WO2006063408A1 - Compositions and methods for treating cardiovascular disorders - Google Patents
Compositions and methods for treating cardiovascular disorders Download PDFInfo
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- WO2006063408A1 WO2006063408A1 PCT/AU2005/001904 AU2005001904W WO2006063408A1 WO 2006063408 A1 WO2006063408 A1 WO 2006063408A1 AU 2005001904 W AU2005001904 W AU 2005001904W WO 2006063408 A1 WO2006063408 A1 WO 2006063408A1
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- optionally substituted
- probucol
- alkyl
- dtbp
- alkenyl
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- 0 ****c1c(*)c(*)c(*)c(*)c1* Chemical compound ****c1c(*)c(*)c(*)c(*)c1* 0.000 description 5
- BOWJCXNBBDVMFA-UHFFFAOYSA-N CCCCc1cc(SSc(cc2C)cc(I)c2O)cc(C)c1O Chemical compound CCCCc1cc(SSc(cc2C)cc(I)c2O)cc(C)c1O BOWJCXNBBDVMFA-UHFFFAOYSA-N 0.000 description 1
- HMHBLLKJEKLJSV-UHFFFAOYSA-N CCCCc1cc(SSc(cc2I)cc(CC)c2O)cc(CC)c1O Chemical compound CCCCc1cc(SSc(cc2I)cc(CC)c2O)cc(CC)c1O HMHBLLKJEKLJSV-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C381/00—Compounds containing carbon and sulfur and having functional groups not covered by groups C07C301/00 - C07C337/00
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- 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
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
- C07C323/10—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton
- C07C323/18—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton
- C07C323/20—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton with singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C381/00—Compounds containing carbon and sulfur and having functional groups not covered by groups C07C301/00 - C07C337/00
- C07C381/04—Thiosulfonates
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C391/00—Compounds containing selenium
- C07C391/02—Compounds containing selenium having selenium atoms bound to carbon atoms of six-membered aromatic rings
Definitions
- the present invention relates to compounds and methods for the treatment of cardiovascular diseases and disorders.
- Heart disease can result from many factors relating to poor functioning of heart tissue which may manifest in commonly known conditions such as angina, stroke, or heart attack.
- the underlying mechanisms of heart disease are not completely understood.
- lipids such as cholesterol
- atherosclerosis i.e., the clogging of arteries, and the build-up of deposits that may eventually lead to heart disease, or stroke.
- Oxidative modification theory' of atherosclerosis it is oxidised lipid, particularly in the form of oxidised low-density lipoprotein (LDL) particles that initiate and contribute to the subsequent development of athero genesis.
- LDL oxidised low-density lipoprotein
- Phenolic compounds are generally known to be good radical scavengers. In vitro, phenolic compounds effectively inhibit the peroxidation of lipids in homogeneous solution (e.g., when the lipids are dissolved in an organic solvent) that itself is a free radical process.
- ⁇ -tocopherol the most active form of vitamin E
- Probucol the initially introduced as a lipid-lowering drug
- ⁇ -tocopherol does not necessarily inhibit, and in some circumstances can even promote, the oxidation of LDL lipids, and this may in part explain why vitamin E supplements have generally failed to provide protection against cardiovascular disease in recent controlled prospective studies in humans.
- inhibition of lipid peroxidation by phenolic compounds does not account for s in vivo protective activity particularly as inhibition of lipoprotein lipid oxidation in the vessel wall and atherosclerosis are two events that can be dissociated from each other.
- EP 1 464 639 (entitled “Succinic acid ester of probucol for the inhibition of the expression of VCAM-I”) discloses analogues of probucol having at least one phenolic residue as inhibitors of both, lipid oxidation and the expression of vascular cell adhesion o molecule-1 (VCAM-I).
- EP 1 464 639 also disclosed the use of phenolic analogues of probucol for the treatment of diseases mediated by VCAM-I, including cardiovascular disorderst
- a B 0 There is a need for alternative therapies for cardiovascular disorders, including treatment and prevention of atherosclerosis and restenosis.
- the present invention relates to compounds of general Formula (I):
- X is selected from S, Se, S(O) and S(O) 2 ;
- Y is selected from S, Se, S(O) and S(O) 2 ;
- A comprises one or more groups selected from optionally substituted C 1-6 alkylene, optionally substituted C 2-6 alkenylene; optionally substituted C 3-1O cycloalkylene; and optionally substituted arylene;
- n is O or 1;
- Z is selected from optionally substituted aryl and optionally substituted heteroaryl, optionally substituted alkyl, optionally substituted alkoxy, and NR 13 R 14 ;
- R 1 , R 2 , R 3 , R 4 , and R 5 may be the same or different and are independently selected from the group consisting of hydrogen, halogen, hydroxyl, thiol, -NR 13 R 14 , nitro, cyano, optionally substituted Ci -I0 alkyl, optionally substituted C 2-I o alkenyl, optionally substituted C 2-10 alkynyl, optionally substituted C 3-10 cycloalkyl, optionally substituted aryl, optionally substituted aryl(C 1-6 alkyl), optionally substituted (Cj -6 alkyl)aryl, optionally substituted heteroaryl, optionally substituted C 3 _io heterocycloalkyl, C(O)R 11 , OR 12 , SR 12 , CH 2 OR 12 , CH 2 NR 13 R 14 , C(O)OR 12 and C(O)NR 13 R 14 ;
- R 11 is selected from OH, C 1-6 alkyl, and Ci -6 alkenyl
- R 12 is selected from the group consisting of hydrogen, optionally substituted C 1- Io alkyl, optionally substituted C 2-10 alkenyl, optionally substituted C 2-10 alkynyl, optionally substituted C 3-J0 cycloalkyl, optionally substituted aryl, -C(O)(C 1-6 )alkyl-CO 2 R 15 , -C(O)(C 2-6 )alkenyl-CO 2 R 15 , and -C(O)NR 13 R 14 ;
- R 13 and R 14 may be the same or different and are individually selected from hydrogen, Cj -6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, C 3-6 heterocycloalkyl, aryl, (Ci -6 )alkylaryl, and heteroaryl; and
- R 15 is H or C 1-4 alkyl; and salts thereof.
- the present invention relates to pharmaceutical compositions comprising at least one compound of Formula (I) as defined in the first aspect of the invention, together with pharmaceutically acceptable excipient, diluents and/or adjuvants.
- the present invention relates to a method of treating a cardiovascular disorder in a vertebrate, said method comprising administering to said vertebrate an effective amount of a compound according to Formula (I) as defined in the first aspect of the invention or a composition according to the second aspect of the invention.
- the invention relates to the use of a compound of Formula (I) according to the first aspect of the invention for the manufacture of a medicament for treating a cardiovascular disorder.
- the invention relates to a process for preparing a pharmaceutical composition comprising homogeneously mixing a compound according to the first aspect of the invention with a pharmaceutically acceptable adjuvant, diluent and/or carrier.
- CE - cholesteryl esters (Cl 8:2 plus C20:4) CE-O(O)H - hydroxides and hydroperoxides of cholesteryl ester
- FIG. 1 Site-specific effect of probucol on atherosclerosis in apoE-/- mice. Lesion sizes at sinus, arch and thoracic/abdominal aorta after six months intervention in probucol- treated mice (•) expressed relative to the corresponding lesion size in control animals (O). Results show mean ⁇ SEM for 17 mice per group and for each site. * Significantly different from corresponding control value (p ⁇ 0.05).
- Figure 2 Site-specific effect of probucol on arterial accumulation of non-oxidized lipids and ⁇ -tocopherol.
- Figure 3 Site-specific effect of probucol on arterial lipid oxidation.
- Aortic sinus lesions of apoE-/- mice fed a high fat diet without (A, C, E) and with 1% (w/w) probucol (B, D, F) were stained for macrophages (A, B) or collagen (C-F) as described in the Methods section. Accumulation of macrophages/macrophage foam cells (brown staining) is evident along the luminal side of the lesions from control and probucol-treated mice. Bright field images (C, D) were used to determine lesion areas, whereas polarization microscopy (E, F) was used to analyze the collagen-containing area that exhibits strong birefringence (red staining). The sections shown are representative of the results seen in six different animals (for control and probucol).
- Calibration bar represents 2 ⁇ m.
- Figure 5 Site-specific metabolism of probucol in aortas of apoE-/- mice. Lesion content of probucol (A) and its proportion present as bisphenol and diphenoquinone (B) after six months intervention. Data in A is given in nmol per mg protein (O) or mmol per mol C+CE (•). Results show mean ⁇ SD from four independent pools each containing 15 respective sections. Where SD cannot be seen, they are smaller than the symbol size. * ' ⁇ Significantly different from corresponding sinus and arch value, respectively (p ⁇
- Tissue cGMP was then expressed relative to that in control rings in the absence of HOCl-treatment, with 100% corresponding to " 454 ⁇ 46 pmol/g wet tissue.
- C Drug content in rings used for relaxation studies. Data show mean ⁇ SEM from rings of 6 animals per treatment. * Significantly different from control (P ⁇ 0.05). s Figure 7 In vitro added probucol and DTBP attenuate HOCl-induced endothelial dysfunction.
- A Aortic rings from rabbits fed normal diet were pre-incubated for 10 min without (D) or with 10 ( ⁇ , O), 25 (A, ⁇ ) or 100 ⁇ M ( ⁇ ,O) probucol (filled symbols) or DTBP (open symbols), washed and then exposed to 400 ⁇ M HOCl prior to pre- constriction and relaxation in response to ACh. *P ⁇ 0.05 for comparison of untreated 0 rings versus rings treated with probucol or DTBP.
- B Tissue cGMP in aortic rings exposed to 400 ⁇ M HOCl for 5 min after pre-incubation in the absence and presence of 100 ⁇ M probucol or DTBP, and (C) aortic drug content before exposure of HOCl.
- cGMP was expressed relative to that in control rings as described in the Legend to Fig. 1, with 100% corresponding to 454 ⁇ 46 pmol/g wet tissue. Data show mean ⁇ SEM from rings S of 6 animals per treatment. * Significantly different from control (P ⁇ 0.05).
- FIG. 8 Oxidation of probucol by HOCl.
- Probucol final concentration 1 mM dissolved in hexane was oxidized with increasing concentrations of reagent HOCl for 60 min at 37 0 C.
- A Consumption of probucol (O) and formation of DTBP (•) and DPQ plus compounds 2, 4, and 6 ( ⁇ ) was monitored by HPLC as described in the Methods Section.
- B Representative chromatograms of reaction mixture before (top) and after oxidant exposure (bottom) monitored at 270 (solid line) and 420 nm (broken line). Eluting products were labelled sequentially 1-7, purified by semi-preparative HPLC and used retrospectively for quantification in panel (A).
- FIG 11 Aortic content of probucol and its metabolites before and after HOCl exposure.
- Aortas from rabbits fed normal diet supplemented with (A) 1% probucol or (C) 0.2% DTBP for 4 weeks were analyzed for probucol and its metabolites before (open bars) and after exposure to 400 ⁇ M HOCl (filled bars) as described in the Methods Section.
- aortic segments obtained from control rabbits were supplemented in vitro with (B) 100 ⁇ M probucol or (D) DTBP and analyzed for probucol and its metabolites before (open bars) and after exposure to 400 ⁇ M HOCl (filled bars).
- Data show mean ⁇ SEM of 3 separate experiments using aortic rings from three different animals. * Significantly different from corresponding vessel without HOCl-treatment (P ⁇ 0.05).
- FIG. 12 DTBP, not BP, inhibits atherosclerosis in apoE-/- mice similar to probucol.
- a Representative cross sections of abdominal aorta from control (Ctrl) and three treatment groups stained for macrophages, indicating respective lesion size (x400).
- NL Total neutral lipids
- LOOH hydroperoxides
- FIG. 13 DTBP, not BP, inhibits intimal hyperplasia in rabbits in response to vessel injury similar to probucol.
- a Representative Verhoeffs hematoxylin-stained cross sections (xlO).
- b I/M ratios of vessels from control and drug-treated rabbits after 6 weeks of ABI (8 serial sections per aortic segment, 100 ⁇ m apart), c and d, Total neutral lipids (NL) and their hydroperoxides (LOOH).
- e Time-dependent changes in plasma cholesterol, with symbols as described in Legend to Figure 12. All results are from 6 rabbits per group. *, PO.05 compared to control.
- Figure 15 Probucol and DTBP, not BP, promote functional re-endothelialization in rabbits after injury, a, Representative longitudinal section with branch orifice (x20) showing denuded and CD-31 + re-endothelialized aortic surface (red arrows) distal and proximal to the branch orifice, respectively (x400), after 6 weeks of injury, b, Re- endothelialization assessed by the length (mm) of section covered by CD-31 + cells from branch orifice for the groups after 6 weeks of injury (3-6 serial sections per segment, 100 ⁇ m apart), c and e, Relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) of pre-constricted aortic ring taken from rabbits of the four groups.
- ACh acetylcholine
- SNP sodium nitroprusside
- HO-I heme oxygenase-1
- Oxidation of the sulfur atoms of probucol to the disulfoxide is a proposed first step in HOCl-mediated conversion of probucol to DTBP. This proposed mechanism is distinct from the oxidation of the phenolic groups of probucol.
- Figure 18 Proposed non-radical mechanism for oxidation of probucol.
- Figure 19 Heme oxygenase- 1 (HO-I) is a target for probucol and DTBP. Blocking heme oxygenase activity via administration of tin protoporphyrin prevented the ability of probucol and DTBP to inhibit intimal thickening in response to arterial balloon injury (Figure 19 ⁇ ), to promote re-endothelialization ( Figure 196), and to inhibit vascular smooth muscle cell proliferation (Figure 19c).
- Vitamin E fails to inhibit intimal hyperplasia and does not promote re- endothelialization or induce HO-I.
- A HO-I mRNA assessed by real time RT-PCR in rabbit aortic smooth muscle cells cultured for 24 hours in the presence of vehicle (control), probucol (50 ⁇ M) or ⁇ -tocopherol (vitamin E, 50 ⁇ M).
- B Re- endothelialization assessed by Evans blue staining for the three groups of rabbits in (A) 3 weeks after injury.
- alkyl and divalent (“alkylene”) straight chain or branched chain saturated aliphatic groups having from 1 to 10 carbon atoms, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms.
- alkyl includes, but is not limited to, methyl, ethyl, 1 -propyl, isopropyl, 1 -butyl, 2-butyl, isobutyl, tert-butyl, amyl, 1 ,2-dimethyl ⁇ ropyl, 1,1- dimethylpropyl, pentyl, isopentyl, hexyl, 4-m ' ethylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methyl ⁇ entyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3- dimethylbutyl, 1,2,2-trimethylpropyl, 1,1,2-trimethylpropyl, 2-ethyl, 1-
- alkenyl group includes within its meaning monovalent (“alkenyl”) and divalent (“alkenylene”) straight or branched chain unsaturated aliphatic hydrocarbon groups having from 2 to 10 carbon atoms, eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms.
- alkenyl groups include but are not limited to ethenyl, vinyl, allyl, 1- methylvinyl, 1- ⁇ ropenyl, 2-pro ⁇ enyl, 2-methyl-l-propenyl, 2-methyl-l- ⁇ ropenyl, 1- butenyl, 2-butenyl, 3-butentyl, 1,3-butadienyl, 1-pentenyl, 2-pententyl, 3-pentenyl, 4- pentenyl, 1,3-pentadienyl, 2,4-pentadienyl, 1,4-pentadienyl, 3-methyl-2-butenyl, 1- hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1 ,4-hexadienyl, 2-methylpentenyl, 1- heptenyl, 2-heptentyl, 3-heptenyl, 1-octenyl, 1-nonenyl, 1-decen
- alkynyl group as used herein includes within its meaning monovalent (“alkynyl”) and divalent (“alkynylene”) straight or branched chain unsaturated aliphatic hydrocarbon groups having from 2 to 10 carbon atoms and having at least one triple bond.
- alkynyl groups include but are not limited to ethynyl, 1-propynyl, 1-butynyl, 2-butynyl, l-methyl-2-butynyl, 3 -methyl- 1-butynyl, 1-pentynyl, 1-hexynyl, methylpentynyl, 1-heptynyl, 2-heptynyl, 1-octynyl, 2-octynyl, 1-nonyl, 1-decynyl, and the like.
- cycloalkyl refers to cyclic saturated aliphatic groups and includes within its meaning monovalent (“cycloalkyl”), and divalent (“cycloalkylene”), saturated, monocyclic, bicyclic, polycyclic or fused polycyclic hydrocarbon radicals having from 3 to 10 carbon atoms, eg, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms.
- Examples of cycloalkyl groups include but are not limited to cyclopropyl, 2-methylcyclopropyl, cyclobutyl, cyclopentyl, 2-methylcyclopentyl, 3-methylcyclopentyl, cyclohexyl, and the like.
- cycloalkenyl refers to cyclic unsaturated aliphatic groups and includes within its meaning monovalent (“cycloalkenyl”) and divalent (“cycloalkenylene”), monocyclic, bicyclic, polycyclic or fused polycyclic hydrocarbon radicals having from 3 to 10 carbon atoms and having at least one double bond, of either E, Z, cis or trans stereochemistry where applicable, anywhere in the alkyl chain.
- Examples of cycloalkenyl groups include but are not limited to cyclopropenyl, cyclopentenyl, cyclohexenyl, and the like.
- heterocycloalkyl includes within its meaning monovalent (“heterocycloalkyl”) and divalent (“heterocycloalkylene”), saturated, monocyclic, bicyclic, polycyclic or fused hydrocarbon radicals having from 3 to 10 ring atoms wherein 1 to 5 ring atoms are heteroatoms selected from O, N, NH, or S. Examples include pyrrolidinyl, piperidinyl, quinuclidinyl, azetidinyl, morpholinyl, tetrahydrothiophenyl, tetrahydrofuranyl, tetrahydropyranyl, and the like.
- heterocycloalkenyl includes within its meaning monovalent (“heterocycloalkenyl”) and divalent (“heterocycloalkenylene”), saturated, monocyclic, bicyclic, polycyclic or fused polycyclic hydrocarbon radicals having from 3 to 10 ring atoms and having at least 1 double bond, wherein from 1 to 5 ring atoms are heteroatoms selected from O, N, NH or S.
- heteroaryl and variants such as “heteroaryl” or “heteroarylene” as used herein, includes within its meaning monovalent (“heteroaryl”) and divalent (“heteroarylene”), single, polynuclear, conjugated and fused aromatic radicals having 6 to 20 atoms wherein 1 to 6 atoms are heteroatoms selected from O, N, NH and S.
- heteroaryl monovalent
- heteroarylene divalent
- heteroarylene single, polynuclear, conjugated and fused aromatic radicals having 6 to 20 atoms wherein 1 to 6 atoms are heteroatoms selected from O, N, NH and S.
- examples of such groups include pyridyl, 2,2'-bipyridyl, phenanthrolinyl, quinolinyl, thiophenyl, and the like.
- halogen or variants such as “halide” or “halo” as used herein refers to fluorine, chlorine, bromine and iodine.
- heteroatom or variants such as “hetero-” as used herein refers to O, N, NH and S.
- alkoxy refers to straight chain or branched alkyloxy groups. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, sec-butoxy, tert- butoxy, and the like.
- amino refers to groups of the form -NR 4 R b wherein R a and R b are individually selected from the group including but not limited to hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted aryl groups.
- aromatic group or variants such as “aryl” or “arylene” as used herein refers to monovalent (“aryl”) and divalent (“arylene”) single, polynuclear, conjugated and fused residues of aromatic hydrocarbons having from 6 to 10 carbon atoms. Examples of such groups include phenyl, biphenyl, naphthyl, phenanthrenyl, and the like.
- aralkyl as used herein, includes within its meaning monovalent (“aryl”) and divalent (“arylene”), single, polynuclear, conjugated and fused aromatic hydrocarbon radicals attached to divalent, saturated, straight and branched chain alkylene radicals.
- heteroaryl includes within its meaning monovalent (“heteroaryl”) and divalent (“heteroarylene”), single, polynuclear, conjugated and fused aromatic hydrocarbon radicals attached to divalent saturated, straight and branched chain alkylene radicals.
- optionally substituted means the group to which this term refers may be unsubstituted, or may be substituted with one or more groups independently selected from alkyl, alkenyl, alkynyl, thioalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, halo, carboxyl, haloalkyl, haloalkynyl, hydroxyl, alkoxy, thioalkoxy, alkenyloxy, haloalkoxy, haloalkenyloxy, nitro, amino, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroheterocyclyl, alkylamino, dialkylamino, alkenylamine, alkynylarnino, acyl, alkenoyl, alkynoyl, acylamino, diacylamino, acyloxy, alkylsulfonyloxy,
- co-antioxidant refers to inhibitors of lipoprotein lipid oxidation that are effective in blood vessel walls in vivo.
- a detailed description of the characterization of co- antioxidants is given in Journal American Chemical Society 1993, 115:6029-6044; Proceedings of the National Academy of Sciences USA 1993, 90:45-49; and Journal of Biological Chemistry 1995,270:5756-5763 which are incorporated herein by reference.
- Co-antioxidants differ from classic "antioxidants” or "radical scavengers” in that the former prevent the pro-oxidant activity of ⁇ -tocopherol in the peroxidation of lipoprotein lipids by inhibiting the process of tocopherol-mediated peroxidation.
- Co-antioxidants may be routinely identified by in vivo analysis of the effects of the inhibitors in blood vessel walls using a suitable animal model such as Watanabe Heritable Hyperlipidemic (WHHL) rabbits, apoE-/- mice, or cholesterol-fed balloon-injured New Zealand White rabbits.
- WHHL Watanabe Heritable Hyperlipidemic
- co- antioxidants may be identified through in vitro assays which are capable of demonstrating such efficacy, such as for example assays described in J Lipid Research 1996, 37:853-867 which is incorporated herein by reference.
- the term “vessels” includes all fluid or air filled vessels of the body which are lined with endothelium, including for example, blood vessels, such as arteries.
- functional endothelium refers to blood vessel containing endothelial cells that to suitable agonists by the production of nitric oxide that itself acts on the underlying smooth muscle cells by activating soluble guanylyl cyclase with resultant formation of cyclic guanosine monophosphate (cGMP) and relaxation of the blood vessel.
- administering and variations of that term including “administer” and “administration”, includes contacting, applying, delivering or providing a compound or composition of the invention to an organism, or a surface by any appropriate means.
- vertebrate includes humans and individuals of any species of social, economic or research importance including but not limited to members of the genus ovine, bovine, equine, porcine, feline, canine, primates (including human and non-human primates), rodents, murine, caprine, leporine, and avian.
- treatment refers to any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever.
- terapéuticaally effective amount and “diagnostically effective amount”, include within their meaning a sufficient but non-toxic amount of a compound or composition of the invention to provide the desired therapeutic or diagnostic effect.
- the exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered, the mode of administration, and so forth. Thus, it is not possible to specify an exact “effective amount”. However, for any given case, an appropriate "effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.
- the present invention relates to compounds, compositions and methods for treating cardiovascular disorders.
- the present invention relates to compounds of general Formula (I):
- Y is selected from S, Se, S(O) and S(O) 2 ;
- A comprises one or more groups selected from optionally substituted Ci -6 alkylene, optionally substituted C 2-6 alkenylene; optionally substituted C 3-10 cycloalkylene; and optionally substituted arylene; n is 0 or 1 ;
- Z is selected from optionally substituted aryl and optionally substituted heteroaryl, optionally substituted alkyl, optionally substituted alkoxy, and NR 13 R 14 ;
- R , R 2 , R 3 , R 4 , and R 5 may be the same or different and are independently selected from the group consisting of hydrogen, halogen, hydroxyl, thiol, -NR 13 R 14 , nitro, cyano, optionally substituted Ci -I0 alkyl, optionally substituted C- 2 - 10 alkenyl, optionally substituted C2- 10 alkynyl, optionally substituted C 3 _io cycloalkyl, optionally substituted s aryl, optionally substituted aryl(Ci -6 alkyl), optionally substituted (Ci -6 alkyl)aryl, optionally substituted heteroaryl, optionally substituted C 3 . 10 heterocycloalkyl, C(O)R 11 , OR 12 , SR 12 , CH 2 OR
- R 11 is selected from OH, Ci -6 alkyl, and C 1-6 alkenyl
- R is selected from the group consisting of hydrogen, optionally substituted Ci -I0 0 alkyl, optionally substituted C 2-1O alkenyl, optionally substituted C 2-I0 alkynyl, optionally substituted C 3-J0 cycloalkyl, optionally substituted aryl, -C(O)(Ci -6 )alkyl-C ⁇ 2 R 15 , -C(O)(C 2 . 6 )alkenyl-CO 2 R 15 , and -C(O)NR 13 R 14 ;
- R 13 and R 14 may be the same or different and are individually selected from hydrogen, Ci -6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, C 3-6 heterocycloalkyl, s aryl, (C 1-6 )alkylaryl, and heteroaryl; and
- R 15 is H or C M alkyl; and salts thereof.
- the spacer group "A” is present. In another embodiment when n is O, the spacer group "A” is absent.
- Q In one embodiment the compound is a compound of Formula (Ia):
- A comprises one or more groups selected from optionally substituted Ci -6 alkylene, optionally substituted C 2-6 alkenylene; and optionally substituted C 3 . 10 cycloalkylene; n is O or 1 ; 0 R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 may be the same or different and are independently selected from the group consisting of hydrogen, halogen, hydroxyl, thiol, -NR 11 R 12 , nitro, cyano, optionally substituted Ci -10 alkyl, optionally substituted C 2-I0 alkenyl, optionally substituted C 2-I0 alkynyl, optionally substituted C 3-1 O cycloalkyl, optionally substituted aryl, optionally substituted aryl(C 1-6 alkyl), optionally substituted (C 1-6 alkyl)aryl, optionally substituted heteroaryl, optionally substituted C 3 . 10 heterocycloalky
- R 11 is selected from OH, Ci -6 alkyl, and C 1-6 alkenyl
- R 12 is selected from the group consisting of hydrogen, optionally substituted Ci.jo alkyl, optionally substituted C 2-10 alkenyl, optionally substituted C 2-10 alkynyl, optionally substituted C 3- Io cycloalkyl, optionally substituted aryl, -C(O)(Ci- 6 )alkyl-CO 2 R 15 , -C(O)(C 2-6 )alkenyl-CO 2 R 15 , and -C(O)NR 13 R 14 ;
- R 13 and R 14 may be the same or different and are individually selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, C 3-6 heterocycloalkyl, aryl, (Ci -6 )alkylaryl, and heteroaryl; and
- R 15 is H or Ci -4 alkyl; and salts thereof.
- X is selected from S, Se, S(O) and S(O) 2 ;
- Y is selected from S, Se, S(O) and S(O) 2 ; and R'-R 10 are as defined for Formula (Ia).
- X is S and Y is Se. In another embodiment, X is Se and Y is S. In a further embodiment, X is Se and Y is Se.
- the optional substituents are independently selected from OH, SH, halogen, C 1-4 alkyl, C 1-4 alkenyl, 0-(C 1-4 alkyl), S-(C 1-4 alkyl), cyano, amino, CO 2 H and C(O)-O(Ci -6 )alkyl.
- R and R may be the same or different.
- R and R are independently selected from hydroxyl, thiol, -NR 13 R 14 , cyano, Ci -6 alkyl, C 2-6 alkenyl, OR 12 , C(O)OR 12 and C(O)NR 13 R 14 , wherein R 12 , R 13 and R 14 are as defined above for Formula (I).
- R 3 and R 8 are independently selected from hydroxyl, O-malonate, O-succinate, O-glutarate, O-adipate, O-maleate and O-fumarate.
- the compound has the formula: wherein each R 12 is independently selected from hydrogen, Ci -1O alkyl and -C(O)(Ci- 6 )alkyl- CO 2 R 15 ;
- R 15 is selected from hydrogen and C 1-6 alkyl
- R 2 , R 4 , R 7 and R 9 are independently selected from methyl, ethyl, propyl, isopropyl, butyl, 1-methylpropyl, 2-methylbutyl, tert-butyl, pentyl, 2-methylpentyl, 3-methylpentyl and hexyl.
- compounds according to the present invention may comprise an optionally substituted phenyl ring linked to an aromatic or alkyl group by a spacer. In some embodiments, compounds according to the present invention comprise an optionally substituted phenyl ring linked to an aromatic group by a spacer.
- the spacer comprises two groups selected from selenium, sulfur, S(O) and S(O) 2 .
- the spacer may further optionally comprise an alkylene, alkenylene, cycloalkylene or arylene moiety between the respective selenium, sulfur, S(O) and S(O) 2 groups.
- the spacer may comprise two adjacent selenium groups, a selenium group adjacent a sulfur group, a selenium group adjacent an S(O) group, and the like.
- respective selenium, sulfur, S(O) and S(O) 2 groups may be linked, for example, via a linear or branched carbon chain.
- the terminal aromatic residues of compounds of Formula (I), i.e, the optionally substituted phenyl ring and "Z" group, may be the same or different.
- the aromatic residues may be substituted or unsubstituted.
- the aromatic residues are each an optionally substituted phenyl ring.
- the aromatic residues may be substituted with one or more hydroxyl group(s).
- the aromatic residues may be substituted, respectively, with 1 , 2, 3 or 4 alkyl group(s), wherein the alkyl group(s) may be the same or different.
- one or more hydroxyl group(s) may be functionalised, eg, as an ether, ester, or carbamate group.
- compounds of Formula (I) may undergo intra-cellular reduction to a substituted or unsubstituted thioaryl compound, or a substituted or unsubstituted selenoaryl compound.
- compounds of Formula (I) may undergo intra-cellular reduction to substituted or unsubstituted mercaptophenol, or a substituted or unsubstituted selenophenol compound.
- Compounds of Formula (I) may be prepared by methods known to those skilled in the art. Suitable methods are generally described, for example, and intermediates thereof are described, for example, in Houben-Weyl, Methoden der Organischen Chemie; J. March, Advanced Organic Chemistry, 4 th Edition (John Wiley & Sons, New York, 1992); D. C. Liotta and M. Volmer, eds, Organic Syntheses Reaction Guide (John Wiley & Sons, Inc., New York, 1991); R. C. Larock, Comprehensive Organic Transformations (VCH, New York, 1989), H. O. House, Modern Synthetic Reactions 2 nd Edition (W. A. Benjamin, Inc., Menlo Park, 1972).
- R 1 , R 2 , R 4 , R 5 and R 12 are as defined above for Formula (I), and R and R 1 are the same or different and are H or an optional substituent as defined herein.
- R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 9 , R 10 and R 12 are as defined above for Formula (I).
- X is S or Se
- Y is S or Se
- R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 9 , R 10 and R 12 are as defined above for Formula (I); and LG and LG' may be the same or different and are each a leaving group.
- suitable leaving groups include, but are not limited to, halides, mesylate, tosylate, trifiate, etc.
- X is S or Se
- Y is S or Se
- R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 9 , R 10 and R 12 are as defined above for Formula (I); m is 0, 1 or 2; and n is 0, 1 or 2.
- counterions include, but are not limited to halides (such as Cl “ , Br “ , I “ ), carboxylates, citrate, acetate, succinate, CF 3 CO 2 " , tosylate, nitrate, BF 4 " , PF 6 “ , and OH “ .
- the counterion(s) may be varied using techniques known to those skilled in the art, including for example, ion exchange and crystallisation.
- Formula (I) should be understood to include, for example, E, Z, cis, trans, (R), (S), (L), (D), (+), and/or (-) forms of the compounds, as appropriate in each case.
- Therapy should be understood to include, for example, E, Z, cis, trans, (R), (S), (L), (D), (+), and/or (-) forms of the compounds, as appropriate in each case.
- Compounds in accordance with the present invention may have in vivo activity associated with one or more of promotion of re-endothelialization, inhibition of smooth muscle cell proliferation, anti-inflammatory activity such as the inhibition of accumulation of pro-inflammatory cells in the affected vessel wall, and induction of heme oxygenase- 1.
- heme oxygenase is a target of compounds of formula (I).
- the heme oxygenase target may be heme oxygenase-1 (HO-I).
- the present invention relates to a method of treating a cardiovascular disorder in a vertebrate, said method comprising administering to said vertebrate an effective amount of a compound according to Formula (I) as defined herein.
- Another aspect of the invention relates to a compound of Formula (I) when used for the treatment of a cardiovascular disorder in a vertebrate.
- the vertebrate is a human.
- a further aspect of the invention relates to the use of a compound of Formula (I) for the manufacture of a medicament for treating a cardiovascular disorder.
- the cardiovascular disorder may be atherosclerosis.
- the cardiovascular disorder may be restenosis.
- a compound of Formula (I) may be administered together with a co-antioxidant.
- Pre-treatment with one or more compounds of Formula (I) may be performed.
- one or more compounds of Formula (I) may be administered 1, 2, 3, 4, 5, 6 or 7 days prior to an intervention, for example, prior to treating restenosis.
- Compounds of Formula (I) may be administered prior to or after angioplasty, PTCA or BA.
- Compounds of Formula (I) may be administered after denudation (removal of endothelial cells).
- compounds of Formula (I) may be administered prior to (e.g., 1, 2, 3, 4, 5, 6 or 7 days prior to) a denudation event, for example, prior to balloon angioplasty.
- Atherosclerosis e.g., 1, 2, 3, 4, 5, 6 or 7 days prior to
- Atherosclerosis i.e., the clogging of arteries, is characterized by the accumulation of cholesterol deposits in macrophages in large- and medium-sized arteries. This deposition leads to a proliferation of certain cell types within the arterial wall that gradually impinge upon the vessel lumen and impede blood flow. This process may be quite insidious lasting for decades until an atherosclerotic lesion, through physical forces from blood flow, becomes disrupted and deep arterial wall components are exposed to flowing blood, leading to thrombosis and compromised oxygen supply to target organs such as the heart and brain.
- heart attack and stroke The loss of heart and brain function as a result of reduced blood flow is termed heart attack and stroke, respectively, and these two clinical manifestations of atherosclerosis are often referred to as coronary artery disease and cerebrovascular disease. Coronary artery disease and cerebrovascular disease are commonly referred to by the collective term, cardiovascular disease.
- cardiovascular disease With respect to the underlying pathology of atherosclerosis, there are a number of environmental and genetic "cardiovascular risk” factors that have proven predictive of the incidence of cardiovascular disease. Traits that are strongly and consistently associated with cardiovascular disease in a manner independent of other traits include age, gender, smoking, obesity, hypertension, diabetes mellitus, and serum cholesterol.
- LDL low-density lipoprotein
- Atherosclerosis manifests itself histological as arterial lesions known as plaques that have been extensively characterized into 6 major types of lesions that reflect the early, developing, and mature stages of the disease. In lesion-prone arterial sites, adaptive thickening of the intima is among the earliest histological changes. As macrophages accumulate lipid, type II lesions form as nodular areas of lipid deposition that are also known as "fatty streaks" and these represent lipid-filled macrophages (i.e., foam cells). Continued foam cell formation and macrophage necrosis can produce type III lesions that contain small extra-cellular pools of lipid.
- Types II and III lesions are readily apparent through the use of fat-soluble dyes that stain cholesterol esters accumulated in macrophages and the extra-cellular space. These early lesions are often evident by age 10 and can occupy as much as 1/3 of the aortic surface by the third decade. Developing lesions represent the next two types of lesions and are characterized by significant areas of extra-cellular lipid that represents the "core" of the atherosclerotic lesion.
- Type IV lesions are defined by a relatively thin tissue separation of the lipid core from the arterial lumen, whereas type V lesions exhibit fibrous thickening of this structure, also known as the lesion "cap.” These type IV and V lesions can be found initially in areas of the coronary arteries, abdominal aorta, and some aspects of the carotid arteries in the third to fourth decade of life.
- Mature type VI lesions exhibit architecture that is more complicated and characterized by calcified fibrous areas with visible ulceration. These types of lesions, commonly referred to as atherosclerotic plaques, are often associated with clinical symptoms or arterial embolization. It was once thought that end-organ damage and infarction was due to gradual advancement of these lesions, but it is now known that the processes involved in precipitating heart attack and stroke are considerably more complex. Plaques contain a central lipid core that is most often hypo-cellular and may even include crystals of cholesterol that have formed in the aftermath of necrotic foam cells. In this late stage of lesion development, residual foam cells may be difficult to see, but have often left the core with an abundant quantity of tissue factor, an important activator of the clotting cascade.
- This lipid core is separated from the arterial lumen by a fibrous cap and myeloproliferative tissue that consists of extra-cellular matrix and smooth muscle cells.
- the junction between the cap and the morphologically more normal area of artery is known as the "shoulder" region of the atherosclerotic plaque. This area is typically more cellular than other areas of the plaque and may contain a variable composition of smooth muscle cells, macrophages, and even T-cells.
- the shoulder region is most prone to rupture and may even contain evidence of previously healed fissures.
- Mature atherosclerotic plaques can be categorized as either stable or vulnerable to rupture.
- Stable plaques tend to be characterized by a smaller lipid core, a thick fibrous cap, and shoulder regions with few inflammatory cells, whereas vulnerable plaques contain considerable lipid in their core, a thin fibrous cap, and a robust population of macrophages and T-cells in their shoulder regions.
- vulnerable plaques may be weaker structurally and more likely to rupture in response to the physical forces of flowing blood. This contention is supported by experimental data linking an increased content of macrophages in lesions to structural weakness.
- Atherosclerosis is characterized by LDL deposition in the arterial wall, a process that is stimulated by environmental and genetic factors such as tobacco use, diabetes and hypertension.
- This LDL deposition occurs primarily within macrophages and ultimately begets the formation of well-defined lesions in the arterial intima.
- the accumulation of macrophages reflects the inflammatory component of atherosclerosis.
- Such lesions then develop and macrophage-rich lesions are prone to rupture and, as a consequence, can precipitate the clinical events such as heart attack and stroke.
- the precipitation of acute vascular events in atherosclerosis involves processes that go beyond plaque vulnerability and rupture.
- endothelium serves as the interface between the vascular wall and flowing blood.
- the endothelium regulates a number of important processes such as vascular tone, platelet adhesion, and leukocyte transit into tissues and the vascular wall.
- the principal factors released by the endothelium that regulate vascular homeostasis on a moment-by-moment basis are prostacyclin, leukotrienes, and nitric oxide.
- Endothelial production of nitric oxide is important in the regulation of vascular tone, arterial pressure, platelet adhesion, and leukocyte trafficking, as mice lacking endothelial nitric oxide synthase (the enzyme that generates nitric oxide in endothelial cells) exhibit spontaneous hypertension, defective vascular remodeling, enhanced vascular thrombosis and leukocyte interactions.
- the "classic" model of bioactivity of nitric involves its binding to the heme group of guanylate cyclase in target cells (e.g., platelets, smooth muscle cells) to increase cellular cGMP and activate cGMP -dependent protein kinase thereby effecting nitric oxide-mediated vasodilation and platelet inhibition.
- endothelial dysfunction refers to a loss of normal homeostatic functions ⁇ e.g., vasodilatation, platelet inhibition). This condition often occurs early in the course of atherosclerosis with one important manifestation being a reduction in the bioactivity of endothelium-derived nitric oxide. Although the loss of nitric oxide bioactivity is not the only manifestation of endothelial dysfunction, it is an independent predictor of future cardiovascular events in patients with atherosclerosis. There are many potential reasons for impaired nitric oxide bioactivity. These range from inadequate nitric oxide production to nitric oxide degradation or an inadequate response to nitric oxide.
- Oxidized LDL is pro-atherogenic by promoting the accumulation of lipids in cells, disturbing the normal vasoregulatory function of endothelial cells, being cytotoxic to endothelial and other cells, mediating the generation of a necrotic core, promoting the recruitment of inflammatory cells, and by inducing thrombogenic tissue factor and the expression of adhesion molecules on endothelial cells. Accumulation of lipid by macrophages can induce the secretion of matrix metalloproteinases and cytokines (e.g., interleukin-8). These thrombotic, adhesive and inflammatory properties of oxidized LDL may be critical for disease progression (whether episodic or continuous) and likely involves episodic damage to the endothelium.
- cytokines e.g., interleukin-8
- endothelial cell injury can itself trigger or contribute to the development of atherosclerosis.
- Restenosis Re-endothelialization is the process whereby an intact endothelial cell layer grows back over a previously denuded area of the blood vessel. Commonly, the re-growth of endothelial cells is initiated at branching points of smaller vessels and cell growth then progresses into the larger vessel. Re-endothelialization is not identical to the process of endothelial cell proliferation. The former is limited to previously damaged areas, whereas endothelial cell proliferation is a more general process required, for instance in angiogenesis which itself can promote rather than inhibit atherosclerosis (Circulation 1999; 99:1726-1732).
- Re-endothelialization is particularly important for the prevention of restenosis after BA (where the endothelial cell layer of large areas of vessels become removed).
- vascular endothelial growth factor a growth factor that specifically promotes the growth of endothelial cells
- Re-endothelialization may also be important in atherosclerosis where injury to endothelial cells occurs, for example as a result of the accumulation and toxic properties of oxidized LDL.
- the endothelium is a cell layer that lines internal body surfaces such as in the heart, blood and lymphatic vessels and other fluid filled cavities and glands. Endothelium must be induced to re-grow if the integrity of the surface is to be maintained. The integrity of endothelium in blood vessels is of central importance to vascular homeostasis in general and processes related to restenosis and atherosclerosis in particular.
- the latter include, but are not limited to, the control of vascular tone via endothelium-dependent relaxing factor (i.e., nitric oxide produced by endothelial nitric oxide synthase), the deposition of matrix by, and proliferation of, smooth muscle cells, the infiltration of the vessel wall by inflammatory blood cells, and the control of coagulation and platelet aggregation.
- Smooth muscle cell proliferation is often implicated in restenosis. Prevention of the proliferation has been effective in inhibiting the progress of restenosis. However, the direct general prevention of smooth muscle cell proliferation may not always be beneficial, as for instance it may decrease the stability of plaques and thereby promote clinical events by promoting plaque rupture. Promotion of re-endothelialization may overcome gross endothelial dysfunction
- Methods of promoting re-endothelialization may also extend to methods of treating conditions associated with endothelial dysfunction, for example, in the control of vascular tone via endothelium-dependent relaxing factor (i.e., nitric oxide produced by endothelial nitric oxide synthase), the deposition of matrix by, and proliferation of, smooth muscle cells, the infiltration of the vessel wall by inflammatory blood cells, and the control of coagulation and platelet aggregation.
- endothelium-dependent relaxing factor i.e., nitric oxide produced by endothelial nitric oxide synthase
- the present invention also relates to pharmaceutical compositions comprising at least one compound of Formula (I) as defined herein together with pharmaceutically acceptable excipients, adjuvants and/or diluents.
- the invention relates to a process for preparing a pharmaceutical composition
- a process for preparing a pharmaceutical composition comprising homogeneously mixing a compound of Formula (I) with a pharmaceutically acceptable adjuvant, diluent and/or carrier.
- compound(s) of Formula (I) when used for the treatment or prevention of cardiovascular diseases or disorders, may be administered alone.
- the compounds may be administered as a pharmaceutical or veterinary formulation comprising one or more compound(s) of Formula (I).
- the compound(s) may be present as suitable salts, including pharmaceutically acceptable salts.
- the compounds of Formula (I) may be used in combination with other known agents and treatment regimes.
- the compounds of Formula (I) may be used in combination with other agent(s) used for treating cardiovascular disease.
- agent(s) may include lipid-lowering drugs such as statins (e.g., simvastatin, pravastatin, lovostatin, and the like), blood pressure-lowering drugs such as Angiotensin Converting Enzyme (ACE) inhibitors (e.g., perindopril, ramipril, etc), beta blockers, diuretics, calcium channel blockers, etc, and agents which promote induction of heme-oxygenase 1 (HO-I).
- statins e.g., simvastatin, pravastatin, lovostatin, and the like
- blood pressure-lowering drugs such as Angiotensin Converting Enzyme (ACE) inhibitors (e.g., perindopril, ramipril, etc), beta blockers, diuretics, calcium channel blockers, etc, and agents which promote induction of heme-oxygenase 1 (HO-I).
- ACE Angiotensin Converting Enzyme
- Combinations of active agents, including compounds of the invention, may be synergistic.
- salt By pharmaceutically acceptable salt it is meant those salts which, within the scope of sound medical judgement, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art.
- suitable pharmaceutically acceptable salts of compounds according to the present invention may be prepared by mixing a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid, or citric acid with the compounds of the invention.
- a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid, or citric acid.
- Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, campliorsulfonate, citrate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate
- alkali or alkaline earth metal salts include sodium, lithium potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, triethanolamine and the like.
- Convenient modes of administration include injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, topical creams or gels or powders, or rectal administration.
- the formulation and/or compound may be coated with a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the therapeutic activity of the compound.
- the compound may also be administered parenterally or intraperitoneally.
- Dispersions of compounds according to the invention may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, pharmaceutical preparations may contain a preservative to prevent the growth of microorganisms.
- Pharmaceutical compositions suitable for injection include sterile aqueous solutions
- the composition is stable under the conditions of manufacture and storage and may include a preservative to stabilise the composition against the contaminating action of microorganisms such as bacteria and fungi.
- the compound(s) of the invention may be administered orally, for example, with an inert diluent or an assimilable edible carrier.
- the compound(s) and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into an individual's diet.
- the compound(s) may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
- such compositions and preparations may contain at least 1% by weight of active compound.
- the percentage of the compound(s) of formula (I) in pharmaceutical compositions and preparations may, of course, be varied and, for example, may conveniently range from about 2% to about 90%, about 5% to about 80%, about 10% to about 75%, about 15% to about 65%; about 20% to about 60%, about 25% to about 50%, about 30% to about 45%, or about 35% to about 45%, of the weight of the dosage unit.
- the amount of compound in therapeutically useful compositions is such that a suitable dosage will be obtained.
- pharmaceutically acceptable carrier is intended to include solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.
- compositions according to the present invention may also be incorporated into the compositions according to the present invention. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
- Dosage unit form refers to physically discrete units suited as unitary dosages for the individual to be treated; each unit containing a predetermined quantity of compound(s) is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
- the compound(s) may be formulated for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in an acceptable dosage unit. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
- the carrier may be an orally administrable carrier.
- Another form of a pharmaceutical composition is a dosage form formulated as enterically coated granules, tablets or capsules suitable for oral administration. Also included in the scope of this invention are delayed release formulations.
- prodrug is an inactive form of a compound which is transformed in vivo to the active form.
- Suitable prodrugs include esters, phosphonate esters etc, of the active form of the compound.
- the compound may be administered by injection.
- the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
- the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
- Prevention of the action of microorganisms can be achieved by including various anti-bacterial and/or anti-fungal agents. Suitable agents are well known to those skilled in the art and include, for example, parabens, chlorobutanol, phenol, benzyl alcohol, ascorbic acid, thimerosal, and the like. In many cases, it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminium monostearate and gelatin.
- Sterile injectable solutions can be prepared by incorporating the analogue in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilisation.
- dispersions are prepared by incorporating the analogue into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
- Tablets, troches, pills, capsules and the like can also contain the following: a binder such as gum gragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin ' or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
- a binder such as gum gragacanth, acacia, corn starch or gelatin
- excipients such as dicalcium phosphate
- a disintegrating agent such as corn starch, potato starch, alginic acid and the like
- a lubricant such as magnesium stearate
- a sweetening agent such as sucrose, lactose or saccharin ' or a flavouring agent such as peppermint, oil of
- tablets, pills, or capsules can be coated with shellac, sugar or both.
- a syrup or elixir can contain the analogue, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
- any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
- the analogue can be incorporated into sustained-release preparations and formulations.
- the pharmaceutical composition may further include a suitable buffer to minimise acid hydrolysis.
- suitable buffer agent agents are well known to those skilled in the art and include, but are not limited to, phosphates, citrates, carbonates and mixtures thereof.
- Single or multiple administrations of the pharmaceutical compositions according to the invention may be carried out.
- One skilled in the art would be able, by routine experimentation, to determine effective, non-toxic dosage levels of the compound and/or composition of the invention and an administration pattern which would be suitable for treating the diseases and/or infections to which the compounds and compositions are applicable.
- an effective dosage per 24 hours may be in the range of about 0.0001 mg to about 1000 mg per kg body weight; for example, about 0.001 mg to about 750 mg per kg body weight; about 0.01 mg to about 500 mg per kg body weight; about 0.1 mg to about 500 mg per kg body weight; about 0.1 mg to about 250 mg per kg body weight; or about 1.0 mg to about 250 mg per kg body weight.
- an effective dosage per 24 hours may be in the range of about 1.0 mg to about 200 mg per kg body weight; about 1.0 mg to about 100 mg per kg body weight; about 1.0 mg to about 50 mg per kg body weight; about 1.0 mg to about 25 mg per kg body weight; about 5.0 mg to about 50 mg per kg body weight; about 5.0 mg to about 20 mg per kg body weight; or about 5.0 mg to about 15 mg per kg body weight.
- an effective dosage may be up to about 500mg/ni2.
- an effective dosage is expected to be in the range of about 25 to about 500mg/m2, about 25 to about 350mg/m2, about 25 to about 300mg/m2, about 25 to about about 50 to about 250mg/m2, and about 75 to about 150mg/m2.
- a compound of Formula (I) may be administered in an amount in the range from about 100 to about 1000 mg per day, for example, about 200 mg to about 750 mg per day, about 250 to about 500 mg per day, about 250 to about 300 mg per day, or about 270 mg to about 280 mg per day.
- Probucol inhibits atherosclerosis in apolipoprotein E gene knock-out mice without inhibition of lipoprotein oxidation in the vessel wall
- mice Male C57BL/6J mice, homozygous for the disrupted apoE gene (apoE-/-) and originally purchased from Jackson Laboratories (Bar Harbor, ME), were used at 8-10 weeks of age and then fed for 24 weeks ad libitum a high fat diet containing 21.2 (w/w) fat and 0.15% (w/w) cholesterol (specifications of the Harlan Teklad diet TD88137), without (controls, 103 mice) or with probucol (1% w/w, 87 mice), as described in Arteriosclerosis Thrombosis and Vascular Biology 2000, 20:e26-e33. The local animal ethics committee approved the study. Aortic sampling for biochemical and histological analyses
- sections at the sinus were taken ⁇ 200 ⁇ m from the first appearance of the leaflets. Sections at the thoracic and abdominal aorta were taken at the branch point of the 3 rd pair of intra-costal arteries and the celiac artery, respectively.
- F 2 -isoprostanes For F 2 -isoprostanes, the thawed aortic segment ( ⁇ 20 mg wet weight) was blotted dry, weighed and F 2 -isoprostanes analyzed by electron capture negative ionization GC/MS after solid-phase extraction and HPLC purification as described in Analytical Biochemistry 1999, 268:117-125, using [D 4 ]-8-iso-prostaglandin F 2 ⁇ (Cayman Chemical) as internal standard. For arachidonate, phospholipids were separated by thin-layer chromatography, the fatty acid methyl esters then prepared and analyzed by GLC, as described in American Journal of Clinical Nutrition 2000, 71:1085-1094.
- the ability of probucol to simultaneously promote and inhibit atherosclerosis provides an experimental model to directly relate the extent of lipoprotein lipid oxidation and atherogenesis in different aortic segments of the same animal. To do. this, the concentrations of the non-oxidized lipids, C and cholesterylesters (CE, defined as the sum of Cl 8:2 plus cholesterylarachidonate, C20:4), and the lipid-soluble antioxidant ⁇ -tocopherol as measures of lipoprotein lipid accumulation were determined. For control and probucol-treated animals, lesions at the sinus contained more C per protein than respective lesions at the arch and thoracic/abdominal aorta (Table 1).
- FIG. 2 is a graphic representation of these results, with data from probucol-treated mice expressed relative to that of control animals for each of the three sites.
- probucol decreased the concentrations of C ( Figure 2A), CE ( Figure 2B) and ⁇ -tocopherol (Figure 2C) in the arch and descending aorta, in parallel with inhibition of disease ( Figure 1).
- probucol did not increase the content of C ( Figure 2A), CE ( Figure 2B) and ⁇ -tocopherol ( Figure 2C) at the sinus.
- Table 1 Total cell density, macrophage and extra-cellular matrix content in aortic sinus lesions of probucol-treated and control apoE-/- mice
- Lesion data show mean ⁇ SEM from 17 mice per group.
- Biochemical data show mean ⁇ SD from four separate pools each containing 19 (control) and 15 (probucol) respective sections, except for F 2 -isoprostanes that show mean ⁇ SD often individual sections.
- CE represents Cl 8:2 plus C20:4.
- a ' b Significantly different from sinus and arch, respectively. Effect of probucol on lipid oxidation in atherosclerotic lesions at different sites
- CE-OOH were more abundant than 7KC and F2-isoprostanes (Table 1).
- tissue 7KC was not different at different sites, irrespective of whether data was standardized to protein or parent lipid.
- protein- and parent lipid-standardized concentrations of CE-OOH and F 2 -isoprostanes were decreased at the thoracic/abdominal site compared with aortic sinus (Table 1).
- Figure 3 compares the parent lipid-standardized content of oxidized lipids at the three sites in control versus probucol-treated mice.
- Probucol inhibits atherosclerosis in apolipoprotein E-/- mice via an anti-inflammatory activity
- Example 1 The materials and methods used were essentially as described under Example 1. In addition, for total cell numbers, nuclei were counted in hematoxylin and eosin-stained sections and expressed per lesion area. At the sinus, sections were taken ⁇ 200 ⁇ m from the first appearance of the leaflets. Sections at the thoracic and abdominal aorta were taken at the branch point of the 3 rd pair of intra-costal arteries and the celiac artery, respectively. For macrophages, 4 ⁇ m paraffin sections were deparaffmized, rehydrated and endogenous peroxidase quenched with 3% hydrogen peroxide (15 min).
- Enzymatic antigen retrieval was performed in trypsin (1 mg/mL) solutions pH 7.7 containing 4 raM CaCl 2 and 200 mM Tris for 30 min at 37 °C, followed by a 20 min incubation in 5% normal rabbit blocking serum.
- Sections were then incubated overnight in a humidified chamber and at 4 0 C with monoclonal rat-anti-mouse F4/80 antibody (Caltag Laboratories; dilution 1:20), followed by biotinylated rabbit anti-rat IgG (Vector Laboratories; dilution 1:200, 30 min), Vectorstain Elite ABC reagent (Vectorstain Elite ABC Kit, Vector Laboratories; 30 min), and 3,3'-diaminobenzedine substrate-chromogen (Dako Corporation) with counterstaining using Harris hematoxylin.
- monoclonal rat-anti-mouse F4/80 antibody Caltag Laboratories; dilution 1:20
- biotinylated rabbit anti-rat IgG Vector Laboratories; dilution 1:200, 30 min
- Vectorstain Elite ABC reagent Vectorstain Elite ABC Kit, Vector Laboratories; 30 min
- ECM Extra-cellular matrix
- the anti-atherosclerotic activity of probucol relates to the extent to which the drug is metabolized.
- probucol is metabolised to io probucol bisphenol and its oxidized form, probucol diphenoquinone, relates to the extent to which the drug inhibits atherosclerosis in apoE-/- mice, consistent with the notion that probucol is a pro-drug.
- the quantity of probucol, BP, and DPQ in aortic homogenates was determined by gradient reverse-phase high-pressure liquid chromatography (HPLC) as described in
- Probucol Probucol 24.4 ⁇ 8.1 7.1 ⁇ 1.6 a 4.9 ⁇ 1.0 a Probucol Probucol 3.9 ⁇ 1.3 1.7 ⁇ 0.4 a 2.5 ⁇ 1.0 metabolites Total Drug Probucol 28.3 ⁇ 9.4 8.9 ⁇ 2.0 a 7.5 ⁇ 1.9 a
- the data show mean ⁇ SD from four separate pools each containing 19 (control) and 15 (probucol) respective sections. a Significantly different from sinus. Importantly, when expressed relative to parent drug, the metabolites were significantly increased, and accounted for nearly one third of the drug, at the descending aorta (Figure 5B), where atherosclerosis was inhibited compared with the aortic sinus where disease was enhanced ( Figure 1). Together, the results show that increased metabolism of probucol was associated with protection against atherosclerosis in apoE-/- mice.
- probucol can be metabolised via a previously unrecognised pathway that yields bioactive intermediate(s) such as 4,4'-dithiobis(2,6-di- tert-butyl-phenol) (DTBP) that may contribute to vascular protection and anti-atherogenic activity.
- bioactive intermediate(s) such as 4,4'-dithiobis(2,6-di- tert-butyl-phenol) (DTBP)
- Probucol was obtained from Jucker Pharma (Stockholm, Sweden), and 3,3',5,5'-tetra-tert-butyl-4,4'-bisphenol (BP), 4,4'-dithiobis(2,6-di-tert-butyl-phenol) (DTBP) and (2,2'-azobis(2-amidino-propane)-hydrochloride (AAPH) from Polysciences (Warrington, PA).
- Authentic DPQ was prepared from BP as described under Example 3, and purified by gradient reversed-phase HPLC (see below). Acetylcholine, lead dioxide (PbO 2 ) and eerie ammonium nitrate (purity 98%, a source of Ce 4+ ) were obtained from Jucker Pharma (Stockholm, Sweden), and 3,3',5,5'-tetra-tert-butyl-4,4'-bisphenol (BP), 4,4'-dithiobis(2,6-di-tert-butyl
- DPBS phosphate buffered saline
- HOCl solutions of HOCl were prepared freshly before use by diluting reagent HOCl (Aldrich) into phosphate buffer (250 mM, pH 7.0) and standardizing with S 235 nm ⁇ 100 M ⁇ cm "1 (hypochlorous acid) and ⁇ 290 nm ⁇ 300 M -1 Cm "1 (hypochlorite), as described in Journal of Physical Chemistry 1966, 70:3798-3805.
- ring segments ⁇ 5 mm in length were mounted in a myobath system (World Precision Instruments, Sarasota, FL) containing 20 mL of Krebs solution aerated at 37 0 C with 5% CO 2 ( g ), and the dilatory response of half maximally norepinephrine pre- constricted rings to incremental doses of ACh (10 "9 -10 "5 mol/L) determined.
- reagent HOCl final concentration 400 ⁇ M was added to the Krebs solution and the rings incubated for 5 min prior to thorough washing, pre-constriction and assessment of vessel relaxation.
- aortic rings were incubated with probucol or DTBP for 10 min, washed thoroughly and then exposed to HOCl prior to assessing endothelium-dependent relaxation. A maximum of three consecutive sequences of constriction/relaxation were performed for each ring. Preparation of aortic homogenates: Aortic rings used in the vascular function studies were removed and immediately cut into small pieces, frozen in liquid nitrogen, and then pulverized and homogenized as described.
- probucol or DTBP
- the aqueous phase was analyzed for the content of sulfate anion (SO 4 2" ) by ion exchange chromatography (see below).
- Analytical analyses Probucol, DTBP, BP and DPQ were analyzed by gradient reversed- phase HPLC and quantified by peak area comparison with authentic standards. Under the conditions used, BP, DTBP, probucol and DPQ eluted at 12.5, 19.2, 20.8 and 28.4 min, respectively. Other oxidation products resolved by this HPLC system were also quantified by peak area comparison, using analytically pure samples obtained from semi- preparative (LC-18, 20 mm x 25 cm, 5 ⁇ m) fractionation of the reaction mixture.
- Ion exchange chromatography was performed on a Waters IC PAK-A column (4.6 x 50 mm x 10 ⁇ m) with an eluent containing sodium gluconate (0.32 g/L), boric acid (0.36 g/L), sodium tetraborate decahydrate (0.5 g/L), glycerol (5.0 mL/L), n-butanol (20 mL/L) and acetonitrile (120 mL/L) at a flow rate of 1.0 mL/min. Eluting anions were detected with a refractive index detector (limit of detection 10 ⁇ M), with sulfate anion eluting at ⁇ 18 min identified by comparison with an authentic standard.
- a refractive index detector limit of detection 10 ⁇ M
- Mass spectrometry Product masses were determined using electrospray ionization mass spectrometry (ESI-MS). Spectra were acquired using an API QStar Pulsar i hybrid tandem mass spectrometer (Applied Biosystems, Foster City CA). Samples (-10 pmol, 1 ⁇ L) were dissolved in water/acetonitrile (1 :4, v/v), loaded into nanospray needles (Proxeon, Denmark) and the tip positioned ⁇ 10 mm from the orifice. Nitrogen was used as curtain gas and a potential of -800 V applied to the needle. A Tof MS scan was acquired (m/z 50-2000, 1 s) and accumulated for ⁇ 1 min into a single file.
- ESI-MS electrospray ionization mass spectrometry
- Precursor masses determined from Tof MS scans were selected by Ql for MS-MS analysis. Nitrogen was used as collision gas and a collision energy chosen that reduced the intensity of the precursor ion by ⁇ 95%. Tandem mass spectra were accumulated into a single file for ⁇ 2 min (m/z 50-1250).
- Electronic spectroscopy Electronic spectra were measured with a Hitachi UV/Vis spectrophotometer. Spectra of authentic compounds and analytically pure oxidation products were obtained in ethanol (purity 99.7%) and maxima determined by manual peak picking.
- Aortas from probucol-fed rabbits contained probucol at ⁇ 100 pmol/mg protein ( Figure 6C), demonstrating the presence of the drug in this tissue. Similar to the situation with in vivo supplemented probucol, preincubation of aortic rings from control animals with increasing amounts of added probucol for 10 min followed by thorough washing also protected the vessels from HOCl- induced loss of response to ACh in a concentration dependent manner, with full protection seen with 100 ⁇ M of the drug (Figure 7). Rings pre-treated with 100 ⁇ M probucol responded to ACh by increased tissue content of cGMP ( Figure 7B), and contained ⁇ 400 pmol drug/mg protein ( Figure 7C).
- probucol As a phenol, probucol is known to scavenge 1-electron (Ie), Le,, radical oxidants, while little is known about its ability to scavenge 2-electron (2e) oxidants such as HOCl. Therefore, the oxidation of probucol by HOCl was examined. Reaction with HOCl resulted in the dose-dependent consumption of probucol as judged by HPLC ( Figure 8A).
- HOCl-induced oxidation of DTBP was examined.
- HOCl dose-dependently oxidized DTBP resulting in a near stoichiometric accumulation of the combined products ( Figure 8C) with a pattern similar to that for probucol ( Figure 8D).
- the aqueous phase of reaction mixtures containing probucol or DTBP oxidized with 5-mol equivalent HOCl also contained SO 4 2" at final concentrations of 139 ⁇ 14 or 204 ⁇ 43 nmol, respectively.
- aortic rings to which probucol was added in vitro subsequent treatment with HOCl significantly decreased .tissue probucol without substantial accumulation of DPQ; BP was not detected ( Figure 1 IB).
- Vascular endothelial cells overlying atherosclerotic lesions contain myeloperoxidase and proteins modified by its principle product HOCl, and blood vessels exposed to HOCl exhibit a defect in endothelium-derived ' NO bioactivity manifested as impaired endothelium-dependent arterial relaxation.
- Impaired endothelial function predicts the occurrence of vascular events and ' NO bioavailability is attenuated by irreversible chemical modification and/or decreased catalytic activity of eNOS. Oxidative reactions are increasingly implied in these processes.
- This study shows that probucol and DTBP scavenge HOCl and that DTBP is an intermediate during HOCl-mediated oxidation of probucol. Probucol and DTBP also react with other oxidants.
- Using Cu 2+ -ions Barnhart et al. ⁇ Journal of Lipid Research 1989, 30:1703-1710) described BP and DPQ as oxidation products of probucol, with a spiroquinone proposed as intermediate.
- the spiroquinone was not detected, independent of whether Cu 2+ , HOCl or other oxidants were employed, including PbO 2 used by Barnhart et al. ⁇ Journal of Lipid Research 1989, 30:1703-1710) to produce spiroquinone standard, and whether the HPLC conditions described here or by Barnhart et al. ⁇ Journal of Lipid Research 1989, 30:1703-1710) were used for detection of products. Notwithstanding this, DTBP was detected consistently and to an extent proportional to the yield of DPQ, independent of whether 2e- or le-oxidants were used (not shown).
- one chemically feasible pathway may be via a non-radical mechanism ( Figure 18).
- DTBP is formed via disproportionation of two molecules of thiosulfinate, or via coupling of the thiophenol and phenylsulfonic acid.
- HOCl oxidized by HOCl to yield the thiosulfonate 4 that oxidizes to the disulfone 6, which becomes hydrolysed to the corresponding sulfonic acid 2.
- the acid product 2 is then chlorinated by another molecule of HOCl to yield the chlorophenol 1 and 2HVSO 4 2" as by-product that was detected in HOCl-mediated oxidations of probucol and DTBP. Finally, the chlorophenol intermediate is converted to DQ. Irrespective of the precise mechanism of its formation, DQ is degraded in the presence of excess HOCl.
- DTBP but not BP
- DTBP has anti-atherosclerotic activity
- This example illustrates that the phenol moiety of probucol is not sufficient for anti- atherosclerotic activity, and that instead the sulfur moieties are required.
- this example illustrates that DTBP at 1/50* of the dose of probucol has anti-atherosclerotic protection comparable to that of probucol yet, unlike probucol, does not lower HDL- cholesterol.
- mice Four groups of male apoE-/- mice (8-10 weeks, Animal Resources Centre, Perth, Australia) were fed a high fat diet based on Harlan Teklad diet TD88137 ⁇ 1% (wt/wt) probucol (96% purity, a gift from AstraZeneca, Sweden),' 0.02% DTBP, or 0.02% BP (Polysciences, Warrington, PA) for 5 months. Tissue harvesting and analyses were done as described in Examples 1 and 3, using 15 and 10 mice of each group for biochemical and histological analyses, respectively. Histology and immunohistochemistry. Aortic lesion assessment was carried out at four sites (sinus, arch, thoracic and abdominal aorta) as described in Example 1.
- Mac-3 macrophages, dilution 1:200, DAKO
- PCNA cell proliferation, dilution 1:500, DAKO
- anti-rat HO-I monoclonal antibody dilution 1:50, Santa Cruz
- Apoptosis was assessed using the TUNEL assay kit (Roche) according to the manufacture's instructions.
- Digital images were taken for quantitative morphometric analysis. Mac-3 areas were determined using Adobe Photoshop V6.0 by tracing.
- Example 1 shows that probucol affects atherosclerosis in apoE-/- mice in a site- specific manner, enhancing lesion size at the aortic root and strongly inhibiting disease at the descending aorta. It also shows that inhibition of atherosclerosis in the thoracic and abdominal aorta by probucol is associated with oxidative metabolism of probucol to BP.
- Example 4 shows that that DTBP is an intermediate in the oxidative conversion of probucol to BP, and that DTBP has biological protective activity in that it inhibits HOCl- mediated endothelial dysfunction. To test whether DTBP has also anti-atherosclerotic activity, the apoE-/- mouse model as described in Example 1 was used.
- the effect of 1% (w/w) probucol was compared with that of 0.02% DTBP and 0.02% BP on atherosclerosis in apoE-/- mice fed a high fat diet for 5 months.
- probucol and DTBP decreased the aortic content of neutral lipids (cholesteryl esters and triglycerides) (Figure ⁇ 2d), independent of the content of oxidized lipids in the affected vessel wall ( Figure 12e). Rather, inhibition of atherosclerosis by probucol and DTBP was associated with a significant decrease in both macrophages lesion area ( Figure 12 ⁇ and/) and proliferating cells (Figure I2g), whereas BP failed to affect these parameters. The results indicate that DTBP is a potential antiatherogenic compound that may not share some of the undesirable side effects of probucol, such as the lowering of HDL-cholesterol.
- Example 6 Probucol and DTBP, but not BP, inhibit intimal thickening following injury
- Rabbit aortic balloon-injury model Four groups of male New Zealand White rabbits (1.8-2.2 kg, M mecanica Farm, Coffs Harbour, Australia), matched for body weight and baseline plasma cholesterol, were fed 100 g per day of normal chow ⁇ 1% probucol, 0.02% DTBP, or 0.02% BP (wt/wt) for up to nine weeks.
- Example 5 As the results in Example 5 showed that DTBP has anti-atherosclerotic activity, the structure-function study was repeated in a rabbit model of intimal hyperplasia in response to injury. To control for probucol's cholesterol-lowering effect, the animals were matched for baseline plasma cholesterol, and fed them a limited amount (100g/day) of standard diet ⁇ the respective drug.
- Sections immediately adjacent sections to those used for lesion assessment were employed for immunohistochemistry, using CD31 (endothelium, dilution 1:50, DAKO), Mac-3 (macrophages, dilution 1:200, DAKO), PCNA (cell proliferation, dilution 1 :500, DAKO) and anti-rat HO-I monoclonal antibody (dilution 1:50, Santa Cruz) with avidin- biotin-horseradish peroxidase for signal detection (Vectorstain Elite ABC Kit, Vector Laboratories). Apoptosis was assessed using the TUNEL assay kit (Roche) according to the manufacture's instructions. Digital images were taken for quantitative morphometric analysis.
- Intimal, medial and Mac-3 + areas were determined using Adobe Photoshop V6.0 by tracing. Re-endothelialization was determined in longitudinal sections as the distance of CD31 + cells from the branch orifice using Scion Image Software (Scion, ML, USA). Total cell profiles, TUNEL + and PCNA + cells were counted manually at high magnification (4Ox objective). Vascular reactivity. Segments (3 mm) of the abdominal aorta at the 2 nd pair of lumber arteries from rabbits were used for isometric tension experiments, and segments extending proximally were analyzed for cGMP content as an index of NO synthase activity, as described in Circulation 2003, 107:2031-2036.
- HMBS Hydroxymethylbilane synthase
- PCR primers 5'- GAGTGATTCGCGTGGGTACC-3'; HMBS reverse, 5'-GGCTCCGATGGTGAAGCC- 3'; HO-I forward, 5'- TGGAGCTGGACATGGCCTTC-3'; HO-I reverse, 5'-TCTGGGCGATCTTCTTAAGG-S '.
- the amount of HO-I mRNA was determined relative to HMBS mRNA using the comparative C ⁇ method described in the ABI 7700 Sequence Detector User Bulletin 2. PCR products were verified by sequence analysis. Heme oxygenase activity was determined in microsomes prepared from homogenized aortic tissue and assessed by HPLC as described in Free Radicals in Biology & Medicine 1998, 24:959-971.
- Re-endothelialization is a key repair process in response to arterial injury that is promoted by probucol. It was then assessed whether DTBP similarly promoted endothelial regeneration using longitudinal sections stained for the endothelial marker CD31.
- endothelium that extended from arterial side-branches ( Figure 15 ⁇ ).
- DTBP and probucol, but not BP significantly enhanced the regeneration of endothelium ( Figure 15b), and they significantly decreased the intima-to- media ratio determined at CD31 -positive sites (not shown).
- Blocking heme oxygenase activity prevents the ability of probucol and DTBP to promote re-endothelialization, inhibit the proliferation of vascular smooth muscle cells, and protect against intimal thickening following injury
- Example 7 showed that inhibition of intimal thickening by probucol and DTBP is associated with the promotion of re-endothelialization and inhibition of proliferation via induction of heme oxygenase- 1 (HO-I), the requirement for heme oxygenase induction for the in vivo protective activities of probucol and DTBP was tested.
- animal received tin protoporphyrin to inhibit heme oxygenase activity in addition to receiving normal chow without (control, Ctrl), or with probucol (P), or DTBP for 9 weeks.
- Blocking heme oxygenase activity via administration of tin protoporphyrin completely prevented the ability of probucol and DTBP to inhibit intimal thickening in response to arterial balloon injury (Figure 19 ⁇ ).
- administration of tin protoporphyrin also completely prevented the ability of probucol and DTBP to promote re-endothelialization ( Figure ⁇ 9b) and to inhibit vascular smooth muscle cell proliferation (Figure 19c).
- Example 9 Classic' phenolic antioxidants, such as the radical scavenger vitamin E, fail to induce heme oxygenase, do not promote re-endothelialization, and also fail to protect against intimal thickening following injury
- Vitamin E ⁇ -tocopherol for cellular studies, ⁇ -tocopheryl acetate for in vivo studies
- Sigma Sigma (St. Louis, MO).
- Heme oxygenase- 1 mRNA was assessed by real time RT- PCR in rabbit aortic smooth muscle cells cultured for 24 hours in the presence of vehicle (control), probucol (50 ⁇ M) or ⁇ -tocopherol (50 ⁇ M) as described in (Circulation 2004; 110:1855-1860).
- Re-endothelialization was assessed by Evans blue staining (Circulation 2003; 107:2031-2036) 3 weeks after injury.
- vitamin E failed to induce HO-I in vascular smooth muscle cells in vitro (Fig. 20A). This was reflected by a lack of ability of the vitamin to promote re- endothelialization (Fig. 20B) and to inhibit intimal hyperplasia in vivo (Fig. 20C). These results show that vitamin E does not share the protective activities identified for probucol.
- Example 4 shows that the oxidation of probucoPs sulphur atoms by 2e-oxidants relates to the extent to which this antioxidant provides vascular protection, while Examples 5-7 show that probucol's sulphur atoms are required for io protection against atherosclerotic vascular disease.
- Example 11 Novel DTBP analogues protect against intimal thickening following injury This example establishes that novel analogues of DTBP provide protection against balloon injury-induced intimal hyperplasia.
- DTBP-s and STBP were synthesized as described in Example 10.
- DTBP at 0.02% provided significant protection against balloon injury-induced intimal hyperplasia (Fig. 21).
- the seleno-analogue STBP (0.02%, wt/wt) protected to a comparable extent (Fig. 21).
- DTBP-s (0.02%, wt/wt) also provided significant protection, albeit less effectively as that seen with STBP (0.02%, wt/wt) and DTBP-s (0.1%, wt/wt) (Fig. 21).
- results shown provide further evidence that the phenolic moiety of probucol and DTBP are not sufficient for in vivo protection against intimal hyperplasia. They also demonstrate that DTBP analogues that contain sulphur or selenium, i.e., moieties that readily engage in 2e-redox reactions, are a novel class of agents that provide protection against atherosclerotic vascular disease.
- composition for parenteral Administration A pharmaceutical composition of the present invention for intramuscular injection could be prepared to contain 1-5 mL sterile buffered water, and 200-300 mg of a compound of Formula (I).
- a pharmaceutical composition for intravenous infusion may comprise 250 ml of sterile Ringer's solution, and 200-300 mg of a compound of Formula (I).
- a pharmaceutical composition of a compound of Formula (I) in the form of a capsule may be prepared by filling a standard two-piece hard gelatin capsule with 200- , 300 mg of a compound of Formula (I), in powdered form, 100 mg of lactose, 35 mg of talc and 10 mg of magnesium stearate.
- a pharmaceutical composition of this invention in a form suitable for administration by injection may be prepared by mixing 1 -5% by weight of a compound of Formula (I) in 10% by volume propylene glycol and water. The solution is sterilised by filtration.
- a lubricating agent such as polysorbate 85 or oleic acid
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3351535A1 (en) * | 2016-10-31 | 2018-07-25 | Uniwersytet Gdanski | Process for the preparation of 5-selenocyanato-uracil |
Also Published As
Publication number | Publication date |
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WO2006063408A8 (en) | 2006-11-02 |
EP1844009A4 (en) | 2010-04-21 |
EP1844009A1 (en) | 2007-10-17 |
US20100004333A1 (en) | 2010-01-07 |
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