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Definitions

• the present invention relates to the use of lipophilic diesters of the chelating agent l,2-bis(2 aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) for inhibition of proteolytic activities of certain metalloproteinases and of calpain.
• BAPTA l,2-bis(2 aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
• MMPs Matrix metalloproteinases
• ECM extracellular matrix
• the most studied MMPs are the gelatinases, which include MMP-2 and MMP-9 that use gelatin, Type IV collagen and fibronectin as preferred substrates. While transcription of MMP-9 genes is transactivated by cytokines and growth factors, MMP-2 is constitutively expressed and unresponsive to phorbol ester and most cytokines. MMP-2 activation is regulated by MT1-MMP, which is a membrane-anchored MMP.
• MMP-9 activation is regulated by a protease cascade involving plasmin and stromelysin-1 (MMP-3).
• Matrix metalloproteinases are responsible for much of the turnover of matrix components and as such are involved in no ⁇ nal as well as in pathological processes.
• MMPs have an important role in maintenance and remodeling of membranes and ECM, for example, in breaking down the extracellular matrix to allow cell growth and tissue remodeling during development and recovery from injury. They also play a role in processes such as ovulation, modulation of capillary permeability and in enabling cell migration to a site of inflammation.
• MMPs are involved in many pathological conditions. For example, they are associated with pathogenic mechanisms in cancer such as invasion, metastasis and angiogenesis [Reviewed by Foda and Zucker (2001) Drug Discovery Today 6: 478-482]. MMPs play a part in progression of iiiflammatoiy conditions and diseases involving degradation of extracellular matrix, such as in stroke, hemorrhage, rheumatic diseases (e.g. arthritis), Crohn's disease, asthma, and in cerebrovascular and cardiovascular disorders [Mun-Bryce and Rosenberg (1998) J. Cerebral Blood Flow Metabolism 18:1163-72; Yong et al. (1998) TINS 21:75- 80; Lukes et al. (1999) Mol. Neurobiol.
• MMPs have been associated with brain damage and ischemia, Guillain-Barre, multiple sclerosis, amyotrophic lateral sclerosis and Alzheimer's disease.
• the current notion is that inflammation leads to the production of cytokines, chemokines, growth factors and hormones that modulate MMPs production.
• Activation of MMPs and plasminogen activators (PAs) is an important regulatory step in the inflammatory response.
• ADAM Disintegrin And Metalloproteases
• TACE is found on the cell surface where it processes the membrane-bound TNF ⁇ , a pro-inflammatory cytokine, to its mature soluble form. Soluble TNF ⁇ , which is released in inflammatory conditions, can induce apoptosis. For example, TNF induces secretion and activation of MMP-9 in macrophages and glial cells and causes neuronal cell death in neuroinflammatory diseases and following brain injuries. TNF has also been shown to play a role in pathological conditions such as rheumatoid arthritis.
• MMPs are tightly regulated at multiple stages, as follows: i) Gene transcription- most MMPs are not constitutively expressed, but their transcription is controlled by various cytokines (e.g. IL-1, TNF) and growth factors (e.g. TGF- ⁇ , retinoic acid, FGF). ii) Pro-enzyme activation- MMPs are normally produced in a latent form (pro-MMP) including a propeptide segment that generally must be removed to activate the enzyme. iii) Inhibition of enzyme activity- There are at least four endogenous MMP- inhibitors known as tissue inhibitors of metalloproteinases (TIMPs), which bind to the enzyme and block its activity. Another known natural inhibitor of MMPs is the serum proteinase inhibitor ⁇ -macroglobulin.
• inhibitors of MMPs have been described in various publications in the scientific and patent literature.
• known inhibitors mainly include synthetic peptides and chelating agents [reviewed by Woessner JF Jr. in AnnN.Y. Acad. Sci. (1999) 30:388-403].
• Some synthetic inhibitors of the MMP active site are peptidomimetics based on the sequence of peptides cleaved in collagen [Masui et al. (1977) Biochem Med. 17:215-21). Peptidic agents based on conserved peptide sequence derived from the pro-segment of human collagenase IV are disclosed by Stelter- Stevenson et al. [Am J Med Sci. (1991) 302:163-70] and in U.S. Patent No. 5,270,447 to Liotta et al. Synthetic peptides isolated from phage display peptide libraries and cyclic peptides with MMP inhibitory activity are described by Koivunen et al. [Nat.
• polypeptides and peptoid compounds useful as metalloproteinase inhibitors are disclosed in U.S. Patent Nos. 4,263,293 and 4,297,275 to Sundeen et al., in U.S. Patents Nos. 4,371,465, 4,371,466 and 4,374,765 all issued to McGregor, and in U.S. Patent Publication No. 2002/0090654 to Langley et al.
• Non-peptidic MMP-inhibitory compounds are disclosed in U.S. Patent No. 4,950,755 to Witiak et al. and in U.S. Patent No. 5,866,570 to Liang et al.
• MMPs inhibitors comprising targeting moieties and chelators are disclosed in International Patent Publication No. WO 01/60820 of Dupont Pharmaceuticals Company, and in International Patent Publication No. WO 02/053173 to Kimberly-Clark Worldwide, Inc.
• Matrix metalloproteinases as well as other members of the ADAM family are inhibited by chelating agents.
• Most of these chelating agents are natural and synthetic hydroxamate compounds and derivatives thereof, such as succinyl hydroxamate, sulfonamide hydroxamate etc. [reviewed by Woessner, J.F. Jr. (1999) in Annals New York Academy of Sciences 30: 388-403].
• the synthetic hydroxamates batimastat (BB-94; Invest New Drags (1996) 14: 193-202) and its orally bioavailable analogue marimastat have been shown to inhibit spread and growth of malignant tumors in animals. These compounds are currently examined in advanced clinical trials.
• MMPs inhibitors include antibiotics such as tetracyclines and their chemically modified analogs (Golub et al. (1983) J Periodontal Res. 18:516-26; U.S. Patent No. 4,704,383 to McNamara et al.; U.S. Patent No. 5,837,696 to Golub et al.].
• Most of the above-mentioned agents are non-specific inhibitors of metalloproteinases and other metal-ion dependent proteases.
• Calpains are members of another family of proteases. These are cytosolic enzymes which are calcium-dependent cysteine proteases. Calpains predominantly exist within cells as inactive proenzymes and are converted into their active forms in the presence of elevated intracellular calcium levels. Upon binding of calcium, the precursor enzyme goes through a self-digestion process that results in release of the activated calpain.
• Calpain serves as substrates for calpain including cytoskeletal, membrane and regulatory proteins. Calpain participates in a number of normal cellular signal transduction systems as well as in pathological conditions. For example, calpain activation has been associated with ischemia and neuronal cell death such as those caused by stroke and traumatic brain and spinal cord injuries [Bartus et al. (1995) Neural. Research 17:249-258]. Calpain proteolytic activity has also been implicated in several neurodegeneration diseases and conditions, including Alzheimer's Disease, Parkinson's disease, Huntington's disease and Pick's disease.
• calpain inhibitors include polypeptides which mimic peptide sequences of the natural inhibitors calpastatin and kininogen [for review, see Wang and Yuen (1994) Trends Pharm. Sci. (1994) 15, 412-419].
• calpam-inhibitors are compounds based on peptide structures that interact with the substrate-binding site of the enzyme. Many of these compounds are non-specific and inhibit a wide variety of proteases in addition to calpain. Moreover, most of the known inhibitors that were active in vitro, were found ineffective in inhibiting calpain in-vivo, in particular in the CNS, as being poor membrane permeants. Furthermore, almost all MMPs-inhibitors tested for treating pathological inflammatory conditions or cancers failed in in- vivo clinical studies.
• Stable hpophilic diesters of the divalent metal ion chelator l,2-bis(2 arninophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) have been disclosed in the International Patent Publication No. WO 99/16741 of the same applicant. Also disclosed in this publication is the use of these compounds in pharmaceutical compositions useful for treating diseases and disorders related to excess of divalent metal ions. Among these diseases and disorders are ischemia, stroke, epilepsy and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.
• DP-BAPTAs diesters of the chelating agent l,2-bis(2 arr_ ophenoxy)ethane-N,N,N',N'- tetraacetic acid
• MMP matrix metalloproteinase
• TACE TNF ⁇ -Converting Enzyme
• the present invention provides, in one aspect, a method of inWbiting protease activity, said protease being selected from metalloproteinase and calpain, the method comprising exposing cells to inhibiting amount of a compound of the general formula (I):
• R is saturated or unsaturated alkyl, cycloalkyl, arylalkyl or cycloalkyl-alkyl radical having from 1 to 28 carbon atoms which may be interrupted by any combination of 1-6 oxygen and or nitrogen atoms, provided that no two oxygen atoms or an oxygen and a nitrogen atom are directly connected to each other; and M denotes a hydrogen or a physiologically acceptable cation.
• certain compounds disclosed herein are novel and in themselves constitute an aspect of the present invention. These compounds include the compound of the general formula (I) wherein R is 2-benzyloxyethyl.
• R is 2-benzyloxyethyl.
• a compound of the general formula (I) which is l,2-bis(2-aminophenoxy)ethane, N,N'-di(2- benzyloxyethyl acetate), N,N'-acetic acid and salts thereof.
• pharmaceutical compositions comprising a therapeutically effective amount of said compound and a pharmaceutically acceptable carrier or excipient.
• the useful compounds for inhibiting the MMP-9 activity are the following compounds and physiologically acceptable salts thereof: l,2-bis(2-aminophenoxy)ethane, N,N'-di(2-octoxyethyl acetate), N,N'- diacetic acid (denoted herein DP- b99), l,2-bis(2-aminophenoxy)ethane, N,N'-di(2-octodecyloxyethyl acetate), N,N'-diacetic acid (denoted herein DP-b 109), l,2-bis(2-aminophenoxy)ethane, N,N'-di(2-benzyloxyethyl acetate), N,N'- acetic acid (denoted herein DP-b440), l,2-bis(2-aminophenoxy)ethane, N,N'-di(2-dodecyloxyethyl
• a pharmaceutical composition containing as an active ingredient a therapeutically effective amount of a compound of the above-mentioned general formula (I).
• a compound of the general formula (I) for the preparation of a medicament for inhibiting the activity of a protease selected from metalloproteinase, calpain and TACE.
• the present invention further provides methods and the use of the compounds of the general formula (I) for the treatment of MMP- or calpain-related diseases, disorders or conditions, which may be selected from the group consisting of cancer (including metastasis cancer), angiogenesis-dependent diseases (e.g. cancerous tumors, arthritis, psoriasis, macular degeneration, chronic inflammation and diabetic retinopathy), ischemic or hypoxic tissue damage, oxidative injury, stroke, trauma, inflammatory conditions and diseases (e.g.
• cancer including metastasis cancer
• angiogenesis-dependent diseases e.g. cancerous tumors, arthritis, psoriasis, macular degeneration, chronic inflammation and diabetic retinopathy
• ischemic or hypoxic tissue damage oxidative injury, stroke, trauma, inflammatory conditions and diseases (e.g.
• arthritides rheumatoid arthritis, osteoarthritis, restenosis, asthma, psoriasis, systemic lupus erythematosus, inflammatory bowel syndrome, Crohn's disease, gingivitis, periodontitis, meningitis, tropical spastic paraparesis, sepsis, bullous skin disorders, acne and inflammation due to infectious diseases), atherosclerosis, thrombotic disorders, arthritis, osteoporosis, diabetes, hemorrhage, autoimmune diseases, rheumatic diseases, ocular pathologies and retmopathies (e.g.
• diabetic retinopathy glaucoma, macular degeneration, cataract, retinal detachment and retinal tears), burns, chronic wounds (e.g. ulcers), neurological and neurodegenerative diseases and disorders (e.g. multiple sclerosis (MS), Alzheimer's disease (AD), motor neuron disease (MND), amyotrophic lateral sclerosis (ALS), Guillain-Barre, Parkinson's disease, Huntington disease, Pick's disease, dementia syndrome, vascular dementia, multiple infarct dementia, HIV-induced neural disorders, brain ischemia (both global and focal ischemia) and neuronal tissue trauma), migraine, cerebrovascular and cardiovascular disorders.
• MS multiple sclerosis
• AD Alzheimer's disease
• MND motor neuron disease
• ALS amyotrophic lateral sclerosis
• ALS amyotrophic lateral sclerosis
• ALS amyotrophic lateral sclerosis
• ALS amyotrophic lateral sclerosis
• dementia syndrome dementia
• vascular dementia multiple infar
• the methods of treatment in accordance with the invention may further comprise treating the patient with additional therapeutic treatment which may be carried out concurrently with, preceding or subsequent to the administration of the pharmaceutical composition comprising a compound of the general Formula (I).
• Figs. 1A-B depict total amount of MMP-9 enzyme (A) and the MMP-9 enzymatic activity (B) in the conditioned media collected from cultured A- 172- glioma cells treated with 10 ng/ml TNF ⁇ , in the absence or presence of 20 ⁇ M or 50 ⁇ M DP-bl09, as indicated. .
• Figs. 2A-B depict gelatin-zymogram gels used for detection of MMP-9 activity in conditioned media collected from cultured C6-glioma cells that were treated with 20 ng/ml TNF ⁇ (A) or 0.1 ⁇ M PMA (B), either in the absence or presence of different concentrations of DP-M09 as indicated. Areas of MMP-9 and MMP-2 protease activity, showed as clear bands, are marked with arrows.
• Figs. 3A-C depict a representative gelatin-zymogram gel (3 A) and analysis of biologically active MMP-9 (3B) and pro-MMP-9 (3C) forms of the enzyme.
• Fig. 4 depicts TNF ⁇ release from primary glial cells treated with 0.5 ⁇ g/ml LPS in the absence or presence of different DP-BAPTA compounds as indicated.
• Figs. 5A-B depict a gelatin-zymogram gel (A) and MMP-9 band analysis
• Fig. 6 depicts a Western blot of cortical primary cells lysates reacted with anti-spectrin antibodies as described in Example 11.
• the cells were pre-tieated with either DP-b99 (15 ⁇ g/ml) or the commercial calpain inhibitor MDL28170 (25 ⁇ M) for one hour prior to induction of oxidative stress (H 2 0 2 , 50 ⁇ M) as indicated.
• the non-cleaved (280 kDa) and calpain-cleaved (150 kDa) forms of spectrin are marked by arrows.
• DP-BAPTAs stable lipophilic diesters of BAPTA
• the synthesis and some utihzation of stable lipophilic diesters of BAPTA have been disclosed in the International Patent Publication No. WO 99/16741 of the same applicant, the teaching and disclosure of which are expressly incorporated herein in their entirety by reference.
• WO 99/16741 publication the neuroprotective effects of DP-BAPTAs were demonstrated in neuronal cell cultures in-vitro, and in ischemia model systems in-vivo.
• the effect of the DP-BAPTA molecules on activities of specific enzymes has not been taught or suggested in that or any other publication. Accordingly, it was neither taught, recognized or suspected that these compounds could be effectively use for the treatment of MMP- and calpain-related diseases and disorders as disclosed in the present application.
• certain diesters of the chelating agent BAPTA are capable of inhibiting the activity of calpain and of certain proteases of the ADAM family, and in particular inhibiting the activity of matrix metalloproteinase-9 (MMP-9).
• MMP-9 matrix metalloproteinase-9
• the useful compounds in accordance with the invention are of the general formula (I) as described above. It is to be understood that within the scope of the invention are included also pharmaceutically acceptable salts of the compounds of the general formula (I) including organic and inorganic cations, as well as various solvates, including hydrates, and other active forms of the compounds of the general formula (I).
• the alkyl chains may be saturated or unsaturated alkyls including one or more double bonds and/or a triple bond.
• the alkyl chain is interrupted by from 1 to 3 oxygen atoms.
• the R moiety of a compound of the general formula (I) includes a monoalkyl ether of ethylene glycols, preferably mono- ,di- or tri-ethylene glycols.
• the alkyl residue at position R of the compound of the general formula (I) may consist of or include cyclic elements that may be aromatic or non-aromatic ring structures.
• the cyclic elements Preferably have 5 or 6 carbon atoms.
• Currently preferred cyclic R radicals comprise aromatic ring which is a phenyl residue.
• Other currently preferred cyclic elements included at position R are saturated or unsaturated cyclopentyl, cyclohexyl or cycloheptyl.
• the cyclic elements may be directly linked to the carboxy moiety of the compound of the general formula (I), or linked via a saturated or unsaturated alkyl chain that may include one or more oxygen and or nitrogen atoms.
• the compound of the general formula (I) includes a monovalent cation at position M.
• Suitable pharmaceutically acceptable cations may include, but are not limited to, H + , Na + , Li + , K + , NH 4 + and mono-alkylammonium.
• divalent cations may be included at position M.
• the choice of the preferred cation at position M of the general formula (I) depends on the intended therapeutic use of the compound, as well as on the specific formulation and route of administration employed. A person skilled in the art will be able to select the appropriate cation as required for the optimal pharmaceutical compositions and way of administration chosen in each particular treated case.
• DP-BAPTA compounds One of the most preferred DP-BAPTA compounds is l,2-bis(2- aminophenoxy)ethane, N,N'-di(2-benzyloxyethyl acetate), N,N'-acetic acid (DP- b440), where the R moieties of the compound of the general formula (I) include an alkylaryl moiety.
• This compound was disclosed generally in the WO 99/16741 publication, but was not specifically claimed and not individually tested.
• DP- BAPTAs can attenuate or block both basal MMP-9 activity and TNF ⁇ - or PMA- induced activation of MMP-9.
• DP-BAPTAs can also inhibit calpain activity.
• DP-BAPTAs may be useful in reducing deleterious protease activities in pathological conditions due, for example, to ischemia and inflammatory responses.
• DP-BAPTA compounds may be useful in preventing, treating or managing diseases and pathological conditions associated with harmful activities of matrix metalloproteinases or calpains.
• the DP-BAPTA compounds in accordance with the invention may be useful in the treatment of a whole range of indications which involve degradation processes carried out or mediated by MMPs, TACE or calpain.
• indications include, but are not limited to, diseases and conditions due to neuronal ischemia (e.g. global brain ischemia and focal brain ischemia), cardiac ischemia, trauma, stroke and inflammatory conditions including neuroi-iflammatory diseases and disorders, rheumatic and autoimmune diseases, neurological, cerebrovascular and cardiovascular diseases and disorders.
• the DP-BAPTA compounds may also be useful in compositions and methods for enhancing wound healing such as, for example, in burns and in chronic wounds (e.g. ulcers).
• rheumatoid diseases e.g. rheumatoid arthritis and osteoarthritis
• asthma e.g. rheumatoid arthritis and osteoarthritis
• psoriasis systemic lupus erythematosus
• inflammatory bowel syndrome e.g. Crohn's disease
• gingivitis e.g. gingivitis
• periodontitis meningitis
• tropical spastic paraparesis e.g. rheumatoid arthritis and osteoarthritis
• sepsis e.g. rheumatoid arthritis and osteoarthritis
• infectious diseases may include, but are not limited to, infectious diseases caused by any type of microorganism such as bacteria, fungi (e.g. candidiatis, aspergilosis) and viruses (e.g. herpes viruses-related disorders, HIV-related diseases), by parasites (e.g. malaria, amebiasis) or by prions (e.g. Creutzfeld- acob Disease).
• infectious diseases caused by any type of microorganism such as bacteria, fungi (e.g. candidiatis, aspergilosis) and viruses (e.g. herpes viruses-related disorders, HIV-related diseases), by parasites (e.g. malaria, amebiasis) or by prions (e.g. Creutzfeld- acob Disease).
• infectious diseases caused by any type of microorganism such as bacteria, fungi (e.g. candidiatis, aspergilosis) and viruses (e.g. herpes viruses-related disorders, HIV-related diseases), by parasit
• Inhibitors of MMPs or calpain can reduce proteolytic damage to tissues such as that caused during inflammatory processes. For example, may limit brain- blood-barrier (BBB) breakdown, inhibit neuroinflammation (e.g. as in meningitis), reduce damage associated with brain or cardiac ischemic injuries, and may diminish proteolytic effects caused by insults such as oxidative stress, burns, infections and central (CNS) and peripheral nervous system (PNS) injuries due to physical causes (e.g. trauma).
• BBB brain- blood-barrier
• neuroinflammation e.g. as in meningitis
• CNS central
• PNS peripheral nervous system
• Elevated MMP or calpain activity has been linked to several neurodegenerative diseases and conditions including, but not limited to, multiple sclerosis (MS), motoneuron disease (MND), amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Guillain-Barre syndrome, Parkinson's disease, Huntington's disease, Pick's disease, dementia syndrome, vascular dementia, multiple infarct dementia, HIV-induced neural disorders, brain ischemia (both global and focal ischemia) and neuronal tissue trauma.
• MMPs have also been associated with pathological conditions such as ischemic or hypoxic tissue damage, oxidative damage, osteoporosis, hemorrhage, arterial restenosis, cardiovascular disorders (e.g. ischemic myocardiac infarction) as well as with various ocular pathologies and retinopathies including diabetic retinopathy, glaucoma, macular degeneration, cataract, retinal detachment and retinal tears.
• MMPs are involved in spread of cancer, and in particular facilitating the metastasis state of the disease.
• MMP-2 and MMP-9 are involved in the breakdown of Type IV collagen, which is a major component of basement membrane, and as such may be key factors in processes involving membrane degradation, for example, in angiogenesis and in tumor invasion and metastasis. Indeed, positive correlation has been found between tumor progression and expression of members of the MMP family. For example, increased expression of MMP-2 and MMP-9 genes has been associated with malignancies of gliomas.
• angiogenesis is believed to be fundamental for primary tumor growth, tumor progression and metastasis.
• the first step in the mechanism of angiogenesis involves degradation of basement membrane so to facilitate the growth of a new capillary sprout.
• degradation and remodeling of the ECM are essential processes for the mechanism of angiogenesis, and methods of inhibiting these processes may be beneficial in blocking angiogenesis and hence dim shing malignancies.
• Angiogenesis is also important in a number of other pathological processes, including arthritis, psoriasis, diabetic retinopathy, chronic inflammation, scleroderma, hemangioma, retrolental fibroplasia and abnormal capillary proliferation in hemophiliac joints, prolonged bleeding etc.
• MMP-inhibitors are expected to be useful for the treatment of these angiogenic-associated diseases.
• the inabihty to control metastasis presents a major problem, as metastases are the leading cause of death in patients with cancer. To date, there is no satisfactory treatment for preventing or limiting metastasis growth.
• the cancer subjected to treatment with DP-BAPTAs may include any type of tumors and neoplastic growths that may be benign or malignant growths including primary tumors as well as secondary tumors.
• the terms "cancer” and “neoplastic growth” are interchangeably used in the specification and claims and mean to cover all kinds of pathological uncontrolled cell growths including invasive and non-invasive neoplasms, solid and non-solid tumors, and including remote metastases.
• treatment means to include therapeutic procedures aiming at preventing, amehorating, palhating, inhibiting or delaying the onset and or development and/or progression of a pathological condition or improving its manifestations.
• a pharmaceutical composition comprising a therapeutically effective amount of a compound of the general Formula (I) is administered to a patient in need thereof.
• the administered pharmaceutical composition may include a compound(s) of the general Formula (I) as the sole active ingredient, or may include said compound(s) in combination with one or more additional agents known to be effective in the treatment of a particular disease or disorder.
• the compound(s) of the general Formula (I) and the additional therapeutically active agent(s) may be included in the same pharmaceutical composition or may be administered in separate compositions.
• the use of DP-BAPTA in combination with another therapeutically active agent (or another therapeutic means) may be concurrently or not.
• the methods of the invention include administration of the DP-BAPTA compound(s) either at the same time, preceding or following exposure to the additional therapeutic agent or procedure.
• Additional agents that may be used in combination with the DP-BAPTA compounds may be therapeutic and prophylactic drags, hormones, immuno- modifying agents etc. and may include other chelating agents, proteins, peptides, carbohydrates, lipidic molecules , DNA and RNA sequences etc. These agents may be selected from, but are not limited to, anti-neoplastic, anti- proliferative, anti-inflammatory, antibiotic, anti-viral, anti-microbial, anti-mycotic, anti-allergic, cardiovascular agents, anti-convulsant, anti-depressant, anti- psychotic, analgesic, neurological agents, neuroprotective agents and bioactive peptides and proteins such as neurotransmitters, immuno-modulators, growth factors, hormones, antibodies etc.
• the DP-BAPTA compound(s) may be used alone or in combination, concurrently or not, with additional anti- cancer treatment.
• the additional anti-cancer treatment may include, but is not limited to, chemotherapy, irradiation therapy, i nmunotherapy, genetic therapy, surgery or any other anti-cancer treatment as known in the art.
• the additional treatment may be carried out concurrently with or consecutively to the adniinistration of the compoimds of the general formula (I), namely the additional treatment may be applied concurrently or successively, either preceding or subsequent to the administration of the compound of the general formula (I).
• the time interval between the two treatments and the overall regimen will be determined by a person skilled in the art taking into account the specific treated disease and the particular condition and response of the treated individual to the treatment.
• Suitable anti-cancer drags may include, but are not limited to, alkaloids (e.g. taxol, vinblastine, vindesine and vincristine), alkylating agents such as alkyl sulfonates, aziridines, ethylenimines, methylmelamines, nitrogen mustards (e.g. cyclophosphamide) and nitrosoureas, antibiotics and analogs (e.g. aclacinomycin, actinomycin, anthramycin, daunorubicin and doxorabicin), antimetabolites such as folic acid analogs (e.g.
• alkaloids e.g. taxol, vinblastine, vindesine and vincristine
• alkylating agents such as alkyl sulfonates, aziridines, ethylenimines, methylmelamines, nitrogen mustards (e.g. cyclophosphamide) and nitrosoureas
• Tomudex ® purine and pyrimidine analogs and platinum complexes (e.g. carboplatin, cisplatin, miboplatin and oxaliplatin).
• the combination treatment with additional therapeutic procedures may be beneficial also in treatment of other diseases and disorders.
• additional therapeutic procedures may be beneficial also in treatment of other diseases and disorders.
• the administration of the DP-BAPTA compounds may be in combination with (either concurrently, preceding or subsequent to) surgery and or treatment with another medicament or therapeutic agent (e.g. antibiotics, antibodies etc.) to remove or kill infectious agents or other pathogenic elements.
• another medicament or therapeutic agent e.g. antibiotics, antibodies etc.
• compositions including these therapeutically effective agents and methods applying them or other medical procedures are also included within the scope of the invention as compositions and methods useful in combination with the compounds of the general formula (I).
• the exact protocol and the additional medicament or therapeutic procedure used will be determined by a person skilled in the art taking into consideration the particulars of the specific medical condition treated, e.g. the stage of the disease or disorder, its severity and progression, as well as the condition of the patient.
• compositions comprising the compound of the general formula (I) may be in a liquid, aerosol or solid dosage form, and may be formulated into any suitable formulation including, but not limited to, solutions, suspensions, micelles, emulsions, microemulsions, aerosols, ointments, gels, suppositories, capsules, tablets, and the like, as will be required for the appropriate route of administration.
• any suitable route of administration is encompassed by the invention including, but not being limited to, oral, intravenous, intraperitoneal, intramuscular, subcutaneous, inhalation, intranasal, topical, rectal or other known routes.
• the useful pharmaceutical compositions are orally or intravenously administered.
• the dose ranges are those large enough to produce the desired proteinase inhibitory effect.
• the dosing range varies with the specific DP-BAPTA used, the treated pathological condition and the route of administration and is dependent on the additional treatment procedure, if such additional treatment is applied.
• the dosage administered will also be dependent upon the age, sex, health, weight of the recipient, concurrent treatment, if any, frequency of treatment and the nature of the effect desired.
• the specific dosage, regimen and means of administration will be determined by the attending physician or other person skilled in the art.
• BAPTA 24 gr.,0.05 mol
• pyridine 8 gr., 0.1 mol
• acetic anhydride 95 ml, 1.0 mol
• the reaction mixture is heated at 90°C for 5 hours with vigorous stirring by magnetic stirrer.
• the temperature is then decreased to 50°C and heating is continued at this temperature for 10 hours longer.
• the reaction mixture is cooled to room temperature and the precipitate is extracted by filtration.
• the BAPTA anhydride of step 1 (10 g, 0.023 Mol) and corresponding absolute alcohol (300 ml) are introduced, under argon atmosphere, into a round-bottom single-neck flask, equipped with reverse condenser and magnetic stirrer.
• the mixture is heated in an oil bath at 90°C (for methyl and ethyl diesters at 70°C) with vigorous stirring.
• After 6 hours about half of the alcohol is distilled from the reaction mixture (high molecular alcohols are distilled under vacuum).
• the obtained mixture is cooled to 0°C and kept at this temperature for 5-8 hours.
• the precipitate is separated from the solution by filtration (glass filter N4) under vacuum and is washed 3-4 times with about 40 ml of ethanol, followed by three washes (100 ml each) of ethyl acetate and finally with three washes (150 ml each) of diethyl ether.
• the product is dried under vacuum for 8 hours.
• Butyl diester of BAPTA Yield 90% (12.1 g..). White powder. M.p. 183°C TLC analysis. Conditions of analyses of diethyl and dibutyl esters of BAPTA are analogous. One spot. R f 0.42. 1H NMR [(CD 3 ) 2 SO]. ⁇ (ppm): 0.74-0.80 (t,6H), 1.09-1.18 (m, 4H), 1.33-1.39 (m, 4H), 3.80-3.86 (t, 4H), 3.98 (s,4H), 4.10 (s, 4H), 4.17 (s, 4H), 6.69-6.92 (m, 8H). Elemental. C 30 H 4 o0 10 N 2 . Calculated: C 61.22%, H 6.80%, N 4.76%. Found: C 61.54%, H 7.10%, 5.03%.
• Octyl diester of BAPTA Yield 70% (11.3 g.). White powder. M.p.l55°C. TLC analysis. Conditions of analyses of diethyl and dioctyl esters of BAPTA are analogous. One spot. R f 0.55.
• Corresponding alkyl diester of BAPTA (0.019 Mol) is introduced into an Erlenmeyer flask (500 ml), equipped with a magnetic stirrer. About 250 ml of a mixture of methanol with water (1: 1 v/v) is added to the ester. This mixture is vigorously stirred, because the ester is not dissolved in the solution. A concentrated solution of NaHC0 3 (0.038 mol, 3.19 g.) or concentrated solution of
• MeONa (0.038 mol) in water is added to the stirring mixture, and after 5-8 hours the mixture becomes transparent. This indicates that the alkyl diester is converted into disodium salt. Methanol and water are evaporated under vacuum. The obtained salt is dried by azeotropic distillation with ethanol and diethyl ether.
• Octyl diester of BAPTA disodium salt. White powder. Yield 90% (15.7 g.).
• Octyl diester of BAPTA calcium salt. White powder. Yield 80% (0.83 g.). C 38 H 54 N 2 ⁇ 10 Ca. Calculated: C 61.79%, H 7.32%, N 3.79%, Ca 5.42%. Found: C 61.94%, H 7.14%, N 4.00%, Ca 5.31%.
• EXAMPLE 2 Synthesis of BAPTA diesters of alkyl or alkylaryl ether of mono-, di- and triethylene glycol and salts thereof
• This first step of obtaining a BAPTA anhydride is identical to step 1 in the procedure for synthesizing the alkyl diesters of BAPTA as described above in Example 1.
• Step 3 Synthesis of BAPTA diesters of monoalkyl ethers of mono-, di- and triethylene glycol
• the BAPTA anhydride of step 1 (1.5 g., 0.0034 Mol) and the corresponding monoalkyl ether of mono-, di- or triethylene glycol of step 2 (10-12 ml) are introduced, under argon atmosphere, into a round-bottom single-neck flask (50 ml), equipped with a reverse condenser and a magnetic stirrer.
• the mixture is heated in an oil bath at 115-120°C with vigorous stirring. After 1-1.5 hours the mixture becomes transparent. Heating is continued for another 1.5 hours, till the reaction is completed.
• the flask is then cooled to room temperature and about 100 ml of petroleum ether (b.p. 60-80°C) is added.
• the formed precipitate is extracted by centrifugation and washed three times with petroleum ether (40 ml each wash).
• the solid product is dried under vacuum for 5 hours and analyzed to verify the product characteristics, as exemplified for the following compounds: BAPTA diester of methylethylene glycol. White solid. M.p.151-152°C. Yield
• BAPTA diester heptyldiethylene glycol are the same. One spot. R f 0.40.
• BAPTA diester of 2-benzyloxyethanol White solid. Yield 80% (2.02g.).
• TLC analysis Conditions of TLC analysis of BAPTA diester of 2- benzyloxyethanol and BAPTA diester of heptylethylene glycol are the same. One spot. R f 0.5.
• Step 4a Preparation of disodium salt of BAPTA diesters of monoalkyl ethers of mono-, di- or triethylene glycol.
• the corresponding BAPTA diester of monoalkyl ether of mono-, di- or triethylene glycol (0.0025 Mol) is dissolved in methanol (arround 10 ml of alcohol is necessary for dissolving 1.0 g. of BAPTA diester) and the obtained solution is introduced into an Erlenmeyer flask (50 ml), equipped with a magnetic stirrer.
• a water solution of sodium bicarbonate (0.005 Mol in 2 ml) is added to a methanol solution of the BAPTA diester and the mixture is stirred for 2 hours at room temperature.
• the solvent is then evaporated under vacuum (30 mm Hg).
• the obtained precipitate is dried three times by azeotiopic distillation with ethanol and two times with diethyl ether. Finally, the obtained product is washed with hexane and is dried under vacuum.
• BAPTA diester of heptyltriethylene glycol, disodium salt White wax. Very hygroscopic. Yield 90% ( 2.2 g.).
• Step 4b Preparation of calcium salt of BAPTA diesters of monoalkyl ethers of mono-, di- or triethylene glycol.
• the corresponding monoalkyl ether of mono-, di- or triethylene glycol diester of BAPTA (0.0025 Mol) is dissolved into 250 ml methanol. About 3-5 ml of water is added to this solution. The obtained solution is introduced into an Erlenmeyer flask (300ml), equipped with a magnetic stirrer. The powder of CaH 2 (0.0025 Mol) is added to this solution with vigorous stirring. The stirring is continued for 3 hours at room temperature. After 3 hours the mixture is filtered through paper filter (Whatman Nl) and the obtained solution is evaporated under vacuum (10-15 mm Hg).
• Methyltriethylene glvcol diester of BAPTA calcium salt. White solid. Yield 80%
• DP-b99 The effect of DP-b99 on MMP-9 activity was tested on cultured C6 glioma cells either in the absence (basal activity) or following treatment with Tumor Necrosis Factor alpha (TNF ⁇ ) to induce gelatinases.
• TNF ⁇ Tumor Necrosis Factor alpha
• C6 rat glioma cells (ATCC; CRL-2199) grown on 100 mm petri dishes were detached by tiypsinization and cultured to a density of 10 5 cells/well on 24- well plates in DMEM + 10% FCS.
• the TNF ⁇ -tieated cells which were stimulated to induce gelatinases were incubated, on the next day after plating, for 18 hours in DMEM without serum in the presence of either 20 ng/ml or 40 ng/ml
• TNF ⁇ (R&D, cat. #410-TRNC).
• Conditioned media (CM) were then collected, centrifuged at 2000 rpm for
• MMP gelatmase activity was determined by using zymogram gels (Invitrogen, cat. #EC6175) which include gelatin as a substrate. Samples were loaded on gels in 62.5 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS and traces of bromophenol-blue. Gels were run in Tris/glycine-SDS running buffer, washed for 30 min. with renaturing buffer (Invitrogen) and for further 30 min. with developing buffer (Invitrogen). MMP activity was developed overnight at 37°C with fresh developing buffer. The gels were then stained for one hour with Coomasie Blue (Brilliant Blue R, Sigma, cat.
• EXAMPLE 4 DP-BAPTA reduces expression /release of TNF ⁇ -induced MMP-9 and inhibits enzyme activity
• TNF ⁇ Tumor Necrosis Factor alpha
• A- 172 human glioma cells (ATCC; CRL-1620) were grown and treated with 10 ng/ml TNF ⁇ in the presence of 0, 20 or 50 ⁇ M DP-M09 following the procedure as described above in Example 3. Cells treated with vehicle without the
• TNF ⁇ treatment served as control groups.
• CM from each treatment group was collected and subjected to zymogram gels (see procedure in Example 3), to MMP-9 immunoassay and to MMP-9 activity assay.
• the total amount of MMP-9 protein was determined by immunoassay using the QuantikineTM (R&D Systems, U.S.A., Cat. # DMP900) kit and following the manufacturer's instructions.
• MMP-9 activity was assayed using the MMP-9 activity assay system
• C6 glioma cells were grown and treated as described above in Example 3, except that cells were stimulated with either 20 ng/ml TNF ⁇ (R&D, cat. #410- TRNC) or 0.1 ⁇ M Phorbol 12-myristate 13-acetate (PMA, Sigma, cat. #P-8139).
• DP-109 decreased the MMP-9 activity induced by either TNF ⁇ or PMA in a dose dependent fashion.
• EXAMPLE 6 Effect of DP-BAPTA on MMP-9 activity in comparison to BAPTA and BAPTA-AM
• DP-BAPTA molecules were screened for their effect on MMP-9 activity and were compared to the effect of the related chelating compounds 1,2- bis(2 aminophenoxy)ethane-N,N,N',N'-tetiaacetic acid (BAPTA) and its hpophilic analog l,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA-AM).
• C6 glioma cells were plated and treated with 20 ng/ml TNF ⁇ following the protocol described above in Example 3, either in the presence or absence of a tested compound as indicated.
• Each of the tested DP-BAPTAs, BAPTA or BAPTA-AM was added at the following final concentrations: 1, 5, 10 and 20 ⁇ M. As controls served cells treated with vehicle only.
• the most potent inhibitors among the tested compounds were l,2-bis(2- aminophenoxy)ethane, N,N'-di(2-benzyloxyethyl acetate), N,N'-acetic acid, disodium salt [DP-b440] and l,2-bis(2-aminophenoxy)ethane, N,N'-di(2- octodecyloxyethyl acetate), N,N'-diacetic acid, disodium salt [DP-M09] with calculated IC 50 of 12 ⁇ M and 10-20 ⁇ M, respectively.
• BAPTA parent chelating compound
• BAPTA-AM The related tetia-ester BAPTA analog, BAPTA-AM, increased rather than decreased the MMP-9 activity in the test model system used (C6 glioma cells stimulated with TNF ⁇ ).
• EXAMPLE 7 DP-BAPTA inhibits MMP-9 activity in primary cultured glial cells.
• Cortical glial cells from rat embryos (day 18 of pregnancy) were seeded on poly-D-lysine coated 24-well plates with MEM medium containing 5% FCS, 5% HS, 2 mM 1-glutamine and 0.6% glucose, at a density of 5xl0 4 cells/well. After 4 days in culture, the medium was changed to MEM + 10% FCS. When glial cells reached confluence (around 17 days after plating), they were exposed for 24 hours to 10 ng/ml or 20 ng/ml human TNF ⁇ in order to induce MMP-9.
• CM Conditioned media
• FIG. 3 A A representative zymogram of MMP-9 activity in conditioned medium (CM) from control (un-stimulated) glial cells and from cultures treated with 10 or 20 ng/ml TNF ⁇ , either with or without DP-b99, is depicted in Figure 3.
• CM conditioned medium
• FIG. 3 A the two forms of MMP-9 were separated on the gel; the upper band represents the pro-MMP-9 with the higher molecular weight, while the lower band (lower MW) represents the biologically active form of the enzyme.
• DP-b99 inhibitory effect on basal MMP-9 activity in glial cells was demonstiated by showing an increase in band intensity of the Pro-MMP-9 on the account of the active MMP-9 form.
• DP-b99 reduced both the pro- and active forms of the enzyme.
• EXAMPLE 8 Effect of DP-BAPTA on TNF ⁇ release from primary glial cells The effect of various DP-BAPTA molecules on the levels of TNF ⁇ released in response to stimulation of primary glial cells with lipopolysaccharide (LPS) was measured.
• LPS lipopolysaccharide
• LPS E.coli 0111 :B4, Calbiochem, cat. #437627
• CM were collected and analyzed for the presence of TNF ⁇ by using an ELISA assay (DuoSet, R&D, cat. #DY510).
• LPS increased the release of TNF ⁇ by around 10 folds over control levels.
• All DP-BAPTA tested compounds mhibited this increase.
• TACE TNF ⁇ Converting Enzyme
• DP-BAPTA molecules may indicate a potential use of these molecules also in treating or ameliorating diseases and conditions related to peripheral inflammatory processes.
• the seven rats were sacrificed after 24 hrs. and each hemisphere of their brains was subjected separately to lysis and extracted for enzymatic activity.
• Brains were minced and solubilized in lysis buffer (25 mM Tris-HCl pH 7.5, 1% IGEPAL CA-630 - a non-ionic detergent from Sigma, 100 mM NaCl, 0.5U/ml aprotinin, 0.01% sodium azide) at a final concentration of 400 mg/ml.
• lysis buffer 25 mM Tris-HCl pH 7.5, 1% IGEPAL CA-630 - a non-ionic detergent from Sigma, 100 mM NaCl, 0.5U/ml aprotinin, 0.01% sodium azide
• Protein concentration was determined by the Bradford assay and 40 ⁇ g protein from each lysate were loaded on gelatin zymogram gels for determination of
• DP-b99 reduced the increase in MMP-9 activity induced by focal brain ischemia in rats.
• the results of this in-vivo study correlate with those of the in vitro experiments (Examples 3 to 7) that showed that DP-BAPTAs reduce MMP-9 activity induced in C6 and A-172 glioma cells and in primary glial cells.
• EXAMPLE 11 DP-b-99 inhibits calpain activity in primary cortical neurons
• Calpain activity in primary cortical neurons was evaluated following activation of the enzyme by H 2 0 2 . Calpain activity was measured by monitoring proteolytic degradation of the calpain substrate ⁇ -spectrin, from the 280 kDa full- length protein to the 150 kDa degradation product.
• Primary cortical neurons were prepared from the brains of embryonic day 16-17 (E16-17) rat fetuses. Cells from embryos of one mother were resuspended in 300 ml of "primary medium" (NBM Gibco, Glasgow, Scotland) with 0.5 mM glutamine, 0.4 units/ml penicillin, 0.4 ⁇ g/ml streptomycin, and B27 supplement (Gibco, Glasgow, Scotland), plus 25 ⁇ M glutamic acid. Cells ( ⁇ 3xl0 5 /ml) were seeded, 1 ml/well, in 24-well plate for viability assay, or 4 ml/well in 6-well plate for analysis of calpain activity.
• primary medium NBM Gibco, Glasgow, Scotland
• H 0 2 was added at the indicated concentiations to cells in "primary medium” from a solution of 10 mM that was prepared in PBS immediately before use. Viability was determined after 18 to 24 hours. DP-b99 was added to the medium 1-2 hours prior to induction of the oxidative stress.
• Assay for Calpain activity Primary cortical neurons in 6-well plates were lysed in lOO ⁇ l RIPA buffer (50mM Tris pH 7.5; 150mM NaCl; 0.5% DOC; 1% Triton X-100; 0.1% SDS; ImM Nappi; 2mM EDTA) plus protease inhibitors (Bohringer, Manheim, Germany). After 10 min. incubation on ice, lysates were cleared by 15 min. centrifugation at 20,000g at 4°C. Samples including 10-50 ⁇ g protein were separated on gradient 4- 12% SDS-PAGE (Nu-PAGE, NO VEX, with MES buffer), and blotted to nitrocellulose paper according to the manufacture instructions.
• ⁇ -spectrin To detect the various forms of ⁇ -spectrin, blots were reacted with anti-spectrin antibodies (Affinity FG6090 1:1000), followed by HRP-second antibodies (Santa Cruz, USA), followed by ECL reaction (Amersham, Buckinghamshire, UK). Detection of bands was performed using the Image Station 440 (Kodak Digital System). The non-cleaved spectiin ran at ⁇ 280kDa, while the calpain cleaved form ran at ⁇ 150kDa. Quantitation of the bands was performed using the Kodak ID software.
• the effect of DP-b99 on calpain activity was studied 4 hours after addition of H 2 0 2 by monitoring the H 2 ⁇ 2 -induced cleavage of spectiin.
• the cortical primary cells were pre-treated with DP-b99 (15 ⁇ g/ml) or with the commercially available calpain inhibitor MDL28170 (25 ⁇ M) for 1 hour before H 2 0 2 (50 ⁇ M) was added.
• Calpain activity was determined by using the "Calpain activity" assay described above.
• DP-b99 inhibits H 2 0 2 -induced calpain activity similarly to the commercial calpain inhibitor MDL28170.
• EXAMPLE 12 DP-b-99 inhibits induced spectrin cleveage in vivo The effect of DP-b99 on calpain activity was further evaluated in vivo in transient focal cerebral ischemia model system in rats.
• MCA Middle cerebral artery
• Sprague Dawley (SD) rats by applying 4-0 silicone-coated nylon monofilament through external carotid artery.
• the animals were anesthetized with 4% halothane and maintained at 1.5% halothane in a 1:2 mixture of nitrous oxide and oxygen without tracheotomy and allowed to breathe spontaneously.
• Via a ventral cutaneous the common, right external and internal carotid arteries (EGA) were exposed.
• a suture was introduced into a ligated right external carotid artery, in a retrograde fashion towards the carotid bifurcation.
• Assay for Calpain activity Each hemisphere was separately lysed by homogenization on ice in 5ml RIPA buffer (50mM Tris pH 7.5; 150mM NaCl; 0.5% DOC; 1% Triton X-100; 0.1% SDS; ImM Nappi; 2mM EDTA) plus protease inhibitors (Bohrrnger, Manheim, Germany). Lysates were cleared by 30 min centrifugation at 3,000g at 4°C, followed by 30 min. centrifugation at 20,000g at 4°C.
• DP-b99 inhibits the increase in calpain activity induced by ischemia in vivo. Calpain inhibition by DP-b99 may be responsible, at least in part, for the neuroprotective effect exhibited by this molecule.

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• 2002
  • 2002-09-25 IL IL15192102A patent/IL151921A0/xx unknown
• 2003
  • 2003-03-16 CA CA2498420A patent/CA2498420C/en not_active Expired - Fee Related
  • 2003-03-16 JP JP2004539399A patent/JP2006500422A/ja active Pending
  • 2003-03-16 EP EP03710190A patent/EP1546085B1/en not_active Expired - Lifetime
  • 2003-03-16 US US10/529,028 patent/US7799831B2/en not_active Expired - Lifetime
  • 2003-03-16 WO PCT/IL2003/000225 patent/WO2004028443A2/en not_active Ceased
  • 2003-03-16 AU AU2003214610A patent/AU2003214610C1/en not_active Ceased
• 2005
  • 2005-03-13 IL IL167406A patent/IL167406A/en not_active IP Right Cessation

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