokine produced primarily by monocytes and macrophages. Beside its physiologic effect, TNF-a is cytotoxic and regulates inflammatory processes through induction for instance of IL-1, IL -6, IL-8, macrophage inflammnatory protein (MIP)-2, granulocyte-macrophage colony stirmulating factor (GM-
CSF) and adhesion molecules. At elevated levels TNF-o. is involved in septic shock syndrorme, autoimmune and inflammatory processes including C rohn’s disease, brain injury, venous thromboses, arteriosclerosis, vasculitis, IBD, MS, EAE, SLE, AD, PD,
AIDS dementia, contact dermatitis, mixed connective tissue disease, arthritis, organ specifics toxic response, hepatic injury during sepsis and reperfusion, chronic inflammatory lung d-seases, muscle wasting and cachexia. The therapeutic immportance of TNF-a blockacie is tremendous, nevertheless it should be kept in mind that T"NF-a is also involved in normal physiological and repair processes and totally eliminating #this cytokine would be detrimental.
By virtue of their anti-inflammatory and immunomodulatorZy properties, resulting from the gene regulation of the pro- and anti-inflammatory mediators selected COX-2, IL- 1B, IL-2, iNOS, TNF-a, MCP-1, IL-10, IL-6, SOCS-1 and SOCS-3, it will be recognized that thee compositions according to the present invention will tbe useful for treating indicat-ions having an inflammatory or autoimmune mechanism inv-olved in their etiology ‘ or pathogenesis. Such diseases or disorders are exemplified Wy multiple sclerosis, amyotrophic lateral sclerosis, systemic lupus erythematosis, myasthenia gravis, Sjogren’s "30 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 includirng uveitis, vitreoretinal disorders, proliferative diabetic retinopathy, allergic conjunctivitis; metabolic diseases that inv-olve abnormalities in lipid peroxisomes and lipid perosxidation and/or oxidative stress; skim related disorders including psoriasis, pemphigus and related syndromes, delayed-type . hypersensitivity and contact dermatitis; respiratory dis-eases including cystic fibrosis, chronic bronchitis, emphysema, chronic obstructive pulrmonary disease, asthma, allergic : rhinitis or lung inflammation, idiopathic lung fibrosis, tuberculosis, and alveolitis; kidney dis eases including autoimmune nephritis, renal ischemia_, nephrites, nephritic syndromes and nephrosis characterized by glomerular nephritides; liver diseases both acute and chronic such as autoimmune hepatitis, cirrhosis, hepatitis and fulminant hepatic failure; gastrointestinal diseases including inflammatory bowel diseases, ulcerative colitis, Crohn’s dis ease and gastritis, polyposis and cancer of the bowel, especially the colon; infectious dis eases generated by certain bacterial, viral and parasitic invasion and sepsis that might result from injury; and post-operative complications fo llowing angioplasty, circulatory recovery techniques, prosthetic implants and tissue or organ transplants, including graft rejection.
When the site of action is the central or the peripheral nervous system, the pharmaceutical compositions comprising as an active ingredient a compound regulating the gene expression of the pro- and anti-inflammatory mediateors selected from COX-2, IL-1 B,
IL-2, iNOS, TNF-a, MCP-1, IL-10, IL-6, SOCS-1 and SOCS-3, act as neuroprotectors. By virtue of their neuroprotective properties, it will be rescognized that the compositions according to the present invention will be useful in treating acute neurological disorders, resulting either from ischemic or traumatic damage, including but not limited to stroke, head trauma and spinal cord injury. The composition of the present invention may also be effective in treating demyelinating disorders and certain c_hronic 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-asseciated neurodegeneration.
By virtue of their analgesic properties it will be r ecognized that the compositions according to the present invention will be useful in treating pain including peripheral, ~ 30 visceral, neuropathic, inflammatory and referred pain.
The compositions of the present invention may also be effective in cardiovascular protection and/or treatment of atheroma, atheroscleromsis, and consequences thereof, restenosis, angioplasty, myocardial ischemia and myocard ial 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.
The pharmaceutical compositions of the present invention, wvhich down-regulate gene expression of at least one the pro-inflammatory mediators COX-22,IL-1B, IL-2, iNOS,
TNF-o, aned MCP-1, comprise as an active ingredient a compound o=f the general formula ®:
Formula I
R4 1 6 2 R, “ay o R; having thes (35,4S) configuration and being essentially free of the (3R,4R) enantiomer, wherein thee dashed line indicates an optional C1-C2 or C6-C1 double bond, and wherein:
R, is selected from the group consisting of a) R’ -where R’ is selected from the group consisting of
A) alinear or branched, saturated or unsaturated, carbon side chain comprising 1-8 car bon atoms optionally interrupted by 1-3 heteroatoms, and
B) a saturated or unsaturated cyclic moiety, an aromatic moiet_y or a heterocyclic moiety; the cyclic moiety having from 5-20 atoms comprising onc or ®two-ringed structures, wherein each ring comprises 3-8 cartoons, optionally interrupted by 1-4 heteroatoms, and optionally further substitu_ted with one or more groups selected from i) a linear, branched or cyclic, saturated or unsaturate=d C,-Cs alkyl, . ii) a linear, branched or cyclic, saturated or unsaturate=d C,-Ce alkoxy, iii) a linear, branched or cyclic, saturated or unsaturate=d C-Cs alkylthio, iv) a halogen, v) carboxyl, vi) —CO0.-Ci-Cy alkyl, wherein the alkyl can be linear, branched or cyclic, saturated or unsaturated,
vii) keto, viii) nitro, iX) a saturated or unsaturated cyclic moi_ety, an aromatic or a . heterocyclic moiety; the cyclic moiety having from 5-20 atoms comprising one or two-ringed structures, wherein each ring ) comprises 3-8 carbons, optionally interrupted by 1-4 heteroatoms, and optionally further substituted with one or more groups selected from i)-viii) as defined above, b) an amine or an amide substituted with at least ome substituent as defined in R’ above, ¢) a thiol, a sulfide, a sulfoxide, a sulfone, a thioester or a thioamide optionally substituted with one substituent as defined in R”> above, and d) a hydroxyl or an ether -OR’ wherein R’ is as defined above;
R; is selected from the group consisting of a) a halogen, b) a linear, branched or cyclic, saturated or unsatur-ated C,-Cs alkyl, and ¢) -OR wherein R is selected from the group consisting of
A) -R", wherein R" is hydrogen or a linear, bran<hed or cyclic, saturated or unsaturated C;-Cs alkyl optionally containing a terminal -OR™ or -OC(O)R™ moiety wherein R™ is hydrogen or a linear, bran ched or cyclic, saturated or unsaturated C;-Cg alkyl, and
B) -C(O)R™ wherein R™ is as previously defined; and
Rj; is selected from the group consisting of a) a linear, branched or cyclic, saturated or unsatur-ated C,-Cz alkyl, b) -OR? in which R”is a linear, branched or cyclic, saturated or unsaturated C,-Cqg alkyl which may be substituted at the terminal carbon atom by a phenyl group, and ¢) a linear, branched or cyclic, saturated or unsatur ated C,-C; alkyl-OR™ wherein R™ is as previously defined; and pharmaceutically acceptable salts, esters or solvates thereof. © 30 The pharmaceutical compositions of the present invention, which up-regulate gene expression of at least one of the anti-inflammatory cytokine IL-10, the protective cytokine
WE) 03/077832 PCT/IL03/00223
IL-6 and of the suppressors of cytokine signaling SOCS-1 aand SOCS-3, comprise as an actives ingredient a compound of the general formula (I):
Form. ula I . R, ‘ 1 2 “uy (8) Rj havin g the (3S,4S) configuration and being essentially free of the (3R,4R) enantiomer, wherein the dashed line indicates an optional C1-C2 or C6-C1 double bond, and wherein:
R, is selected from the group consisting of a) R’ where R’ is selected from the group consisting of
A) a linear or branched, saturated or unsaturated, carbom side chain comprising 1-8 carbon atoms optionally interrupted by 1-3 heteroatomss, and
B) a saturated or unsaturated cyclic moiety, an aromatic moiety or a heterocyclic moiety; the cyclic moiety having from 5-2€) atoms comprising one or two-ringed structures, wherein each ring comprises 3--8 carbons, optionally interrupted by 1-4 heteroatoms, and optionally further substituted with one or more groups selected from i) a linear, branched or cyclic, saturated or unsaturated C,-Cs alkyl, ii) a linear, branched or cyclic, saturated or unsaturated C-Cs alkoxy, iii) a linear, branched or cyclic, saturated or unsaturated C;-Cs alkylthio, iv) a halogen, v) carboxyl, vi) —CO0,-C,-C; alkyl, wherein the alkyl can be Linear, branched or } cyclic, saturated or unsaturated, vii) keto, viii) nitro, ix) a saturated or unsaturated cyclic moiety, an aromatic or a heterocyclic moiety; the cyclic moiety havingg from 5-20 atoms comprising one or two-ringed structures, wherein each ring comprises 3-8 carbons, optionally interrupted by 1-4 heteroatoms, and optionally further substituted with one or more groups selected from 1)-vii1) as defined above, } b) an amine or an amide substituted with at least one substituent as defined in R’
Ss above, c) a thiol, a sulfide, a sulfoxide, a sulfone, a thioester or a thioamide optionally substituted with one substituent as defined in. R” above, and d) ahydroxyl or an ether —OR’ wherein R’ is as defined above;
R; is selected from the group consisting of a) a halogen, b) a linear, branched or cyclic, saturated or unsaturated C;-C¢ alkyl, and ¢) -OR wherein R 1s selected from the group comsisting of
A) -R", wherein R" is hydrogen or a linear, baranched or cyclic, saturated or unsaturated C,-Cs 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;-Cg alkyl, and
B) -C(O)R™ wherein R" is as previously defined; and
R; is selected from the group consisting of a) a linear, branched or cyclic, saturated or unsaturated C,-C;, alkyl, b) -OR? in which R®is a linear, branched or cyc lic, saturated or unsaturated C,-Cy alkyl which may be substituted at the terminal carbon atom by a phenyl group, and ¢) a linear, branched or cyclic, saturated or unsaturated C,-C; alkyl-OR" wherein R™ is as previously defined, and pharmaceutically acceptable salts, esters or solvates thereof.
Currently more preferred compounds are those wherein Ra is hydroxy or lower acyloxy and wherein Rs is dimethylheptyl or a dimx ethylalkyl radical with a total of at least 7 carbon atoms. ) According to currently preferred embodimerats of the present invention Rj is a heterocyclic moiety selected from the group consistirag 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 C4 alkyl, C4
WNO 03/077832 PCT/IL03/00223 alky’loxy, C,.; alkylthio, keto, carboxy, nitro, sasturated or unsaturated cyclic moi eties or aromatic or heterocyclic moieties whesrein each ring comprises 3-8 carbbons optionally interrupted by 1-4 heteroatoms, said heteroatoms each independently selected from the group consisting of IN, O, and S, wherein each ring opti onally is further substituted with one or more groups selected from the group consisting of Cj alkyl, C.¢ alkyloxy, C,¢ alkylthio, keto, carboxy, or nitro, whesrein C, alkyl, C, alkoxy and C,_¢ alkylthio ares intended to include saturated and unsaturated linear, branched and cyclic structures.
According to more preferred embodiments of the present invention Ry is selected from the group consisting of hydroxyl, imidazole, pyrazole, oxazole, isoxazole, tetrachydropyridine, pyrazoline, oxazoline, pyrrolidine, imidazoline, 2-thio-imidazole, 2- methylthio-imidazoline, 4-methyl-2-imidazoline, 4,4-dimethyl-2-imidazoline, methyl sulfLde, methylsulfoxide, acetamido, benzamide, cyarao, 1,2,4-triazole, 1,3,4-triazole, 1,2,3 4-tetrazole, 1,2,3,5-tetrazole, thiophene, phenyl, morpholine, thiomorpholine, thia=olidine, glycerol, piperazine, 4-piperidinopiper-idine, 4-methylpiperidine and tetra_hydropyran.
According to additional more preferred embodimemats of the present invention Ry is selected from the group consisting of mono or di-ssubstituted amines wherein the subsstituent is selected from the group consisting of am Cis alkyl, Ci alkyloxy, Cie alkylthio, imidazolyl, an imidazolinyl, a morpholino, a piperidyl, a piperazinyl, a pyra.zolyl, a pyrrolyl, a pyrrolidinyl, a triazolyl, and aa tetrazolyl, wherein each cyclic moiety may optionally be further substituted with at least one substituent selected from the gromp consisting of Cy.s alkyl, Cys alkyloxy, C;. alkylth io, keto, carboxy, nitro, saturated or wnsaturated cyclic moieties or aromatic or heterocy-clic moieties wherein each ring com prises 3-8 carbons optionally interrupted by 1-4 heteroatoms, said heteroatoms each inde-pendently selected from the group consisting of MN, O, and §, wherein each ring optionally is further substituted with one or more gsroups selected from the group ) consisting of Cis alkyl, Ci. alkyloxy, Ci.¢ alkylthio, keto, carboxy, or nitro, wherein C4 alky-1, Ci. alkoxy and Cj. alkylthio are intended to iraclude saturated and unsaturated linear, branched and cyclic structures.
According to another currently preferred embodimeznt, we disclose a pharmaceutical com_position which down-regulates gene expression of at least one the pro-inflammatory mediators COX-2, IL-1f, IL-2, iNOS, TNF-a 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 signalimg SOCS-1 and SOCS-3, comprising as an : active ingredient a compound of the general formula (I) wherein R; is OH, Rs is 1,1- dimethylheptyl, there is a double bond between C6 and Cl, and Ry is selected from the group consisting of hydroxyl, 2-mercaptoimiidazole, imidazole, pyrazole, 4-methyl- piperidine, and 4-piperidino-piperidine.
Specific pharmaceutical 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.
The 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 ard 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 invemtion.
For water soluble compounds standard formulations will be utilized. Solid compositions for oral administration such as tab Jets, 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 polyethyl eneglycol succinate, sodium lauryl sulfate, polyethoxylated castor oils, non-ionic surfactants, stearic acid, magnesium stearate, dicalcium phosphate and gums as pharmaceutically acceptable diluents. The tablets or pills _ can be coated or otherwise compounded with plmarmaceutically acceptable materials known in the art, such as microcrystalline cellulose and cellulose derivatives such as - 30 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,
WOR (3/077832 PCT/IL03/00223 intrathecal, intralesional, adjacent to or into tumors, and other parenteral routes of administration. The liquid compositions include aqueous solutions, with or without organic cosolvesnts, aqueous or oil suspensions including but not limited to cyclodextrins as : suspencling agent, flavored emulsions with edible oils, tariglycerides and phospholipids, as well as elixirs and similar pharmaceutical vehicles. In 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 adh esive film with pharmaceutically acceptable excipients including but not limited to propylene glycol, phospholipids, monogRycerides, diglycerides, triglycerides, polysosrbates, surfactants, hydrogels, petrolat-um or other such excipients as are known in the amt.
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 mgz to about 50 mg per kg body weight, in a regimera of 1-4 times a day. The preferred range o-f dosage is from 0.1 mg to about 20 mg per kg body weight. However, it is evident to the ran skilled in the art that dosages would be deteromined 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 cosmpounds are administered locally rather than systematically, and for prevention or chronic treatment rather than for acute therapy~.
A= farther aspect of the present invention provides aa method of preventing, alleviating or treaking a patient by regulating pro- and anti-inflarmmatory mediators selected from
COX-2 , IL-1p, IL-2, iNOS, TNF-a, MCP-1, IL-10, IL-6, SOCS-1 and SOCS-3, by administering to said patient a therapeutically effective amount of pharmaceutical compossition containing as an active ingredient a com pound of general formula (I) as previously defined. : Aw further aspect of the present invention relates to the use for the manufacture of a medicazment for preventing, alleviating or treating a dissease by regulating pro- and anti- + 30 inflamymatory mediators selected from COX-2, IL-1, IL-2, iNOS, TNF-a, MCP-1, IL-10,
IL-6, S«OCS-1 and SOCS-3, of a compound of general formula (I) substantially as shown in the specification.
The principles of the present invention will be more fully understood in the following examples, which are to be construed in a non-limitative manner. } PHYSIOLOGICAL EXAMPLES : Unless otherwise indicated the test compounds are parepared 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 comcentration of 0.1% DMSO. For in vivo assays the test compounds are first diluted in crermophor: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.
During this study, we concentrated our attention toward 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-1(, IL-2, TNF-a, 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 ggene to be tested the following procedure were followed for quantitation.
A- Impact of tricyclic dextrocannabinoids on gene expression.
Example 1.
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 ug of total RNA were combined with an oligo (dT);s primer, 0.5 mM dNTP mix, 8 units of reverse transcriptase and other reaction components up to a final volume of 20 pl, accordirg to the kit instructions. The reaction mixture was incubated at 42°C for 45 min and inactivated at 70°C for 15 minutes.
Quantitative real-time RT-PCR includes 1 ul of tihe cDNA, 300 nM of the appropriate ’ forward and reverse primers (see below) and 7.5 ul 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 pl. Gene amplification was obtained using the GeneAmp 5700 sequence detection system (Applied Biosystems). Amplification included one stage of 10 minutes at 95°C followesd by 40 cycles of a 2-steps loop: 20 seconds at 95°C, and 1 minute at 60°C. During ezach annealing step, the amount of the amplified product is measured by the fluorescence of the double strand DNA binding dye, syber Green. The cycle of threshold (Cr), 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 Cris translated into a two-fold decrease in starting template molecules and vice versa. The c=hanges in the Cr of the specific gene product are normalized to the changes in the Ct 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 limes or vehicle “treated” animals. In all cases, results are also normalized to either one of thee reference house-keeping genes.
Primer sequences used:
Mouse COX-2 forward 5’-TTCCGTTTCTCGTGG TCACTT-3’
Mouse COX-2 reverse 5’- AGCGCTGAGGTTTTCCTGAA-3’
Mouse haptoglobin forward 5’-GCTGGGATCCTGRAGCTTTGA-3’
Mouse haptoglobin reverse 5°- TTGGCCATGGTTTCCTGAAC-3’
Mouse IL-1p forward 5'-ACACTCCTTAGTCCTC GGCCA-3'
Mouse IL-1 reverse 5'-CCATCAGAGGCAAGGAGGAA-3'
Mouse IL-2 forward 5-GAAACTCCCCAGGATG-CTCAC-3'
Mouse IL-2 reverse 5'-GCCGCAGAGGTCCAAGITC-3'
Mouse IL-6 forward 5'-AGAAGGAGTGGCTAAGGACCAA-3'
Mouse IL-6 reverse 5-GGCATAACGCACTAGG TTTGC-3'
Mouse IL-10 forward 5'-GCCCTTTGCTATGGTGTCCTT-3'
Mouse IL-10 reverse 5'-TCCCTGGTTTCTCTTC(CCCAA-3'
Mouse iNOS forward 5'-TTCACCTCACTGTGGCCCGT-3'
Mouse iNOS reverse 5'- GCACTCTCTTGCGGACCATC-3'
Mouse MCP-1 forward 5'-TC. ACAGTTGCCGGCTGG-3'
Mouse MCP-1 reverse 5'-TCTTTGGGACACCTGCTGCT-3'
Mouse SAA-3 forward 5’-CAGAAGTTCACGGGACATGGA-3’ . Mouse SAA-3 reverse 5’- CCAGCAGGTCGGAAGTGGT-3’
Mouse SOCS-1 forward 5'-GCATCCCTCTTAACCCGGTACT-3' : Mouse SOCS-1 reverse 5-AATAAGGCGCCCCCACTTA-3'
Mouse SOCS-3 forward 5'-AGGCACTCCCCGGGAGTAC-3'
Mouse SOCS-3 reverse 5-GGCCACGTTGGAGGAGAGA-3'
Mouse TNF-a forward 5'-AA GGACTCAAATGGGCTTTCC-3'
Mouse TNF-a reverse 5'-CCTCATTCTGAGACAGAGGCAAC-3'
Mouse cyclophilin A forward 5-TCGCCATTGCCAAGGAGTAG-3'
Mouse cyclophilin A reverse 5-GGTCACCCCATCAGATGGAA-3'
Mouse GAPDH forward 5'-G-GTTGTCTCCTGCGACTTCAA-3'
Mouse GAPDH reverse 5'-GTAGGCCATGAGGTCCACCA-3'
Human COX-2 forward 5’- TCCTGCCTACTGGAAGCCAA-3’
Human COX-2 reverse 5°- AGCCCTTCACGTTATTGCAGAT-3’
Human IL-2 forward 5’- GGGACTTAATCAGCAATATCAACGT-3’
Human IL-2 reverse 5°- TTC TACAATGGTTGCTGTCTCATCT-3’
Human cyclophilin A forward 5-GCATACGGGTCCTGGCATC-3'
Human cyclophilin A reverse 5'-“-TGCCATCCAACCACTCAGTCT-3'
Human GAPDH forward 5'-ACCCACTCCTCCACCTTTGA-3'
Human GAPDH reverse 5'-C TGTTGCTGTAGCCAAATTCGT-3'
Example 2.
Quantitation of protein usimg ELISA.
The technique used to quantify the amount of a given protein in a liquid samples, either tissue culture supernatant or body fluid, is based on Enzyme Linked Immuno-Sorbent
Assay (ELISA) methodology. Either commercially available or established in ho-use, the assay is based on the capture of the protein of interest by specific antibodies bound to the ~ 30 bottom of an ELISA plate well. Unbound material is washed away, the captured pwotein is then exposed to a secondary antibody generally labeled with horseradish peroxidases (HRP) or alkaline phosphatase (ALP). Again the unbound material is washed away, the samples are then incubated with the appropriate substrate yielding a colorimetric reacti-on. The reaction is stopped and reading is carried out in a spectrophotometer at the appropriamte wavelength. Samples are tested at least in duplicate and the appropriate standard curve, consisting of serial dilutions o»f the recombinant target protein, is incorporated on eaech . plate. Concentration of the protein in the sample is calculated from the standard curve. . 5 Example3.
Quantitation of COX-2 gene expression in LPS activated macrophages and LIPS injected mice brains.
We previously showed that dexanabinol and its analogs reduce the levels of secreted
PGE, in LPS activated mouse macrophages (RAW 264.7) cells in vitro (WO 01/9828).
The ICs for inhibition of PGE secretion were determined and found to be 10 pM, 10 pl, 4 pM, and 8 pM, for dexamabinol, PRS-211,092, PRS-211,095 and PRS-211,2220, respectively. For comparison, the ICs for inhibition of PGE, secretion for the known amti- inflammatory drugs Celecoxib, Rofecoxib and NS-398 were respectively 5 nM, 100 mM and 100 nM in the same experimental setup. We assumed then that this phenomenson observed at the level of PGE, secretion was due to direct inhibition of COX-2 enzymaatic activity. To check this hypothesis we have now tested in vitro the enzymatic activity of recombinant COX-2 on its substrate in the presence of dexanabinol and its analogs PRRS- 211,092, PRS-211,095 and PRS-211,220. The assay reproduces in vitro the true enzymamtic activity, the test compounds, wp to 10 uM, were preincubated for 5 minutes with purif3ed
COX-2, then the natural substrate AA was added and the reaction was allowed to proceed for 2 minutes. HC] was used te stop the enzyme catalysis and saturated stannous chlor-ide was added to stabilize the P(GH, product into PGFs. The concentration of PGF, vwvas measured using an Enzyme Immuno Assay (EIA), a standard curve was prepared and results were derived from this standard curve. Whereas classical COX-2 inhibitors such as
VIOXX®, Indomethacin and INS-398 strongly inhibited COX-2 enzymatic activity in Chis assay, no inhibitory effect was observed for dexanabinol and its analogs. Therefore, we decided to test whether dexanabinol and its analogs have an unexpected impact on COX-2 gene expression and for this purpose we utilized LPS stimulated RAW 264.7 cells us ing real-time RT-PCR. © 30 RAW 264.7 macrophag=es, a mouse cell line (ATCC # TIB 71), were grown: in
Dulbecco's modified Eagle's medium (DMEM) with 4 mM L-glutamine adjusted to con@iain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, and 10% heat inactivated fetal bovine seraim.
Cells were grown in tissue culture flasks and seeded at appropriate density into 24 w ells tissue culture plates. 0.5 x 10% Raw cells in one milliliter were stimulatesd with 2 pg/ml
Lipopolysaccharide ¥8.coli 055:B5 (DIFCO Laboratories). The mouse macrophages were pre-treated for one hour with controls or 10 pM of dexanabinol and its analogs, unless : indicated otherwise, and later on activated with LPS. RNA samples weres extracted from the cells 2.5 hrs after activation and COX-2 gene expression levels were analyzed by real- time RT-PCR as previously described.
The results of his experiment are expressed as fold activation of CCOX-2 over non- activated macrophages, after normalization to cyclophilin A expression. When cells are treated with vehicle only we observe a maximal 6-fold increase in COX—2 expression. A decrease of 67% in RRNA levels of COX-2 is observed when the activation. is carried out in presence of 10 uM dexanabinol. At this concentration all three analogs (PRS-211,092,
PRS-211,095 and PR_S-211,220) cause an inhibition of at least 50%. Together with the fact that COX-2 enzyme activity is not directly affected by these compounds, these results indicate that PGE, concentrations, that were originally monitored, were reeduced as a result of decrease in COX -2 gene expression and not as previously thought “by inhibition of enzymatic activity. Flowever, at this stage we do not know if COX-2 gene regulation is directly achieved by our compounds, or results indirectly from the reg-ulation of other genes with secondary” impact on COX-2 through feedback mechanisms.
To test if the gene transcription regulatory effect of compounds of the invention was of physiological consequence in the whole animal, we injected either PBS or 100 ng LPS in vivo into mice berains intra cerebral ventricular (i.c.v.). Each treatrment group was composed of at least five C57/BL male mice (6-8 weeks old, 25 g avera ge body weight,
Harlan, Israel). The mice were anesthetized with a mixture of 35 mg/kg peratal and 8 mg/kg xylazine. LPS was di ssolved in saline at 20 ng/pl and 5 pl were injected ira each ventricule at arate of 1 pl/min with the help of a syringe pump and a brain infusieon canula. After each injection, the cannula is left in situ for one more minute to avoid refJux. The various treatment groups, comnirols including the cremophor:ethanol vehicle, dexzanabinol and its . analogs (20 mg/kg), -were injected i.p. (0.1 mI/10 g body weight) simultareously with the i.c.v injection of LPS. Six hours following LPS injection, the animals were sacrificed by + 30 ip. injection of 100 rmg/kg pentobarbitone sodium and their brains were resmoved and kept at —80°C until next step. RNA was extracted from each whole brain amd COX-2 gene expression levels were analyzed by real-time RT-PCR as previously described. The results of this experiment are expressed as fold activation of COX-2 in LPS versus PBS injected brains. LPS injected brain s treated with vehicle only show a 5.3 fold increase in COX-2
RNA level, mice treated =with 20 mg/kg dexanabinol display a reduction of 53% in the level of COX-2 activation. ) From these experimental results, it is evident that the active ingredients of tke present invention are effective im reducing COX-2 RNA level both in vitro and in vivo.
Consequently, the compo unds may be therapeutically effective in the wide wariety of
COX-2 related disorders.
Example 4.
Quantitation of secretion and gene expression in LPS activated macrophages .
To test if COX-2 is the sole gene whose transcription is affected by comppounds of the invention, additional genes were further analyzed by real-time RT-PCR in tthe above- described model of activated macrophages. This part of the study was performed with
PRS-211,092 at doses up to 20 uM. The level of TNF-a and of MCP-1 was el evated by 19-fold and 55-fold respectively in activated as compared to resting macrophages, but
PRS-211,092 did not affect the level of expression of any of these genes at the doses tested. The levels of iNOS- and IL-1P were significantly elevated in activated macrophages by 266-fold and 18,960-fo1d respectively. PRS-211,092 inhibited gene expressiom in a dose dependent manner with an ICsp of 4 pM for iNOS and of 17.5 pM for IL-1 B. In this experiment COX-2 gene -expression was elevated 70-fold by activation and urder these conditions the dose related effect of PRS-211,092 yielded an ICsp of 21.5 uM.
Moreover, it was comfirmed that the reduction in IL-1B gene expression correlates to a reduction in TL-1p secretion. The ICso of dexanabinol, PRS-211,092 and PRS-211,220 for inhibition of IL-1B se cretion are 6 uM, 3 pM and 4 pM, respectively. PRSS-211,095 was tested at the single concentration of 10 pM and found to inhibit IL-1 se«retion by 70%, similarly to PRS-21 1,092 and PRS-211,220 that both yielded 76% inhibitdon at that same concentration.
From these experimental results, it is evident that the active ingredients of t he present invention are effective not only in affecting COX-2 RNA level but also in specifically reducing RNA levels of aclditional inflammatory related genes such as IL-1f and iNOS. As expected the impact on gene expression correlates with a decrease in secretion of the relevant inflammatory nmediators. The fact that COX-2 is not the sole gene whose transcription is affected provide a significant advantage to compound of the invention over classical anti-inflarmmatory therapies which are designed to block one mediator at a time.
A compound that can reduce the expression and secretion of multiple inflammatory - mediators simultaraeously harbors in a single molecule the approach of combiraed therapy now used to replace the single target classical anti-inflammatory strategies.
Example 5.
Quantitation of IML-1P gene expression and activated microglia in LPS inj ected mice brains.
The local i-c.v. injection of LPS in mice brain stimulates a wide range of inflammatory responses and cascades where various cytokines and chemrokines are implicated. Following the results obtained in examples 3 and 4, we decided to assess the effect of additional active compounds of the invention on other genes involved in the inflammatory response. Specifically, we tested the impact of dexanabinol and PRS- 211,092, both at 20 mg/kg, on the expression of pro-inflammatory IL-1( cy€okine. The protocol was basically as previously described with minor modifications. The amount of
LPS injected in th_e mice brain was raised to 250 ng and the animals were sacrificed 24 hours after LPS and treatment injection for mRNA analysis, carried out as previously described. Moreover, some animals were kept alive and sacrificed 72 hours after LPS and treatment injectiom for immunohistochemical analysis. For this purpose, after <uthanizing the animals by inj ecting i.p. 100 mg/kg of sodium pentobarbitone, brains wexe removed and fixed in 4% formaldehyde for at least 72 hours. Brains were then washed with PBS and transferred fox cryoprotection in a solution of 30% sucrose in PBS, until they sank.
After the brains sank in the sucrose they were frozen using the cryostat special fast- freezing techniques (-60°C). The brains were cryosectioned (18 pm) at the Level of the entire hippocampus. Immunohistochemistry staining was carried out using po lyclonal 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 (Vantana, France). Quantitative analysis was carried out by . 30 counting the number of immunoreactive cells/mm? at the level of the hippocampus.
Statistical analysis.
Results are expressed as meantSD. Data “were analyzed using analysis of variance (ANOVA) 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-1 mRNA, normalized to cyclophilin. LPS injected animals have 18 fold higher IL-1 mRNA levels than saline injected animals. Treatment with 20 mg/kg of PRS-211,092, administered simultaneously with the LPS stimnulation, reduced the amount of IL-1 mRNA fold activation by 56% as compared to its vehicle.
Immunohistochemical analysis for the glial cell marker revealed that i.c.v. injection of LPS caused massive gliosis, restricted to the hippocampus. Sham operated animals displayed a baseline of zero activated microglia while LPS i.c.v. injected animals had on average as many as 488 immunoreactive cells/amm?. Treatment with 20 mg/kg of PRS- 211,092 significantly reduced the amount of activated microglia by 64%, while 20 mg/kg of dexanabinol caused a reduction of 40%, both as compared to animals treated with vehicle alone.
Altogether, these results show that active compounds of the invention act through modulation of inflammatory mediators in in vive models of neuroinflammation as well as in in vitro models of activated cells of the immure system, such as the macrophages. Their 2€ effect is not only expressed by modulation of gene expression of soluble inflammatory mediators, but also by a significant decrease in the amount of activated cells involved in the inflammatory process.
Example 6.
Quantitation of gene expression and protein secretion in activated T cells.
IL-2 gene expression.
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 (pho1bol-12-myristate-13-acetate) and calcium ' ionophore. 39 The human acute lymphoma T cell line Jurkat (ATCC # TIB 152) was used to test the possible immunosuppressive effect of dexan abinol 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 serumm. Cells were grown in tissue culture . flasks and seeded at appropriate density into 24 well s tissue culture plates. 2 x 10° cells in one milliliter were stimulated using 10 ng/ml of PM_A (Sigma) and 1 uM A23187 calcium ) jonophore (Sigma). Cyclosporin A (Sandoz), a known immunosuppressive drug, was used as positive control. The controls and test commpounds were added at indicated concentrations one hour before stimulation. RNA samples were extracted from the cells 6 hrs after activation and TL-2 gene expression levels wwere analyzed by real-time RT-PCR as previously described.
Figure 1A depicts the result of this experimerat 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 uM. At this concentration 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-2 11,220 by 84% (the latter not shown).
Cyclosporin A completely blocked IL-2 transcriptioxh 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 ckange is stopped by acidification. The absorbance measured at 450 nm is proportional to tthe 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 1B, where the am ount of secreted IL-2 (ng/ml) is plotted for each treatment group. When the activated cells are treated with vehicle only the maximal level of IL-2 secretion is 8.73 ng/ml. In correlation with gene expression inhibition, dexanabinol and its analogs PRS-211,092, PRS-211,095 and PRS-211,220 inhibited the secretion of IL-2 in a dose dependent mmanner at ICso of 8 pM, 0.4 uM, 1 pM and 2 pM respectively. For comparison, the positive control Cyclosporin A inhibited IL-2 concentrations in the growth medium with an ICs of 0.06 nM in the same experimental setup. h Taken together, these results indicate that dexanabinol and its analogs down-regulate . 5 IL-2, either directly or indirectly, both at the level of its RNA and at the level of its secretion, as was shown in activated Jurkat T cells. This implies that compounds of the invention may have a beneficial therapeutic ixnpact on T cell mediated disorders.
COX-2 gene expression.
In addition, we used this experimental system to test the impact of dexanabinol and its analogs on COX-2 gene expression. Following activation of Jurkat cells with PMA and calcium ionophore we obtained elevated CO X-2 gene expression levels. The impact of the test compounds on COX-2 was assessed as previously described for IL-2. Figure 2 depicts the results of this experiment as fold activation of COX-2 over non-activated T cells. The results are plotted after normalization to GAPDH expression. We observe that COX-2 gene expression was inhibited by dexanabinol, al so known as PRS-211,007, and PRS-211,092,
PRS-211,095, and PRS-211,220, by 88%, &7%, 85% and 92% respectively. The positive control cyclosporin A was also very potent in this assay. COX-1 RNA levels were not changed throughout the experiment (data no¥ shown) supporting the specificity of action of dexanabinol and its analogs.
From these experimental results, we confirm that the active ingredient of the present invention is effective in reducing COX-2 RINA level in another in vitro system, composed of T cells instead of macrophages. Moreover, these results prove that the effect of the compounds of the present invention is not Limited to down-regulation at the level of gene expression, since the decrease in RNA is correlated to a decrease in protein secretion, as shown for IL-2. Consequently, the compournds may be therapeutically effective in the wide variety of COX-2 and IL-2 related disorders .
Example 7.
Quantitation of gene expression and protein secretion in activated Mast cells. . Mast cells are multifunctional bone m_arrow derived cells that upon activation release many potent inflammatory mediators. Rel ease is done either from preformed granules, trough the process of degranulation, or fol lowing 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) axe grown in EMEM medium with Earle’s . 5 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° cells in one milliliter are seeded. Following overnight incubation, the plated cells are preincubated for half an hour with test compounds and 10» controls and then stimulated with 10 ng/ml of PMA (Sigma) and 1 uM 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, secretion, and
IL-4 and TNF-a gene expression and secretion. For COX-2 gene expression RNA is collected one hour after stimulation. The Src family inhibitor PP2 or the PKC inhibitor
GF109203X (both from Calbiochem) are used as positive control. 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. Inhibition is calculated versus vehicle treated cells. In parallel, RNA samples are extracted from the cells at the appropriate precletermined 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.
Transient MCAo in mice : This model corresponds to cerebral ischemia as observed in stroke. Mice (C57/BL, male, 25 gr average body weight, Harlan, Israel) were anacsthetized with halothane in 30% " 30 oxygen and 70% nitrogen (4% for induction in an anesthesia chamber, and 1-2% in a facemask for maintenance). A midline incisiomx 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 (ECA) 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 (Asst, 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 eaclm 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.
Following the operation, the animaals were allowed to wake up in the cage. One-hour after insult initiation animals were clinically tested to verify the success of MCA occlusion.
The evaluating system was based on woetks by Belayev et al., (Stroke 27: 1616-23, 1996;
Brain Res. $33: 181-90, 1999). It consisted of two tests: the postural reflex test and the fore limb-placing test. The postural reflex was evaluated while the animal was suspended by the tail, whereas the fore limb-placing test was performed while the animal was held by the stomach. Table 1 summarizes the tests arad their scoring system.
Table 1: Neurological evaluation of mice with MCAo.
Item Normal Deficit
Postural reflex test (hang test) =* 0 2
Placing test (performed on eack side) #
Visual placing
Forward 0 2
Sideways 0 2
Tactile placing
Dorsal surface of paw 0 2
Lateral surface of paw 0 2
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 tol2 were included in the study. Ninety h minutes after initiation of the insult, 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 Upj ohn). Eighteen hours later, animals were sacrificed by i.p. injection of pentobarbitone sodium 100 mg/kg. Brains were then removed, and total 1S 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 (MCA0) 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 S-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 braiins.
Chemokines are low molecular weight, secreted proteins that chemoattractant and activate specific subpopulations of leukocytes. Monocyte chemoattractant protein-1 (MCP- 1) is highly specific for monocytes, which are recruited to the site of injury, become activated and secretes inflammatory. Increased MCP-1 RNA levels following MCAo were previously reported ((Che et al., Brain Research 902: 171-7, 2001). Immunolnistochemistry studies showed thak both ischemic neurons (after 12 hours of ischemi-c insult) and . astrocytes (two dayss after insult) expressed MCP-1. We tested the effect o_f dexanabinol and PRS-211,092 treatments on MCP-1 RNA levels in mice brains after- 18 hours of . MCAo. Vehicle trea ted animals displayed a 16 fold increase in MCP-1 geme expression versus sham operatec] animals. Treatment with dexanabinol and PRS-211,0922 reduced this outcome by 41% ard 63% respectively, in comparison to the vehicle treated and the untreated groups (Figure 3A).
IL-2 gene expressiomn in MCAo brains.
Dexanabinol amd its analogs were already shown to be effective in meducing IL-2 both at the level of gene expression and at the level of secretion in actiwated T cells.
Therefore, we wishe d to verify their impact in vivo and we assessed IL-2 FRNA 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. Tr-eatment 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).
I1.-10 gene expression in MCAo brains.
IL-10 is a potent anti-inflammatory cytokine strongly related to tke previously described pro-inflammatory genes. Moreover, it has already been reported th at IL-10 gene expression levels increase in rat brain following MCAo (Zhai et al, J. Newmrol. Sci. 152: 119-24, 1997) and th_at I1.-10 administration reduces rat brain injury followin_g focal stroke (Sperat et al, Neurosci. Lett. 251: 189-92, 1998). We wish to check that this: phenomenon can be repeated in whe mice model of MCAo and that our compounds h_ave a further positive impact on the expression of this anti-inflammatory cytokine. We tessted the effect of dexanabinol and its analogs on IL-10 RNA levels in mice brains after~ 18 hours of
MCAo. Vehicle trea ted animals displayed a 35 fold increase in IL-10 gerie expression . versus sham operatezd animals. Treatment with dexanabinol and PRS-21 1,092 further increased this outcormie by 4.4-fold and 2.3-fold respectively, in comparison to the vehicle © 30 treated and the untreated groups (Figure 3B).
Moreover, mamy more genes encoding for instance cytokines, adhesion molecules, or transcription factors, were reported to display abnormal levels of expression in cerebral ischemia. Out of a total of twenty four such agents screened in this study only the four genes previously described were affected by compounds of the invention. Surprisingly, in this model IL-1B, IL-6 and iNOS gene expression were not modulated by dexanabinol or . PRS-211,092, at least not at the time point selected for analysis.
From these experimental results, we confirm that 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 immune/inflammatoxy 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.
Example 9.
Quantitation of gene expression in liver and spleen following ConA injection in mice.
The ConA model for T cell mediated injury.
The most common causes of life threatening T cel 1 mediated liver damage in humans are infections with hepatitis B or C viruses and autoimmune hepatitis. Different animal models of autoimmune liver injury have been develop ed, including acute liver failure in mice induced by intravenous injection of the T cell stim ulatory plant lectin concanavalin A (ConA). ConA has high affinity for the hepatic sinus. Treatment of mice with ConA activates T cells that accumulate in the liver and relea se cytokines (IL-6, IL-10, TNF-c,
INF-y, IL-2) that regulate liver damage. Pretreatment with the immunosuppressor drugs such as cyclosporin A or FK506 completely prevents liver injury caused by ConA injection, demonstrating the major role of T cell activation in this model. We have shown that human T-lymphocytes (Jurkat cell line), activated by PMA and calcium ionophore, are inhibited by dexanabinol and its analogs (PRS-211,092, PRS-211,095 and PRS-211,220).
Therefore, we decided to test in vivo the potency of these compounds to regulate T cell ’ induced cytokine expression levels and reduce liver infl ammatory damage using the ConA model.
Each experimental group contained at least twenty” BALB/c inbred female mice (25 g average weight, Harlan, Israel). The negative control group was composed of mice injected with saline instead of ConA. The injection of ConA (Sigma) was done i.v. at the base of the tail at the dose of 15 mg/kg in saline. The treatments were injected i.v. at 5 mg/kg , 30 minutes before ConA injection, unless indicated otherwise. Compounds were diss«olved in cremophor:ethanol, further diltated with saline before injection, and vehicle omly was . included as an internal controk. Analysis was performed at predetermined times points following ConA injection.
Impact of treatment was nnonitored at three levels. First, blood samples (200—~400 pl) were collected at predetermined time points after ConA injection, using retro-orbital puncture. After short centrifugation (5000 rpm for 2 min) serum was recovered and stored at —80°C until further use for cletermination of cytokines concentrations by ELESA and aminotransferase release from thae liver as a marker for liver injury.
In parallel, the concentration of cytokines was also determined in the or-gans of interests. For this purpose, the maice were killed by dislocation of the cervical vertebrae, at predetermined time points following ConA injection. The spleen and the liv-er were removed. Part of the liver was fixed in 4% formaldehyde for histology and the other part was kept at —80°C for protein oT RNA extraction. The spleens were weighted andh a small part of the spleen was fixed ira 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. Large tissue fragnments are removed by gravity sedimentation and the supernatants are collected. Cells are washed 3 times with 5 ml of erythrocyte lysis buffer (Boehringer), resuspended in 4 mm] RPMI medium supplemented with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 1 0 mM HEPES, 1.0 mM sodium pyruvate, and 140% heat inactivated fetal bovine serum, and plated in a 6 wells culture dish. Cells are incutoated for 24 hours and cytokine concentrations in the supernatant were determined by ELISA as previously described.
ALT release from ConA injured liver.
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 znd cirrhosis. The level of its release into the bloodstream ’ 30 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 h ours. At this time point the .ALT concentration measured was about 1700 units/l and 5 mg/kg of
PRS-211,092 when administered 30 minutes before ConA injection significaratly reduced
ALT concentrationss by 57%. We used this assay to preliminarily assess tkne temporal . window of PRS-211,092 efficiency and the compound was administered eith.er 30 or 60 minutes after indu ction of injury. At both time points PRS-211,092 still reduced ‘ significantly ALT concentrations by 61% and 51% respectively. FK-506 veas used as positive control and at the dose of 1 mg/kg, it reduced ALT concentrations by 92%, 93% and 86% when injescted at the three previously described time points. Th us, the test compound has an important beneficial impact on liver injury as assessed by tie reduction of ALT, a robust marker of the liver injury. This therapeutic effect is mainztained even when the treatment is administered up to at least one hour after injury.
IL-2 gene expressioen in liver and spleen, and secretion, in the ConA model.
The induction of IL-2 expression in the liver of ConA injected mice and the effect of immunosuppressors in such a model have already been reported (Okam oto T. and
Kobayashi T., Jpn. J. Pharmacol. 77: 261-3, 1998). The level of pro-inflamrmatory IL-2 expression was assezssed in the liver at four time points: 15 minutes, 1, 4 &nd 8 hours following ConA injection. The effect of the compound is compared to its velaicle, which has no effect of its own on gene expression as assessed by the fact that vehicle treated animals yield resultss similar to saline treated animals (data not shown). Increzase in IL-2 gene expression is detected only from one hour on (Figure 4A). At this time point, vehicle treated animals displayed a 428-fold increase in IL-2 RNA levels versus sal ine injected animals. Treatment with 5 mg/kg i.v. of PRS-211,092 reduced this outcome by 37%. When the analysis is carried out 4 and 8 hours following ConA injection, the increase in IL-2 expression is correspondingly 817 and 549-fold and the test compound reduce ®hese effects by 56% and 53% respectively. The effect of PRS-211,092 is statistically significant at all time points from one hour on.
Similarly, the level of IL-2 gene expression was assessed in the spleen Of the ConA injected mice, one and four hours following injury. In this organ, vehicle treated animals displayed at each tirme point respectively a 309-fold and a 997-fold increase ir IL-2 RNA levels versus saline dnjected animals. Treatment with 5 mg/kg i.v. of PRS-211,8092 reduced this outcome by 46% when tested one hour after injury and by 32% when tested at four hours after injury.
In parallel, the spleen was removed and splenocytes weres cultured for 24 hours to allow the assessment of secretion levels. Saline injected anirmals yielded undetectable backgrovand concentrations of IL-2, ConA injected animals treated with vehicle only . yielded 1 ng/ml IL-2 while ConA injected animals treated with PRS-211,092 produced only 0.6 ng/ml a 40% reduction in secretion from splenocytes. Moreover, the : concentration of IL-2 was assessed in the plasma, PRS-211,092 succeeded to reduce the vehicle treated concentrations from 0.88 ng/ml down to 0.52 ng/ml, i.e. a 41% reduction.
In this im vivo model we show that the reduction in IL-2 RN_A levels is supported and correlated with a reduction in protein secretion. This was previously shown in the in vitro model of activated T cells.
MCP-1 gene expression in liver in the ConA model.
Animals were sacrificed at the four time points following ConA injection previously described and the level of pro-inflammatory MCP-1 expression. was assessed in the liver.
Increase in MCP-1 gene expression is detected only from one hour on (40-fold increase over saline injected animals), but at this time point treatment with 5 mg/kg PRS-211,092 has no effect (Figure 4B). Four and eight hours following ConA injection, the increase in
MCP-1 expression is correspondingly 111 and 287-fold and the test compound reduces these effects by 29% and 55% respectively. The effect of PIRS-211,092 is statistically significant at both time points.
TNF-a gene expression in liver in the ConA model.
TNF-a, a cytokine produced mainly by activated macrophages, has pleiotropic effects both beneficial, as in liver regeneration, and deletexious, when it has direct cytotoxic role in human hepatocytes. TNF-a 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 h_epatitis. In most of these disorders, the concentration of TNF-« correlate inversely with patients survival.
Animals were sacrificed at the four time points previously described and the level of pro-inflammatory TNF-a expression was assessed in the liver. Increase in TNF-a gene . expression is detected immediately, as early as 15 minutes following ConA injection (21- fold increase over saline injected animals), and already at this £ime point treatment with 5 mg/kg PPRS-211,092 has an inhibitory effect of 32%. One, four: and eight hours following
ConA injection, the increase in TNF-a. expression is correspondingly 162, 81 and 38-fold.
The test compound reduces these effects by 27% and 17%, and thesn maintains the same level of TNF-a expression (Figure 4C).
Moreosver, it was confirmed in vitro that the reduction in TINF-o gene expression ' observed in vivo is indeed in correlation with a reduction in TNF-a secretion. For this purpose, the previously described experimental system of LPS activated macrophages was used. The IC 5, of dexanabinol, PRS-211,092, PRS-211,095 and PRS—211,220 for inhibition of TNF-a. se=cretion are all between 10 to 20 pM.
IL-1P gene «expression in liver in the ConA model.
Animals were sacrificed at the four time points previously desscribed and the level of pro-inflammmatory IL-1B expression was assessed in the liver. In crease in IL-1 gene expression iss detected immediately, as early as 15 minutes followin_g ConA injection (14- fold increases over saline injected animals), but at this time point treatment with 5 mg/kg
PRS-211,092 has an enhancing effect of 64% (Figure 4D). One, four and eight hours following CeonA injection, the increase in IL-1B expression is corre=spondingly 35, 20 and 15-fold. The= test compound reduces these effects by 52%, 46% and 227%, respectively. The effect of PR_S-211,092 is statistically significant at all time points. “The effect of cytokine modulation nmight be in some cases a time dependent issue. At certain period, under given circumstancees, the presence of a cytokine might be beneficial ~while it will become deleterious i n another time window. The fact that IL-1§ is first up-megulated may have an initial positi=ve effect, such as induction of SOCS-1, further sustained by its later inhibition, as suggestecl by the decrease in ALT at the end of the study that supports the overall hepatoprotective activity of the test compound.
IL-6 gene expression in liver and spleen in the ConA model.
A bim_odal role has been suggested for IL-6 in the ConA inducced model of hepatitis.
Some studies supported the claim that IL-6 production favors the d_evelopment of hepatic injury, while in other studies IL-6 predominantly displayed a hepatoprotective activity.
These contradictory observations have been reconciled when a thorough time course : experiment ~was carried out with neutralizing antibodies. The results of this study indicate that IL-6 pwesence in the early phase of the disease is critical for evoking a strong hepatoprotective effect, while continued high concentrations of thes cytokine are harmful for the liver (Tagawa Y-Let al., J. Leukoc. Biol. 67: 90-6, 2000).
Animals were sacrificed at the four time points previously described and the level of expression of the protective IL-6 cytokine was asses sed in the liver. Increase in IL-6 gene expression is detected 15 minutes following ConA injection (4-fold increase over saline . injected animals), and at this time point treatment with 5 mg/kg PRS-211,092 has an enhancing effect of 470% (Figure 4E). One, fouar and eight hours following ConA : injection, the increase in IL-6 expression is correspondingly 90, 38 and 10-fold. The test compound continues to have an enhancing effect om IL-6 gene expression but reduced to 38% at one hour after injury. At four hours 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. As observed in the case of IL-1B, PRS-21 1,092 has a bimodal effect on IL-6 expression. The fact that IL-6 is first up-regulated whnen it acts as a protective mediator and then inhibited when its hepatotoxic role predominates, supports the overall hepatoprotective activity of the test compound.
Preliminary studies carried out with the spleeras of the ConA injected mice suggest that similarly to the situation observed in the liver, IL-6 gene expression is during the first hour after injury initially up-regulated by treatment with 5 mg/kg i.v. of PRS-211,092.
Again, this up-regulation of IL-6 when the cytokine acts predominantly as a protective mediator is in line with the overall anti-inflammatox-y and liver protective activity of the test compound.
IL-10 gene expression in liver and spleen, and secr-etion, in the ConA model.
The roles of the anti-inflammatory cytokine IE.-10 in ConA model of murine liver injury have already been reported and it has been shown that administration of anti-IL-10 antibodies results in aggravated liver injury (Kato et al, Hepatology Research 20: 232-43, 2001). Animals were treated with 5 mg/kg i.v. of PRS-211,092 and euthanized 15 minutes, 1, 4 or 8 hours following ConA induced liver injury. Vehicle treated animals displayed on average a 9-fold increase in IL-10 liver or spleen RNA levels versus saline injected animals at all time points tested. 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 . 30 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 fixst up-regulated in the spleen then in the liver. In parallel to RNA quantitation in the whole organ, the spleen was removed at one hour after injury and splenocytes were cultured for 24 hours to allow the assessment of secretion_ levels. Saline injected animals yielded undetectable backgrowund levels of IL-10,
ConA injected animals treated with vehicle only yielded 331 pg/mL IL-10 while ConA injected animals treated with PRS-211,092 produced 549 pg/ml, a 1.7 fold increase in : secretion. from splenocytes, which correlated with the increased RN A expression in the spleen. Last, the level of IL-10 was determined in plasma 1 hour following ConA injection : and 5 mg/kg i.v. of PRS-211,092 significantly (p=0.03) increased thae level of this anti- inflammatory cytokine from 493 pg/ml in vehicle treated animals to 726 pg/ml in PRS- 211,092 treated animals, a 1.5 fold increase which also correlates with the increase in gene expression observed in the spleen at this early time point. In this in vivo model we show that the -increase in IL-10 RNA levels is supported and correlated with an increase in protein secretion.
Fro-m these experimental results, we confirm in a second in wivo model that the active ingredient of the present invention is effective in reducing the RNA level of pro- inflammatory mediators while it is increasing the RNA level of th e anti-inflammatory cytokine IL-10. Consequently, it further supports the fact that compounds of the invention may be -therapeutically effective in the wide variety of immune/irflammatory related disorders _
Expression of genes involved in cytokine signal transduction, in the ConA model.
The purpose of this study was to determine if a common mechamism lye beyond the observations that active compounds of the invention are efficient mociulators of anti- and pro-inflarmmatory mediators. In recent years, progress was made in -unraveling cytokine signal transduction. This work led to the recognition of Janus kinases (JAKSs) and signal transduce rs and activators of transcription (STATSs) as positive regulators; and of suppressoers of cytokine signaling (SOCS), protein inhibitors of activated STATs (PIAS) and the S H-2 containing phosphatase, as negative regulators of this si gnaling pathway. In this study~ we checked the effect of ConA induced liver injury on the levels of SOCS-1 and
SOCS-3, as previously described. Both SOCS-1 and SOCS-3 have irmportant recognized roles in the liver. SOCS-1 deficient mice suffer from three mmajor abnormalities: lymphopenia, macrophage infiltration of several organs including the liver, heart, lung . 30 and skin, and severe fatty degeneration of the liver. On the other hand, SOCS-3 is the main
SOCS geme induced in the liver by GH. Thus, we tested if PRS-211,092 had an effect on the expresssion of these negative regulators of gene expression.
As can be seen from Fi gures 4F and 4G, the pattern of impact on gene expression is a mirror image of the impact of PRS-211,092 on the cytokine themselves. While PRS- 211,092 lowered the almost linear slope of MCP-1 expression over time from liver injury, : it symmetrically increased the slope of SOCS-1 gene expression. Similarly the pattern of § increase in SOCS-3 due to PRS-211,092 treatment is the mirror image of the decrease in ’ IL-2, TNF-a, IL-1p and IL-6. These results, showing activity of the test compound on the modulation of regulators of cytokine signal transduction, support the fact that the active compounds of the invention act through regulation of transcription of genes involved in inflammatory processes.
Moreover, when taken together with the results obtained with the downstream regulated cytokines and chemokines, we can make the following observation. From the graphs displayed in Figure 4 we see that generally speaking PRS-211,092 has a clear moderating effect on the expression of inflammatory related genes, or their regulators, that are activated during the course of liver injury in the ConA model. PRS-211,092 down- regulates the expression of positive inflammatory mediators and up-regulates the expression of negative mediators. The cytokines and chemokine tested often act in concert and cross regulate one another to yield the final physiological outcome. When the ultimate effect of the above-described gene modulation is tested by a biological marker of liver injury, such as ALT, SAA-3 and haptoglobin, we conclude that the overall end result is indeed hepatoprotection.
Expression of genes involved in acute phase response, in the ConA model.
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). As previously stated activated macrophages and other leukocytes release pro-inflarnmatory cytokines such as TNF-o, IL-1 and IL-6. These 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). Measurement of acute phase protein level is used to monitor the severity of the innate response and thus predict the prognosis of the disease. The purpose «30 of this study was to determine if indeed the modulation of anti- and pro-inflammatory mediators in the liver lead to the appropriate down-regulation of positive APPs, both at the level of gene expression and secretion. In this study we checked the effect of ConA induced liver injury on the levels of SAA-3 and haptoglobin, and we tested if PRS-211,092 had an effect on the expression of these acute phase proteins, as previously described.
Administration of 5 mg/kg PRS-211,092, 1 mg/kg FK-506 or vehicle thirty minutes before ConA had the following effects on the APPs tested four and eight hours after induction of liver injury. 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. These results furt her strengthen the fact that the modulatory 1d effect of active compound of the invention on gene expression of the cytokine network of pro- and anti-inflammatory mediators has an overall therapeutic benefit, as monitored by the significant decrease in the APPs, SAA-3 and haptoglobin.
Example 10.
Hepatoprotection through inhibition of apoptosis in HepG2 cell lines. 1S The purpose of this study is to assess wwhether the hepatoprotective effect of the active compounds of the invention is achieved. through inhibition of apoptotic events in cells of hepatic lineage. The proteolytic cle avage of caspases and poly-ADP-ribose polymerase (PARP) is a known marker of apoptosis, and is investigated by immunoblotting or immunohistochemistry in HLepG2 cells. Apoptosis is induced in these cells either by TNF-a, anti-CD95 or ethanol.
HepG2 (ATCC # HB-8065) are human hepatocellular carcinoma cells and are grown in Eagle’s Minimum Essential medium, supplermented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential armino acids, and 1.5 g/l sodium bicarbonate.
A total of 1 x 10° cells/well are seeded in 6-wrell plates and are treated with either anti-
CD95 (0.5 mg/ml) or with TNF-a (10 ng/ml), and cycloheximide (10 mg/ml). For ethanol induced apoptosis, the range of the inducer is 1020 to 400 uM and cells must be cultured for 24 hours under those conditions. Test compournds and controls are added to the cells one ’ hour before the induction of apoptosis, unless otherwise stated. After 6 hours for TNF-a or anti-CD95 or 24 hours for ethanol induction, cel Is are washed in cold PBS and lysed in 1%
Triton X-100, 50 mM Tris-HCI, 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. After centrifugation (10 minutes, 13,000 rpm, 4°C), 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 washexd 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 fact ors in activated T cells.
We have established that [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 cletermination 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.
Two different lines were established, im each case Jurkat cells were cotransfected with a plasmid containing a luciferase reportex gene controlled by either NF-AT (Clontech cat# S2088) or AP-1 (Clontech cat S2087) transcription elements and with a plasmid containing the neomycin resistance gene. Transfection was carried out using the cationic lipid reagent method (Dimeri-C, Invitrogen L.ife Technologies). In order to isolate stable clones, the transfected human T cells were submitted to gentamicin selection for four weeks, and positive clones were identified. The stable clones, transfected with the various transcription elements controlling luciferase, were grown as previously described for
Jurkat cells, with the addition of 300 pg/ml gentamicin. On the experimental day, the cells containing each reporter were plated in a 24 well plate at 10° cells/mlUwell. Test compounds were resuspended in DMSO, and added for one hour before cell activation, at predetermined doses wherein DMSO final coracentration is 0.1%. Cyclosporin A was used . 30 as positive control at the dose of 100 nM. Cells were then stimulated with 10 ng/ml PMA and 2 uM calcium ionophore for 6 hours. At the end of the experiment, cells were collected, rinsed in PBS and lyzed for 15 minutes on ice in 50 pl luciferase lysis buffer (Promega). Cell debris were removed by spirming down the cell lysate for 5 minutes at high speed at 4°C. Luciferase activity was measured in a black 96 well plate, where 10 pl of cleared cell lysate were combined with 90 pl of luciferin substrate (Promega).
Luminescence was immediately measured in the appropriate reader (X-flour, Tecan).
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 cxpression. PRS-211,092 has an ICs of 1.5 pM and
PRS-211,220 has an ICs of 3.9 uM. In this experimental setup, 100 nM of cyclosporin A caused a total inhibition. Similarly, we tested the activity of 10 pM PRS-211,092 on cells stably transfected with the AP-1 reporter. Cell activation increased the level of AP-1 driven luciferase expression from 182 LU to 19391 LU, however neither PRS-211,092 nor cyclosporin A affected this outcome. These results further support that the active compounds of the invention act through regulation of transcription of genes involved in inflammatory processes.
Example 12.
DNA arrays.
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 exarmined 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. We have used membrane-based : focused macroarrays each consisting of gene families representing a biological regulatory pathway such as cytokines or chemokine arrays, commercially made by SuperArray Inc. ) 30 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 PCCR, as previously described.
B- Impact of tricyclic dextrocannabi noids on animal models for various diseases.
Example 13. © 5 Effect of the compounds in carrageeman induced paw edema.
The purpose of this study is toe test in vivo the anti-inflammatory activity of the compounds in paw edema induced by injection of 1% carrageenan in the animal hind paw.
Female Balb/c mice (20 gr average b-ody weight, Harlan, Israel) are anesthetized with a combination of xylazine and pentobarbitone diluted in sterile saline, 15 and 6 mg/kg 1.p. respectively. Anesthetized mice are ikjected 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 frorm 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 1.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, 0.0mm) ard 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 p aws of the same animal, either as A Paw Volume (APV) in millimeters cube or as A Paw Thickness (APT) in millimeters. Each group comprises at least 10 animals. Results can be further normalized to the APV and APT values of each treatment group at 0 m g/kg (vehicle only). At the end of the study, animals are euthanized with an i.p. injection of 100 mg/kg pentobarbitone.
The differences between APV amd APT among various treatment groups are analyzed by analysis of variance (ANOVA) fol lowed by post-hoc Fisher test. A value of p<0.05 1s considered to be statistically significamt. : 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.
These results are statistically significant as compared to vehicle treated animals. At these doses, known anti-inflammatory drugs such as Celecoxib and Dexamethasone (DXM) yield respectively 24% and 26% reduction in paw thickness at 0.1 mg/kg, 28% andl 31% at 0.25 mg/kg and both 33% at 0.5 mg/kg. It should be kept in mind that these comrmercially : available drugs display serious side effects that prevent chronic uses without complementary protective: medication. The fact that compounds of the invention h_ave anti- inflammatory activity cormparable to these drugs is very encouraging since comp ©ounds of this family have the advantage of being devoid of side effects, thus makimg them interesting candidates fox the replacement of existing anti-inflammatory drugs. These results support that the active compounds of this invention, which act through modulation of pro/anti-inflammatory mediators, have an anti-inflammatory effect that may be relevant to a wide range of human conditions with inflammatory components.
Example 14.
Effect of compounds in cancer chemoprotection.
In vitro.
Cells from several tumor-derived cell lines are tested for their proliferation capacity in presence of our test coxnpounds. Pancreatic tumor cell lines were obtained from ATCC.
Panc-1 (ATCC # CRL-1469) were cultured in DMEM supplemented witim 4 mM
Glutamine, 4.5 g/L. glucose, 1.5 g/L bicarbonate, antibiotics (penicillin, nystatin and streptomycin) and 10% heat inactivated 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° cells/ml/well) and grown overnight. The cells are incubated with the test compounds (1-100 pM) or vehicle (0.1% DMSO final concentration). Cell viabwility 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% form aldehyde in PBS for 10 minutes. Following fixation the cells are washed three times with PBS and . 30 250 pl of 0.5% (w/v) crystal violet is added to each well and the plates were incu bated for minutes at room temperature with gentle agitation. The stained cells were them washed three times with double distilled water and the color was extracted by adding to each well 250 pl of 10% acetic acid. The plates were agitated for 15 minutes at room temperature and 100 pl were transferred ir duplicate to a 96 well plate for reading. Optical density (OD) of the cells was measured at 620 nm in an ELISA reader and results are expressed a-s % viable cells. Absorbance of untreated cells is recorded as 100%. The experimental : results show that Aspc-1 proliferation was not affected by the presence of dexanabinol up to 15 uM whereas Panc-1 «ells proliferation was inhibited by 26% at this sam € concentration. The ICs (dose inhibiting cell growth by 50%) is determined.
Moreover, 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 495 formaldehyde in PBS, for 10 min. Cells are washed twic € with PBS-0.1% Tween20 (PBS-T) and permeabilized with cold methanol for 20 min. Th.e cells are washed twice with PEBS-T and incubated with 1 ml blocking solution (3% BSAa,
PBS-T) for 30 min. The primary antibody (rabbit anti- cleaved caspase 3 (asp175) Cell
Signaling Technology, dilutecl 1:50 with blocking solution) is added and the cell s incubated for 60 min. at 37°C. The cells are washed twice with PBS-T. The secondarZy antibody (HRP conjugated anti—rabbit IgG diluted 1:200 with blocking solution) is added teo the wells and incubated for 60 min. at RT. Cells are washed twice with PBS-T aned incubated for 10 min with a fluoresceine tyramide reagent (NEN, diluted 1:50 witih amplification diluent). Cells are washed twice with PBS-T and the signal visualized by fluorescence or confocal microscope. Beside monitoring activated caspase-3, th-e expression of apoptosis-related. genes in cells treated with dexanabinol and its analogs is compared to that in untreated cells. The procedure for real-time RT-PCR is as previously described. For each gene, a pair of specific PCR primers is designed and the reaction 1s done according to the ABI pro tocols. The quantification of each gene expression level 1s normalized to a housekeeping geene and compared to RNA samples from non-treated cells.
Invivo.
Once we have shown tha t dexanabinol and its analogs were inhibitors of tumor cell proliferation in vitro, we wished to test if our compounds were as efficient in vivo. For this purpose the tumor cells LoVo (originating from a colorectal tumor; ATCC # CCL-229») were grown in DMEM contairaing 4.5 g/L ghicose, 2 mM Glutamine, 1% Pen/Strep aned 10% heat inactivated fetal calf serum. The cells were harvested using Trypsin-EDTA, th ¢ detached cells were rinsed in PBS, and counted. Predetermined amounts (1x10° cells im constant volume of 0.12 ml/aniamal) were injected s.c. above the right femoral joint in nud-e
CD-1 male mice (average weight 20-25 gr, Harlan, Israel). Each treatment group wa_s composed of at Keast 7 animals. Each animal was clinically monitored daily. The growth of the tumor was also monitored during the daily visits but actual nneasurements 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 raange of 2.5 to 10 mg/kg/day.
Thirty-six out of the 40 implanted mice developed a visible tuzmor within 5 to 6 weeks from tumor implantation. All animals were treated first on the 36™.42" day of tumor implantation and the treatment lasted 8 weeks. One animal died in each treatment group between the 5™ and the 7" 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 (mw) seems more efficient than 5 mg/kg (MA), itself better than 10 mg/kg (x), which has very little effect, if at all, as compared to vehicle (--¢--). By the end of the treatment, 2.5 mg/kg dexanabinol reduced the tumor growth by 89%, 5 mg/kg by 61% and 10 mg/kg by 21%. These experimental results strongly suggest that the active compound of this invention, which acts through modulation of prro/anti-inflammatory mediators, may be therapeutically effective against certain types of tumors.
Example 15.
Treatment of neurodegenerative disorders: the MPTP model
Parkinson’ s disease (PD) is a neurodegenerative disorder characterized by tremor, slowness of mov-ements, stiffness and poor balance. Most, if not all, of tknese disabilities are due to a profourad reduction in striatal dopamine content caused by loss of dopaminergic neurons in the Substantia Nigra pars compacta (SNpc) and of their proj ecting nerve fibers in the striatum. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP is a well known neurotoxin that can cause depletion of dopamine content in the striatum_ and a reduction in the number of migrostriatal dopaminergic neurons in several species including humans (Turski L. et al, Nature 349: 573, 1991). Neuroinflammatory phenome=na are observed in this model. The zim of the present study is to examine the effect of compounds involved in . 30 gene regulation of pro- and anti-inflammatory mediators on the progeression of MPTP- induced dopaminergic toxicity. The study is carried out in two time win:dows. In the short- term model, the neurological outcome is assessed, namely by measurirag the effect of the test compounds on tae number of immunoreactive cells. In the long-term model, the functional outcome is assessed in the rotarod system following various treatmerts.
Animal treatment and procedure: the short-term model for neurological outcom e. ’ The mice (C577/BL male mice, average weight 30 g, Harlan, Israel) were administered ip. with 4 injections of MPTP (Sigma, USA) (20 mg/kg, 5 ml/l<g) in saline (Teva Medical Israel) at 2 hours interval on day 1. The test compounds and vekaicle 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 mg/kg and PRS-211,220 was tested at 0.5, 1, and 5 mg/kg. Each treatmenf 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 (&TS, Israel) and their brains are removed for tyrosine hydroxylase (TH) detection using immunohistochemistrsy.
TH immunoreactivity at the level of the SNpc
Brains were fixe=d by immersion in 4% formaldehyde for at least 72 hourss. The brains were then washed wit’h 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 ffast-freezing technique (-60°C). Thee brains were cryosectioned (20 um) at the level of the striatum and at the level of the substantia nigra (SN). Immunohistochemistry staining wass carried out using Rabbit anti-tyrossine hydroxylase (1:250, Calbiochem, USA). The slides were stained using the 3,3-diamino benzidine tetrahydrochloride (DAB) chromagen detection kit of the automated immunostaining system (Vantana, France). Quantitative analysis was carried out by counting the rmumber of TH-immunoreactive cells/mm? at the widest area of the
SNpec.
Statistical analysis.
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 consiadered to be } statistically significant.
The results are depicted in Figure 8A, where we can see that MPT P injections © 30 resulted in a reductiom of about 65% in the number of TH-IR cells at the SINpc level as compared to the saline injected animals. Treatment of the animals with the test compounds results in a dose-dependent preservation of the TH-IR cells. Percent pre=servation is
WO (»3/077832 PCT/IL03/00223 calculated by dividing the number of rescued cells (treatmexrat-MPTP) by the maximal possible mumber of rescued cells (saline-MPTP). Best rescue were observed with 20 mg/kg of dexanabinol, which preserved about 25% of the TH-IR cellss, increasing the dose to 30 : mg/kg having no significant effect, and 5 mg/kg of PRS—211,220, which preserved approximately 50% of the TH-IR cells. Vehicle alone had no effect on TH-IR preservation.
These two doses of test compounds were further used to determine the functional outcome.
Animal treatment and procedure: the long-term model for functi onal outcome.
The mice (C57/BL male mice, average weight 30 g, Harlan, Israel) were administered i.p. with MPTP (Sigma, USA) (40 mg/kg, 5 ml/k g) in saline (Teva Medical,
Israel) at 2 weeks interval for four times. The test compounds and controls were injected ip. once just before the first MPTP administration at a —volume dose of 5 mlkg.
Dexanabdinol 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.
Seven days following each MPTP injection, the anirmals were submitted to a functional test using the rotarod apparatus as described by Roz as et al. (Rozas G. et al., J. of Neuroscience Methods 83: 165-75, 1998). The performamce of the animals in the rotarod s ystem, which measures overall locomotor ability, reflects their ability to achieve functions generally affected in PD. The animals were trained for 4 days before beginning the experiment. Their task was to stay on the accelerating rod without falling for 12 minutes (3 minutes at each speed). The tested speeds were: 15, 19, 23 and 27 rpm. Animal performance on the rod was scored as follows: each animal cold obtain a maximum of 3 points (1 for each minute) for full walking on the rod at each speed. Therefore, an animal could get a maximum score of 12 points (3 for each speed). Catching the circling beam of the rod without walking subtracted 0.5 points for every 3 circles circled by the animal. The first 3 circles did not affect the score. Fifty-two days followings initiation of study animals were euthanized by i.p. administration of 100 mg/kg pentobarbitone sodium (CTS, Israel).
The results are shown in Figure 8B, where we can see “that each additional MPTP ] injection generate a further decrease in the animals ability to perform the rotarod test.
Though this trend can be observed as early as in the first sessioen following the first MPTP . 30 injection. the effect of MPTP on animal performance becomes statistically significant only from the third session and on. Indeed in the two first sessions we did not observe a significant effect of any of the treatment group on the rotarod score of the animals (data not shown). Xn the following sessions, we observe that the vehicle mas no effect of its own, that dexanabinol at 20 mg/kg shows a positive trend at the fourth session and that PRS-21 1,220 at 5 mg/kg yields a statistically significant improvement in functional outcome at both later sessions when «compared to vehicle. ’ Another parameter that was measured in this experiment 1S mortality, which was relatively high in this model. Animals that received only saline, arad 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 ©% mortality (0/7 animals).
Altogeth-er the results of this study show that the active compounds of this invention, which act thro~ugh modulation of pro/anti-inflammatory mediators. may be therapeutically effective agaist the neuroinflammatory component of neurodeg-enerative discases. The effect of these compounds is not only major on the mortality rate but above a certain level of TH-IR cells rescue, also significant on the functional abilities of the survivors.
Example 16.
Peripheral no-xious pain: the formalin test.
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). First the test compounds are injected i.p. Then formalin is injected s.c. in the pl antar surface of the hind paw of a mousse 90 min after the test compound. Immediately after formalin administration pain is assess ed (every 5 min for 1 hr) by the number of tim es the animal licks the formalin-injec ted paw.
Example 17.
Neuropathic gpain: attenuation of mechanical allodynia.
The aim of this study is to assess the potential analgesic effescts of our compounds in an animal mo del of neuropathic pain. A peripheral monopathy wvas induced in the right hind limb of rzats following a chronic constriction of the sciatic nexve (Bennet G.J. and Xie . Y-K. Pain 33 : 87-107, 1988). The development of mechanical allodyna was monitored using an estab lished behavioral test (Von Frey filaments).
Pre-surgsery baseline values are ascertained as the mean of” two pre-surgery values.
Once the baseline values had been established, the animals are surgically prepared by con stricting the right sciatic nerve with 4 chromic cat gut loeose ligatures. On day 11 post- operation, the animals that have developed mechanical allod-yna 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 raats, are allowed to acclimatize to tthe behavioral testing equipment before testing. On thes 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 130 minutes a series of Von Frey filaments (pre-calibrat-ed 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 filamemt handle no. 3.61), and the withdrawal threshold for both the ipsilateral and contralateral hind paws is evaluated. Each filarment is indented on the mid-plantar surface of the foot to €he point where it just starts to bend]; this is repeated approximately 8-10 times per filament at a frequency of approximately 1 Hz. The withdrawal threshold is defined as “being the lowest force of two or more consecutive Von Frey’s filaments to elicit a reflex wi thdrawal response (i.e. a brief paw flick) and is measured in grams.
Example 18.
Visceral pain.
The ability of the tricyclic dextrocannabinoid to decrease visceral pain is tested using the acetic acid induced model. ICR male mice (average body weight 30 g, Harlan, Israel) are pre-treated with iv. injesction of either vehicle (Cresmophor:Ethanol 70:30 w/w further diluted 1:20 in®o saline; 5 ml/kg), or test com pounds. Each treatment group comprises at least fiv-e animals. Fifteen minutes later, the mice are injected i.p. with 10 ml/kg of 0.6% a_cetic 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 thes body and extension of the hind. limb. Results are expressed as mean number of wwrithes = 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.
Example 19.
Diabetes type I: the NOD mice rnodel.
The purpose of the present study is to establish a model in non-obese diabeti < (NOD) : mice to test the protective activity of dexanabinol and its analogs in an experimemtal setup relevant to human insulin-dependent diabetes mellitus.
NOD/It female mice (70-80 days old at study onset, Harlan, Israel) are wedghted at day 1. Their baseline glucose level is established using a drop of blood obt=zained by sectioning the tip of the tail anal a glucometer with the appropriate glucosticlkss (Elite,
Bayer). Mice are then injected i.gp. 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 muRtistick (Bayer). When this test indicates that the animals reach glucourea, then the level of glucose in the blood is reassessed during two corasecutive days after overnight starvation. Animals are defined as diabetic if their gluco se blood levels are above 300 mg/dl. Three days following the diagnostic of diabetes, thes animals 1S are sacrificed by i.p. injection of 100 mg/kg pentobarbitone. Their spleen and pan creas are removed for further study including FACS analysis of the T cells subpopulatioms in the spleen and histo- and immuno-pathological evaluation of the pancreas.
The histopathological evahation 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 thes level of mononuclear infiltrate: 0- no infil tration, 1- periductular infiltrate, 2- peri-islet infaltrate, 3- intra-islet infiltrate, 4- intra-islet infiltrate associated with B-cell destruction. T he mean : score for the pancreas of each znimal is calculated by dividing the total score by the number of islets examined.
Example 20.
Renal ischemia.
The purpose of the present study is to test the nephro-protective ac-tivity of dexanabinol and its analogs in an acute renal ischemia model in rats.
Male Sprague Dawley rats (250 gr average body weight, Harlan, Israel) are © 30 anesthetized with a combinatiora of xylazine and pentobarbitone 8 and 35 mg/kg ip. respectively. Then a 45-minutess ischemia is induced bilaterally on both kidn eys. The sedated animals are positioned own their backs. The abdomen skin is shaved and cleaned with 70% ethanol. A miciline skin incision is performed (2-3 cm long) and the abclomen 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 iso-lated by blunt dissection from thes surrounding fat, and occluded together with the renal veims in the ' kidney hilus by arterial micro clips (FST Canada). Kidneys that become pale imnediately after artery occlusion are considered ischemic. Only animals showing that both kidneys are ischemic are included im the study. During the ischemic insult the intestines are returned into the abdominal cavitsy. The wound is covered with wet sponges (they were kept wet by rinsing warm saline).In 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 Par-mer model 8402-00).
Forty-five minutes after the ischemia initiation, the artery clips are removed.
Reperfusion is verified by the return of the pink color of the kidney. The wound is then closed with 3-0 silk suture material (Assut, Switzerland) in two layers (abdomen ~wall and skin). At 1, 3 and 7 days post ischemic insult animals are lightly anesthetizesd in an anesthesia chamber with. ether and blood samples are collected after an infra orbi tal sinus puncture. Blood is collected into eppendorf tubes, and centrifuged (4000 rpmm for 5 minutes). Serum is then separated and kept at -20°C before evaluation of blood Jevels of creatinine and blood urea nitrogen (BUN). At the end of the study, animals are euthanized with pentobarbitone sodi um 100 mg/kg i.p. Kidneys are removed, weighted and ke=pt in 4% formaldehyde solution for possible further usage.
Treatments are adrministered 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 Aschemic (vehicle treated) and shamm (the same procedure, without renal artery occlusion).
Statistical analysis.
The blood levels of BUN and creatinine are compared using ANOVA followed by
Duncan’s post-hoc test.
Example 21.
Inflammatory bowel disease: the acetic acid-induced model.
The purpose of this study is to evalua te the activity of test compounds in a masked . study of acetic acid-induced inflammatory bowel disease in rats.
Male Sprague Dawley rats (10 week s old, 200-250 gr, Harlan, Israel) are lightly anaesthetized by ip. injection of a ketamine:rrompun combination (100:10 mg/lxg respectively). A polyethylene catheter (outer diameter 1.7 mm) is inserted through the rectum 5 cm into the colon. And 2 ml of 5%% acetic acid are then slowly administered into the colon. Fifteen seconds later the colon is washed with 3 ml saline and 15 seconds later with additional 3 ml of saline. Inmediately~ after, each group of animals are treated with either one of the appropriate treatments. Al] treatments are administered once daily for 7 days. Animals are clinically followed for 1 week. During this period, the followirg parameters are daily monitored and recorded: body weight, presence of blood in the sto-ol and stool consistency. These findings are scored according to table 1 (Murthy SN. et al
Dig. Dis. Sci. 38: 1722-34, 1993).
Table 1: Criteria for Scoring Disease Activity Index (DAT¥) of IBD.
Score | Weight Loss Stool Consistemcy * | Occult Blood or
ET
CC LC RL
EE LR OR
EI CL CN LS
# 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 oo the anus; and diarrhea - liquid stools that sticks to the anus.
Seven days post disease induction animals are sacrificed with pentobarbital 100 mg/kg ip. The whole colon is excised, slit longitudinally and examined under a magnifying glass, and any visible damage is recorded and scored according to table 2 (Wong etal, J. Pharm. Exp. Ther. 274: 475-80, 1995).
Table 2: Gross Pathology Scoring Method for Evaluating the Severity of IBD.
ER ee
More then 1 site of erosior/or ulcer, or 1 erosion site or ulcer
CEST
Statistical analysis.
The clinical outcome is analyzed using analysis of variance (ANOVA) followed by
Duncan’s post-hoc test. A non-parannetric test (Wilcoxon Rank Sum Test) is used for evaluating the gross pathology findirgs.
Example 22.
Experimental autoimmune diseases: CIA, EAE and DTH.
Autoimmune diseases are assoeciated with elevated levels of inflammatory cytokines.
The rodent models most commonly studied are experimental allergic encephalomyelitis (EAE), a model for multiple sclerosis in the human, experimental autoimmune arthritis, a model for rheumatoid arthritis in thee human and delayed type hypersensitivity (DTH), a model for allergic reactions in the human. EAE is an autoimmune neurological disease elicited by sensitization of the aninmals to myelin basic protein from the central nervous system, which is also known as bassic 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 dimitrofluorobenzene 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.
Collagen Induced Arthritis.
Adult DBA/1 male mice (20 g average body weight, Harlan, Israel), at least eight per treatment group are used in this studly. Bovine collagen type 2 is dissolved in 0.05 M acetic acid at a concentration of 2 mg/ml by stirring ON at 4°C. The collagen solution is further emulsified in an equal volume of Complete Freund’s Adjuvant (CFA). Each animal is admministered with 100 pg collagen type 2 in 0.1 ml CFA emulsion. The collagen is administered s.c. at the base of the tail. Twenty-one diay after priming, the mice receive an : intradermal booster injection of 100 pg collagen in Inecomplete Freund’s adjuvant.
The volume of each hind paw is measured using a plethysmometer (Hugo Basill,
Italy), and the thickness using a dial, constant pressure gauge, (Mitutoyo, Japan).
Measurements are performed before collagen adrministration and every second day throughout the designated follow-up period. All treatments are administered intraperitoneally. At the end of the treatment permiod the animals are sacrificed with pemtobarbital 100 mg/kg i.p.
Statistical analysis.
The differences between the severity of the paw swelling among various treatment groups are compared using analysis of variance AN®VA followed by post-hoc t-Test. A value of p<0.05 is considered to be statistically signifacant.
Experimental Autoimmune Encephalomyelitis.
Various animal models of autoimmune encepohalomyelitis are known in the art, degpending on the method of induction, the strain of tlme animal and the antigen employed to incluce 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 appearan_ce 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 an3mals (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 enulsified 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 chromatographmy. Each animal receives 50 pg of the . puxified 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 mmycobacterium tuberculosis, and each © 30 an-imal receives 100 pl (50 pl 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 consescutive 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.
Statistical analysis.
The differences between the severity of the clinical outcomes among various treatment groups was analyzed by analysis of variance (ANOVA) followed by post-hoc
Fisher test. A value of p<0.05 is considered to be statistically significant.
Delayed Type Hypersensitivity in mice model.
Adult female BALB/c mice (20 g average body weight, Harlan, Israel) were sensitized on day 0 and day 1 by application of 30 pl of 0.15% Dinitrofluorobenzene (DNFB) diluted in acetone on the shaved skin of the abdomen. On day 6 the animals were challenged by application of 10 pl of DNFB in acetone on one ear. The contralateral ear was not challenged but received the applic ation of 10 pl acetone. Test compounds were administered at increasing doses from 0 to 15 mg/kg ip. 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 sec ond 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 treatrment group to the response generated by the appropriate vehicle only.
Although the present invention has b een described with respect to various specific i embodiments presented thereof for the sake of illustration only, such specifically disclosed . embodiments should not be considered limiting. Many other such embodiments will occur to those skilled in the art based upon applic-ants’ disclosure herein, and applicants propose to be bound only by the spirit and scope of their invention as defined in the appended claims.

Claims (20)

1. A pharmaceutical composition for decreasing the transcription of at least one of the pro-inflammatory mediators COX-2, IL-1, IL-2, iNOS, TNF-a and MCP-1, the composition comprising as an active ingredient a compound of the general formula (I): Formulal R4 1 6 2 R> “uy o Rs having the (3S,4S) configuration and being essentially free of the (3R,4R) enantiomer, wherein the dashed line indicates an optional C1 -C2 or C6-C1 double bond, and wherein: R; is selected from the group consisting of a) R’ where R’ is selected from the group consisting of A) a linear or branched, saturated or unsaturated, carbon side chain comprising 1-8 carbon atoms optionally interrupted by 1-3 heteroatoms, and B) a saturated or unsaturated cyclic moiety, an aromatic moiety or a heterocyclic moiety; the cyclic moiety ha ving from 5-20 atoms comprising one or two-ringed structures, wherein each rirag comprises 3-8 carbons, optionally interrupted by 1-4 heteroatoms, and optionally further substituted with one or more groups selected from i) alinear, branched or cyclic, saturated or unsaturated C;-C, alkyl, ii) a linear, branched or cyclic, sa turated or unsaturated C,-Cg alkoxy, ni) a linear, branched or cyclic, saturated or unsaturated C,-Cq alkylthio, iv) ahalogen, v) carboxyl, vi) —CO,-C,-C4 alkyl, wherein thes alkyl can be linear, branched or cyclic, saturated or unsaturated, vii) keto, viii) nitro, 66 Amended sheet 21/09/2005
1X) a saturated or unsaturated cyclic moiety, an aromatic or a heterocyclic moiety; the cyclic moiety having from 5-20 atoms comprising one or two-ringed structures, wherein each ring comprises 3-8 carbons, optionally interrupted by 1-4 heteroatoms, and optionally further substituted with one or more groups selected from i )-viii) as defined above, b) an amine or an armide substituted with at least one substituent as defined in R’ above, c) athiol, a sulfide, a sulfoxide, a sulfone, a thioester or a thioamide optionally substituted with oene substituent as defined in R’ above, and d) ahydroxyl or an ether —OR’ wherein R’ is as defined above; R; is selected from the group consisting of a) a halogen, b) a linear, branched or cyclic, saturated or unsaturated C,-C¢ alkyl, and c¢) -OR wherein R is selected from the group consisting of A) -R", wherein R" is hydrogen or a linear, branched or cyclic, saturated or unsaturated C,-Ce¢. 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¢ alkyl, and B) -C(O)R"™ wherein R™ is as previously defined; and Rj is selected from the group consisting of a) a linear, branched or cyclic, saturated or unsaturated C,-C,, alkyl, b) -OR? in which R? is a linear, branched or cyclic, saturated or unsaturated C,-Cy alkyl which may bee substituted at the terminal carbon atom by a phenyl group, and c) a linear, branched or cyclic, saturated or unsaturated C,-C; alkyl-OR" wherein R"™ is as previously de fined; and pharmaceutically acceptable salts, esters or solvates thereof.
2. A pharmaceutical «<omposition for increasing the transcription of at least one of the anti-inflammatory cytokirae IL-10, the protective cytokine IL-6 and of the suppressors of cytokine signaling SOCS-1 and SOCS-3, the composition comprising as an active ingredient a compound of the general formula (I): 67 Amended sheet 21/09/2005
Formula I R4 1 6 2 R, “uy (0) R; having the (3S,4S) configuration and being es sentially free of the (3R,4R) enantiomer, wherein the dashed line indicates an optional C1—-C2 or C6-C1 double bond, and wherein: Ris selected from the group consisting of a) R’ where R’ is selected from the group consisting of A) a linear or branched, saturated or unsaturated, carbon side chain comprising 1-8 carbon atoms optionally interrupted by 1-3 heteroatoms, and B) a saturated or unsaturated cyclic moiety, an aromatic moiety or a heterocyclic moiety; the cyclic moiety ha-ving from 5-20 atoms comprising one or two-ringed structures, wherein each rirmg comprises 3-8 carbons, optionally interrupted by 1-4 heteroatoms, and optiomally further substituted with one or more groups selected from i) a linear, branched or cyclic, saturated or unsaturated C;-Cs alkyl, i1) a linear, branched or cyclic, saturated or unsaturated C,-C¢ alkoxy, iii) a linear, branched or cyclic, saturated or unsaturated C;-Cs alkylthio, 1v) a halogen, v) carboxyl, vi) -C0,-C,-C4 alkyl, wherein thes alkyl can be linear, branched or cyclic, saturated or unsaturated, vii) keto, vill) nitro, ix) a saturated or unsaturated cyclic moiety, an aromatic or a heterocyclic moiety; the cyclic moiety having from 5-20 atoms comprising one or two-ringed sstructures, wherein each ring comprises 3-8 carbons, optionally interrupted by 1-4 heteroatoms, 68 Amended sheet 21/09/2005 and optionally further substituteed with one or more groups selected from i)-viii) as defined above, b) an amine or an amide substituted with at le=ast one substituent as defined in R’ above, c) a thiol, a sulfide, a sulfoxide, a sulfone, a thioester or a thioamide optionally substituted with one substituent as defined in R’ above, and d) a hydroxyl or an ether —OR’ wherein R’ is as defined above; R; is selected from the group consisting of a) a halogen, b) a linear, branched or cyclic, saturated or un saturated C,-C¢ alkyl, and c) -OR wherein R is selected from the group consisting of A) -R", wherein R" is hydrogen or a linear, branched or cyclic, saturated or unsaturated C,-Cs alkyl optionally containimng a terminal -OR™ or -OC(O)R"™ moiety wherein R"™ is hydrogen or a linear, branched or cyclic, saturated or unsaturated C,-Cg alkyl, and B) -C(O)R"™ wherein R" is as previously defined; and Rj is selected from the group consisting of a) a linear, branched or cyclic, saturated or unssaturated C;-C;; alkyl, b) -OR? in which R"is a linear, branched or csyclic, saturated or unsaturated C,-Co alkyl which may be substituted at the termiral carbon atom by a phenyl group, and ¢) a linear, branched or cyclic, saturated or unsaturated C,-C; alkyl-OR" wherein R" is as previously defined; and pharmaceutically acceptable salts, esters or sol~vates thereof.
3. The composition according to any of claimss 1 or 2 wherein R, is OH, Ryis OH R; is 1,1-dimethylheptyl and there is a double bond between C6 and C1.
4. The composition according to any of claims 1 or 2 wherein Ry is 2- mercaptoimidazole, Ry is OH, Rs is 1,1-dimnethylheptyl and there is a double bond between C6 and C1.
5. The composition according to any of claims 1 or 2 wherein R; is imidazole, R; is OH, R; is 1,1-dimethylheptyl and there is a edouble bond between C6 and Cl. 69 Amended sheet 21/09/2005
6. Thae composition according to any of claims 1 o r 2 wherein R, is pyrazole, R; 1s OH Rj; is 1,1-dimethylheptyl and there is a double bond between C6 and C1.
7. Thee composition according to any of claims 1 or 2 wherein Ry is 4-methyl piperidine, R; is OH, Rj; is 1,1-dimethylheptyl ard there is a double bond between C6s and C1.
8. Th.e composition according to any of claims 1 or 2 wherein R; is 4-piperidino- piperidine, R; is OH, Rj is 1,1-dimethylheptyl an d there is a double bond between C6s and C1.
9. Th e composition according to any one of claims 1 to 8 further comprising a pharmaceutically acceptable diluent or carrier.
10. Th. e composition according to claim 9 wherein the diluent comprises an aqueous cossolvent solution comprising a pharmaceutically, acceptable cosolvent, a micellar solution or emulsion prepared with natural Or synthetic ionic or non-ionic sur-factants, or a combination of such cosolvent an d micellar or emulsion solutions.
11. Th e composition according to claim 9 wherein thhe carrier comprises a solution of ethwanol, a surfactant and water.
12. Th< composition according to claim 9 wherein the carrier is an emulsion cormprising triglycerides, lecithin, glycerol, an emwlsifier, and water.
13. Thee composition according to any one of claims 1 to 8 which is in unit dosage foram.
14. Thee composition according to claim 13 which is smitable for oral administration.
15. The composition according to claim 13 which is suitable for parenteral adrministration.
16. A pharmaceutical composition according to any ome of claims 1 to 8 for preventing, alleviating or treating a disease or disorder by regulating pro and anti-inflammatory mediators selected from COX-2, IL-1f, IL-2, iNOS, TNF-a, MCP-1, IL-10, IL-6, SO CS-1 and SOCS-3. 70 Amended sheet 21/09/2005
WO (3/077832 PCT/IL.03/00223
17. The composition of claim 16 wherein the diseases characterized by abnormal production of any of COX-2, IL-1, IL-2, iNOS, TN_F-a, MCP-1, IL-10, IL-6, SOCS-1 and SOCS-3 are selected from the group cormprising inflammatory and immune disorders, pain, allergic inflammation, diseases characterized by monocyte infiltration, sarcoidosis, Wegener’s granulomatosis, tuberculosis, atherosclerosis, rheumatoid arthritis, vasculitis, interstitial lung disorder s, inflammatory pulmonary diseases, asthma, inflammatory bowel diseases, pancmreatitis, inflammatory skin diseases, osseous inflammation, tumor growth or metas-tasis, neurological diseases involving immune-mediated or post-traumatic infflammation, inflammatory demyelinating neuropathies, multiple sclerosis, neurodegenerative disorders, Alzheimer’s disease, Parkinson’s disease, bacterial, parasitic or viral infections, sepsis, renal disorders, diabetic nephropathy, liver disorders, postoperative complications in cardiovasvular surgery, in transplants or organs or tissue replacements and in prosthetic implants, transplant rejection.
18. The composition of claim 16 which is suitable to be administered orally, parenterally, intravenously, intramuscularly, intralessionally, subcutaneously, transdermally, intrathecally, rectally and intranasally.
19. Use for the preparation of a medicament for preventimg, alleviating or treating a disease or disorder by regulating pro and anti-inflam-matory mediators selected from COX-2, IL-1P, IL-2, iNOS, TNF-a, MCP-1, LL-10, IL-6, SOCS-1 and SOCS-3, of a pharmaceutical composition according to any one of claims 1 to 8.
20. The use of claim 19 wherein the diseases characterized by abnormal production of any of COX-2, IL-1B, IL-2, iNOS, TNF-a, MCP-1, IL-10, IL-6, SOCS-1 and SOCS-3 are selected from the group comprising irmflammatory and immune disorders, pain, allergic inflammation, diseases characterized by monocyte infiltration, sarcoidosis, Wegener’s granulomatosis, tulberculosis, atherosclerosis, rheumatoid arthritis, vasculitis, interstitial lung disorderss, inflammatory pulmonary diseases, asthma, inflammatory bowel diseases, pancreatitis, inflammatory skin diseases, osseous inflammation, tumor growth or metastasis, neurological diseases involving immune-mediated or post-traumatic inflammation, inflammatory demyelinating neuropathies, multiple sclerosis, newrodegenerative disorders, Alzheimer’s disease, Parkinson’s disease, bacterial, parasitic or viral infections, 71 Amended sheet 21/09/2005
WO €3/077882 PCT/IL03/00223 sepsis, renal disorders, diabetic nephropathy, liver disorders, postoperative complications in cardiovascular surgery, in transpslants or organs or tissue replacerments and in prosthetic implants, transplant rejection.
72 Amended sheet 21/09/2005
ZA200407182A 2002-03-18 2004-09-08 Dexanabinol and dexanabinol analogs regulate inflammation releated genes. ZA200407182B (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
IL14873602A IL148736A0 (en) 2002-03-18 2002-03-18 Dexanabinol and dexanabinol analogs which regulate inflammation related genes

Publications (1)

Publication Number Publication Date
ZA200407182B true ZA200407182B (en) 2006-02-22

Family

ID=27840273

Family Applications (1)

Application Number Title Priority Date Filing Date
ZA200407182A ZA200407182B (en) 2002-03-18 2004-09-08 Dexanabinol and dexanabinol analogs regulate inflammation releated genes.

Country Status (7)

Country Link
EP (1) EP1485083A4 (en)
JP (1) JP2005526768A (en)
AU (1) AU2003214608A1 (en)
CA (1) CA2479676A1 (en)
IL (1) IL148736A0 (en)
WO (1) WO2003077832A2 (en)
ZA (1) ZA200407182B (en)

Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0326393D0 (en) * 2003-11-12 2003-12-17 Ares Trading Sa Cytokine antagonist molecules
EP1774328A1 (en) * 2004-07-23 2007-04-18 Novartis AG Biomarkers for rheumatoid arthritis (ra)
US20080262079A1 (en) * 2004-08-09 2008-10-23 Bernard Mach Cannabinoid Compositions and Methods of Use Thereof
JP2008533107A (en) * 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 Use of 5'-methylthioadenosine (MTA) in the prevention and / or treatment of autoimmune diseases and / or transplant rejection
EP1752149A1 (en) * 2005-07-29 2007-02-14 Laboratorios Del Dr. Esteve, S.A. CB1 Antagonists or inverse agonists as therapeutical agents for the treatment of inflammation involving gene expression
JP2009514890A (en) * 2005-11-07 2009-04-09 マーティ・ファーマシューティカルズ・インク Improved delivery of tetrahydrocannabinol
CN102851295B (en) * 2006-08-28 2015-01-07 长春华普生物技术有限公司 Toll-like receptor regulatory oligonucleotide and application thereof
GB0713116D0 (en) * 2007-07-06 2007-08-15 Therapeutics Ltd E Treatment of melanoma
GB0719771D0 (en) * 2007-10-10 2007-11-21 Therapeutics Ltd E Dexanabinol in combination with inhibitors of BRAF or MEK for the treatment of melanoma
GB0915877D0 (en) * 2009-09-10 2009-10-14 E Therapeutics Plc Cancer cell apoptosis
GB201207305D0 (en) * 2012-04-26 2012-06-13 E Therapeutics Plc Therapy
JP6226278B2 (en) 2012-08-03 2017-11-08 国立大学法人愛媛大学 Immune cell activation inhibitor and use thereof
JP2017031120A (en) * 2015-08-05 2017-02-09 オリザ油化株式会社 TNF-α AND IL-6 PRODUCTION INHIBITORS, AND MUSCLE INFLAMMATORY INHIBITORS USING THE SAME
WO2017068349A1 (en) 2015-10-23 2017-04-27 E-Therapeutics Plc Cannabinoid for use in immunotherapy
JP6470879B1 (en) * 2017-03-08 2019-02-13 日清オイリオグループ株式会社 Anti-inflammatory agent, anti-inflammatory pharmaceutical composition, anti-inflammatory food composition

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL115245A (en) * 1995-09-11 2002-12-01 Yissum Res Dev Co Tumor necrosis factor inhibiting pharmaceuticals
JP2004501145A (en) * 2000-06-22 2004-01-15 ファーモス　コーポレイション Novel cannabinoids without psychotropic effects

Also Published As

Publication number Publication date
AU2003214608A1 (en) 2003-09-29
JP2005526768A (en) 2005-09-08
IL148736A0 (en) 2002-09-12
WO2003077832A2 (en) 2003-09-25
CA2479676A1 (en) 2003-09-25
EP1485083A2 (en) 2004-12-15
EP1485083A4 (en) 2008-11-19
WO2003077832A3 (en) 2003-12-31

Similar Documents

Publication Publication Date Title
EP1604665B1 (en) C-kit kinase inhibitor
ZA200407182B (en) Dexanabinol and dexanabinol analogs regulate inflammation releated genes.
JP2022504601A (en) MST1 kinase inhibitors and their use
JP2021183640A (en) Prevention, treatment and reversal of disease using therapeutically effective amounts of activated fatty acids
AU2012224499B2 (en) New pyridazinone and pyridone compounds
EP1014980A1 (en) Use of nitroxides for the treatment of essential hypertension
JP2010265322A (en) Pyrazole derivative modulator of hepatocyte growth factor (scatter factor) activity
JP2007277273A (en) Medicinal preparation including halogenated aromatic for inhibiting unwanted proliferation of mammalian cell
US20050137251A1 (en) Dexanabinol and dexanabinol analogs regulate inflammation related genes
JPH06234635A (en) Use of leflunomide for inhibiting interleukin-1 beta
EP3458448A1 (en) Methods of using fasn inhibitors
JP5081890B2 (en) Combination drugs of antithrombotic drugs and pyrazolone derivatives
JPH06234637A (en) Use of leflunomide for inhibiting tumor necrosis factor alpha
US6756394B1 (en) Small molecule inhibitors of necrosis
CN113181160A (en) Application of isothiocyanate compounds
JPWO2003024446A1 (en) Oxidative stress inhibitor and method for measuring oxidative stress
MXPA06004434A (en) Novel medical uses of compounds showing cb1.
CN111356764B (en) Compositions and methods for reducing serum cholesterol and PCSK9
US20130338203A1 (en) TREATMENT OF ARTERIAL AGEING BY COMBINATION OF RAAS INHIBITOR AND HMG-CoA REDUCTASE INHIBITOR
JP7290223B2 (en) IL-1β inhibitor
US20160279079A1 (en) Compositions and methods for treating bone diseases
WO1999004777A1 (en) Carcinostatics
KR20210094753A (en) Novel compound and uses of the same
KR101453607B1 (en) Composition comprising 4―Dimethylamino―2―methoxy―6―（（methyl―［2―（4―nitrophenyl）ethyl］amino）methyl）phenol for anti―inflammation
WO2004092163A1 (en) Phenylazole compound, production process, and antioxidant drug