WO1999004803A1 - Heteropolytungstate angiogenesis inhibitors - Google Patents

Heteropolytungstate angiogenesis inhibitors Download PDF

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Publication number
WO1999004803A1
WO1999004803A1 PCT/AU1998/000578 AU9800578W WO9904803A1 WO 1999004803 A1 WO1999004803 A1 WO 1999004803A1 AU 9800578 W AU9800578 W AU 9800578W WO 9904803 A1 WO9904803 A1 WO 9904803A1
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Prior art keywords
angiogenesis
solution
inhibition
polyoxometallate compound
compounds
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PCT/AU1998/000578
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French (fr)
Inventor
Barry Matthews
George Holan
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Biomolecular Research Institute Limited
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Priority to AU87189/98A priority Critical patent/AU8718998A/en
Publication of WO1999004803A1 publication Critical patent/WO1999004803A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/42—Phosphorus; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/26—Iron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/30—Zinc; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/32—Manganese; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/34—Copper; Compounds thereof

Definitions

  • This invention relates to inorganic, and more particularly polyoxometallate, compounds which are effective in inhibition of angiogenesis and accordingly can be used instead of sulfated polysaccharides such as heparin to inhibit the angiogenetic process.
  • Angiogenesis the formation and growth of new blood vessels, plays an important role in a variety of physiological processes such as wound healing, corpus luteum and endometrium formation and embryonic development.
  • the angiogenetic process participates in many pathological processes such as diabetic retinopathy, arthritis and inflammation. It has also been suggested that angiogenesis is essential for the growth of solid tumours, in restenosis (narrowing of heart arteries, following angioplasty due to proliferation of fibrobiasts), or in the attachment of metastases of tumours. Accordingly, anti-angiogenetic agents may be useful for therapy of these syndromes or diseases.
  • Heteropolytungstate compounds have been known for over 100 years. Most of their applications stem from their redox chemistry and also their high ionic weights and charges. Their redox chemistry has led to their use as catalysts for the oxidation of organic substrates such as, for example, propylene to acrylic acid, ethylene to acetaldehyde. In the biological field, heteropolytungstates have found use as electron dense stains for electron microscopy, as analytical reagents for proteins and several have also been shown to inhibit viral DNA and RNA polymerases. (J.C. Chermann, et al , Biochem. Biophys. Res. Commun., 1975, 65:1229; M. Herve, et al., ibid, 1983, 116, 222).
  • the heteropolytungstates within the scope of the present invention include 5 the Keggin and Dawson (also known as the Wells-Dawson) type structures and compounds based on these structures in which one or more of the tungsten atoms are removed and, in the majority of cases, exchanged by other metal atoms.
  • Vacancies in the structures are most often created by the extraction of WO 10 or W 3 0 6 6+ from the Keggin (XW 12 O 40 n" ) or Dawson (P 2 W 18 O 62 6 ⁇ ) species. Isomers of these unsaturated (lacunary) polyanions are possible, a consequence of the location of the vacancy. (R. Massart R. Contant, J. M. Fruchart, J. M. Ciabrini, M.
  • Ciabrini J. Chem. Res. (S), 1977, 222; R. G. Finke, M. W. Droege and P. J.
  • the position of the vacancy in P2 17 0 61 is defined by the prefix c - for a belt vacancy or ⁇ 2 - for a cap vacancy.
  • the rotation of W 3 -oxide triads in the structures leads to a number of isomers.
  • a 60° rotation of a W 3 triad cap can convert, for example, an ⁇ - isomer to the ⁇ - isomer.
  • Unsaturated heteropolyanions can behave as ligands by bonding, at their 30 vacant site, with metal ions. These metal ions, when not sterically crowded, can carry ligands such as water, organic coordinating species or organometallic groups.
  • Organometallic moieties can also react with exposed oxygen atoms on, for example, trisubstituted Keggin or Dawson structures (R. G. Finke and M. W. Droege, J. Am. Chem. Soc, 1984, 106, 7274 and R. G. Finke, B. Rapko and P. J. Domaille, Organometallics 1986, 5, 175).
  • An oxygen atom on the Keggin structure can also be alkylated with reagents such as trimethyloxonium salts (W. H. Knoth and R. L. Harlow, J. Am. Chem. Soc. 1981 , 103, 4265).
  • heteropolyanion species are formed by reaction of two W 5 0 18 H ions with metal ions such as the lanthanoids (R. D. Peacock and T. J. R. Weakley, J. Chem. Soc. A, 1971 , 1836).
  • metal ions such as the lanthanoids
  • Heteropolyanions having PW 7 phosphotungstate groups, generally bridged by phosphate group(s) are known (J. Fuchs and R. Palm, Z. Naturforsch. 1988, 43b, 1529 and R. Acerete, J. Server-Carrio, A. Vegas and M. Martinez-Ripoll, J. Am. Chem. Soc, 1990, 112, 9386).
  • the central atom in the compounds can vary widely, especially in the case of the simpler Keggin type structures.
  • the central atom in the Dawson type structures is most often phosphorus.
  • Heteropolytungstate species are often more stable in solution than the corresponding heteropoiymolybdates.
  • Heteropoly compounds of other metals, such as niobium and vanadium, have also been made but often are stable only over a more limited pH range.
  • heteropolytungstate polyanions containing a "central" species are active as angiogenesis inhibitors as shown in the tests described below using the inhibition of growth of blood vessels in rat aorta rings.
  • a method of prophylactic or therapeutic inhibition of angiogenesis in a human or non-human animal patient which comprises administration to the patient of an effective amount of a polyoxometallate compound selected from the group of compounds of
  • the compounds of Formulae 1 to 14 may be prepared by the literature methods or adaptions thereof, varying reactants and conditions as required to obtain the target compound.
  • General review articles, describing the preparation, structure and properties of many of the compounds, include P. Souchay, "Ions Mineraux Condenses", Masson, Paris, 1969; M. T. Pope, “Heteropoly and Isopoly Oxometalates", Springer-Verlag, Berlin, 1983; T. J. R. Weakley, Structure and Bonding, Springer-Verlag, Berlin, 1974, 18, 131 ; M. T. Pope and A. M ⁇ ller, Angew. Chem. Int. Ed. Engl. 1991 , 30, 34.
  • the compounds of the invention useful as active angiogenesis inhibitors are listed as Formulae 1-14 below along with appropriate methods of preparation for each sub-type.
  • X B m , P v , Si ⁇ v , Ge ⁇ v , Zn", Co", Co 1 ", Fe 1 ", Ga 1 ", Ti ⁇ v , or Zr ,v .
  • M Mn", Mn MI , Fe", Fe 1 ", Cu”, Co", Co” 1 , Ga” 1 , Ni", Zn", Ti ⁇ v ,
  • Nb v Y ligand (e.g. H 2 0, OH “ , O 2" , NH 3 , NCS “ , N0 2 " , CN “ , SO 3 2” , aromatic/aliphatic amines, or cyclopentadienyl group.)
  • M Ti ⁇ v , Zr ⁇ v , V v , Zn", Co", Fe", or Fe IM .
  • M V v , Fe 1 ", Nb v , Cr 1 ", Ti lv or Zr lv
  • M Eu , Ce m , Ce ⁇ v , Sm'", or other stable lanthanoid metal ion.
  • M V v , Ti ⁇ v , Mo vl , or Nb v
  • These compounds may be made by reflux of an aqueous mixture of ⁇ -Na 8 HPW 9 O 34 with an Fe(lll) acetate species in a 1 :2 molar ratio as described in International Patent Application PCT/AU91/00280 (WO 92/00078).
  • the structure of the polyanion is not known.
  • M Mn
  • Fe Co
  • Ni Cu
  • Zn Zn
  • M Co", Co'", Ni", Zn", Mn' ⁇ Mn'", Fe'", Al"' or Ga"'.
  • A is a cation
  • n is the number of such cations necessary for electrical neutrality of the molecule.
  • the oxygen on the transition metal atom(s) may be either doubly protonated (H 2 0), singly protonated (OH), or completely deprotonated (O).
  • the acidity of these protons, and the compounds that are obtained, as is known to one skilled in the art of heteropolytungstate chemistry, depends on the nature of the transition metal atom, its oxidation state, the basicity of the polyanion formed and the basicity of the solution from which the compounds were isolated.
  • oxygen atoms are necessarily oxo groups and the charge (and hence the number of counter cations (A)) on the polyanion will depend on the number of protons attached to the oxygen atom(s).
  • compounds containing groups such as, for example, FeOH, may dimerize by an intermolecular condensation reaction. Dimers, where formed, of the compounds listed, are also included in the invention.
  • the charge on the polyanions can vary, depending upon the extent of protonation of the polyanions, as noted earlier, and upon the oxidation states of the metal atoms.
  • the number of associated counter cations (A) will vary correspondingly.
  • A may be a proton, an alkali metal ion, an alkali earth ion, or ammonium or alkyl ammonium ion of type R 4.n H ⁇ N + , where R is an alkyl chain of from 1 to 6 carbon atoms.
  • the required cation is generally introduced into the compound either by use of an ion exchange resin or by precipitation with excess of a salt of that cation. It is to be noted that, as one skilled in the art of heteropolyanion chemistry would know, not all combinations of the elements given in Formulae 1 to 14 are stable. This invention does not include such unstable compounds.
  • the method of the present invention includes inhibition of angiogenesis in a patient, treatment of conditions where growth of new blood vessels is involved such as chronic inflammation, diabetic retinopathy, psoriasis and rheumatoid arthritis, as well as treatment of related disorders and conditions including, but not limited to, prevention of restenosis by inhibition of vascular smooth muscle cell proliferation, acceleration of wound healing by activation of the release of active growth factors stored in the extracellular matrix, and inhibition of tumour cell metastasis.
  • the present invention provides a pharmaceutical or veterinary composition for prophylactic or therapeutic anti-angiogenetic treatment of a human or non-human animal, which comprises a polyoxometallate compound as broadly described above, in association with at least one pharmaceutically or veterinarily acceptable carrier or diluent.
  • Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal 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, and it is described, by way of example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the human subjects to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and/or diluent.
  • the specifications for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active ingredient for the particular treatment.
  • this invention provides the use of a prophylactic- or therapeutic-effective amount of a polyoxometallate compound as broadly described above in the prophylactic or therapeutic anti-angiogenetic treatment of, or in the manufacture of a medicament for prophylactic or therapeutic anti-angiogenetic treatment of a human or non-human animal.
  • a variety of administration routes are available. The particular mode selected will depend, of course, upon the particular condition being treated and the dosage required for therapeutic efficacy.
  • the methods of this invention may be practised using any mode of administration that is medically acceptable, meaning any mode that produces therapeutic levels of the active component of the invention without causing clinically unacceptable adverse effects.
  • modes of administration include oral, rectal, topical, nasal, inhalation, transdermal or parenteral (e.g. subcutaneous, intramuscular and intravenous) routes.
  • Formulations for oral administration include discrete units such as capsules, tablets, lozenges and the like.
  • Other routes include intrathecal administration directly into spinal fluid, direct introduction such as by various catheter and balloon angioplasty devices well known to those of ordinary skill in the art, and intraparenchymal injection into targeted areas.
  • compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing the active component into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active component into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active component, in liposomes or as a suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, or an emulsion.
  • compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active component which is preferably isotonic with the blood of the recipient.
  • This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in polyethylene glycol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono-or di-glycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • the active compound may also be formulated for delivery in a system designed to administer the active component intranasally or by inhalation, for example as a finely dispersed aerosol spray containing the active component.
  • Other delivery systems can include sustained release delivery systems.
  • sustained release delivery systems are those which can provide for release of the active component of the invention in sustained release pellets or capsules.
  • Many types of sustained release delivery systems are available. These include, but are not limited to: (a) erosional systems in which the active component is contained within a matrix, and (b) diffusional systems in which the active component permeates at a controlled rate through a polymer.
  • a pump- based hardware delivery system can be used, some of which are adapted for implantation.
  • the active component is administered in prophylactically or therapeutically effective amounts.
  • a prophylactically or therapeutically effective amount means that amount necessary at least partly to attain the desired effect, or to delay the onset of, inhibit the progression of, or halt altogether, the onset or progression of the particular condition being treated. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition and individual patient parameters including age, physical condition, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgement. It will be understood by those of ordinary skill in the art, however, that a lower dose or tolerable dose may be administered for medical reasons, psychological reasons or for virtually any other reasons.
  • daily oral doses of active component will be from about 0.01 mg/kg per day to 1000 mg/kg per day.
  • Small doses (0.01-1 mg) may be administered initially, followed by increasing doses up to about 1000 mg/kg per day.
  • higher doses or effective higher doses by a different, more localised delivery route
  • Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.
  • the compounds according to the invention may also be presented for use in the form of veterinary compositions, which may be prepared, for example, by methods that are conventional in the art.
  • veterinary compositions include those adapted for: (a) oral administration, external application, for example drenches (e.g. aqueous or non-aqueous solutions or suspensions); tablets or boluses; powders, granules or pellets for admixture with feed stuffs; pastes for application to the tongue;
  • parenteral administration for example by subcutaneous, intramuscular or intravenous injection, e.g. as a sterile solution or suspension; or (when appropriate) by intramammary injection where a suspension or solution is introduced into the udder via the teat;
  • topical application e.g. as a cream, ointment or spray applied to the skin;
  • Figure 1 shows the results of angiogenesis inhibition assays using compounds of the present invention and the standard angiogenesis inhibitor PAMPS.
  • H3PO3 (85% 30ml ) was added dropwise to a boiling solution of Na2WO . 2 H 2 O (20. Og) in water (70 ml). The solution was refluxed for 13 h. It was then cooled and the product precipitated by adding 20g of solid KCI. The light green solid was collected by filtration and air-dried (15.2g). Recrystallisation from water after leaving the solution to stand overnight at 4° gave a light green solid. A portion of this was purified by washing and after drying (1.376g) of clean material was obtained.
  • the ⁇ , ⁇ -K 6 P 2 W 18 0 62 (1.3 g) was dissolved of water (5ml) with heating. Bromine, 1 drop, was added to the stirred warm solution causing the light green colour to change to light yellow. The solution was cooled to room temperature and KHCO3 (1 M ) was slowly added with stirring. A white solid precipitated and the mixture was stirred for an additional 30min before HCI (1 ml, 6M) was slowly added to give a slightly cloudy yellowish solution. This was filtered and KCI (2g) added to the filtrate to precipitate a yellowish solid. The suspension was kept overnight at 4° and the solid was then collected by filtration; dried, yielding 0,91 g of product.
  • ⁇ - K 6 P 2 W 18 O 62 (0.9g) was dissolved in water (2.5ml) with gentle heating. The solution was cooled to room temperature and NaCIO4 (0.75g) was added to the stirred solution. A white precipitate was formed and the reaction mixture was stirred for an additional 1 h and then filtered. An solution of Na2CO3 (10ml; 1M) was added dropwise to the filtrate. At pH8.5 a white precipitate began forming. The pH was adjusted to 9.0 and kept there by periodic addition of the sodium carbonate solution. The suspension was then filtered and the white solid washed with sat. NaCI, ethanol, ether; then air-dried to give the product - ⁇ -Na 12 P 2 W 15 O 56 (0.60g) as a white powder.
  • Titanium tetrachloride (0.55ml; O. ⁇ mmol) was added to a vigorously stirred solution of Na 2 W0 4 .2H 2 0 (18.2g; 5.5mmol) in water (100ml). The pH of the solution was adjusted to 4.5 with glacial acetic acid and the mixture then boiled until a clear solution was obtained. A solution of NdCI 3 (627mg; 0.25mmol) in water (5ml) was then added dropwise with vigorous stirring to the above solution at 70°C. The solution was cooled to room temperature and a saturated solution of KCI (5ml) was added. Upon standing at 0°C a solid material separated from the solution. The solid was collected by filtration and recrystallised from water (3X) to give a white powder.
  • Ni (NO 3 ) 2 .6H 2 O (1.95g; 6.7mmol), NaHPO 4 (0.48g; 3.4mmol) and 6N HCI (3.1 ml;
  • 6NHC1 (26ml) was added to a stirred solution of Na 2 W0 4 .2H 2 O (36.4g) and sodium silicate (2.2g) in water (40ml) and the solution boiled for 1 hr and concentrated to ca. 30ml. The solution was cooled and the precipitated silica residue removed by filtration. A solution of anhydrous sodium carbonate (10g) in water (40ml) was added and Na 10 [a-SiW g O 34 ] precipitated from the gently stirred solution. The solid was collected by filtration and air dried.
  • the rat-aorta-ring model initially described by Nicosia and Ottinetti [Cell Dev. Biol. 26 119-128, (1990)] was used. Briefly, a sterile 1.5% solution of agarose was poured into culture dishes and allowed to gel (Agarose, Pharmacia Biotech, Uppsala, Sweden). Rings were obtained by punching two concentric circles, with diameter of 10 and 17 mm, respectively, in the agarose gel. The excess agarose inside and outside the rings was removed. The rings were transferred to a 6 well plate (Nunclon, Roskilde, Denmark), each well containing three rings. Thoracic aortas were obtained from 3-month-old male Wistar rats.
  • the aortas were immediately transferred to a culture dish with serum-free MEM.
  • the fibroadiposic tissue around the aorta was carefully removed in order not to damage the aortic wall.
  • Thin slices (0.5 mm thick) of aortic rings were sectioned and extensively rinsed in 12 consecutive washes of serum free medium.
  • the bottom of each agarose well was coated with 150 ⁇ l of clotting fibrinogen. After the fibrin gel had formed, the aortic ring was transferred to the agarose well and positioned in the centre of the agarose well. Then the agarose wells were completely filled with clotting fibrinogen.
  • Partially purified bovine fibrinogen (Sigma) was dissolved in serum-free medium in order to obtain a concentration of 3mg/ml. Clotting was obtained by a adding 20 ⁇ l of a 50 U/ml bovine thrombin solution (Sigma) to 1 ml of fibrinogen solution. The fibrin gel formed within 30 seconds at room temperature. After fibrin gelation, 6 ml of medium, supplemented with 20% FCS (Gibco), 10 mM Hepes (Gibco) and 1 mM L-glutamine (Gibco), were added to each well of the 6-well plate and the test compounds were added to the medium at the appropriate concentration.
  • microvessels were examined daily and scored under an inverted microscope. The growth of microvessels is represented by means of a microvascular growth curve. Formation of more than 200 to 250 microvessels is common due to the three-dimensional complexity of the microvascular network. The margin of error for the observer who is counting the microvessels is high: therefore, the formed microvessels were scored on a scale from 0 (no vessels) to 10 (maximum vessel number). The results are shown in Figure 1.
  • the potent angiogenesis inhibitor PAMPS (Sandra Liekens, Johan Neyts,
  • All test compounds inhibited angiogenesis.
  • a complete inhibition of angiogenic growth of microvessels for the 11 day period of the test was shown in aortal rings treated with BRI 6174 at a concentration of 100 ⁇ g/ml of medium while BRI 6175, BRI 6176 and BRI 6178 have shown no higher score than 1 for the same period of the tests at a concentration of 10O ⁇ g/ml. This compares with the score of 1.75-2.75 after 6 days of the test for the standard angiogenesis inhibitor PAMPS. A noticeable inhibition of angiogenesis was shown by BRI6174 even at concentration of 4 ⁇ g/ml.

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Abstract

A method of prophylactic or therapeutic inhibition of angiogenesis in a human or non-human animal patient comprises administration to the patient of an effective amount of a heteropolytungstate selected from one of the following formulae: AnM1-4X1-4WqOr; AnYMXW11O39; An[CFeOA)4P2W18O68]; An[Co(OH)3(H2O)6(HPO4)2(P3W27O102)]; or AnP2W15O56; where A = a cation; n = number of cations for electrical neutrality; Y = a ligand; X = B, P, Si, Ge, Zn, Co, Fe, Ga, Ti, Zr, V, or Cu; M is selected from various metals or combinations thereof; q = 9-11, 15-18, 22, 30 or 34, and r = 39, 40, 56, 62, 65, 68, 78, 102, 112 or 122.

Description

INORGANIC ANGIOGENESIS INHIBITORS
FIELD OF THE INVENTION
This invention relates to inorganic, and more particularly polyoxometallate, compounds which are effective in inhibition of angiogenesis and accordingly can be used instead of sulfated polysaccharides such as heparin to inhibit the angiogenetic process.
BACKGROUND OF THE INVENTION
Angiogenesis, the formation and growth of new blood vessels, plays an important role in a variety of physiological processes such as wound healing, corpus luteum and endometrium formation and embryonic development. In addition, the angiogenetic process participates in many pathological processes such as diabetic retinopathy, arthritis and inflammation. It has also been suggested that angiogenesis is essential for the growth of solid tumours, in restenosis (narrowing of heart arteries, following angioplasty due to proliferation of fibrobiasts), or in the attachment of metastases of tumours. Accordingly, anti-angiogenetic agents may be useful for therapy of these syndromes or diseases.
Heteropolytungstate compounds have been known for over 100 years. Most of their applications stem from their redox chemistry and also their high ionic weights and charges. Their redox chemistry has led to their use as catalysts for the oxidation of organic substrates such as, for example, propylene to acrylic acid, ethylene to acetaldehyde. In the biological field, heteropolytungstates have found use as electron dense stains for electron microscopy, as analytical reagents for proteins and several have also been shown to inhibit viral DNA and RNA polymerases. (J.C. Chermann, et al , Biochem. Biophys. Res. Commun., 1975, 65:1229; M. Herve, et al., ibid, 1983, 116, 222).
The heteropolytungstates within the scope of the present invention include 5 the Keggin and Dawson (also known as the Wells-Dawson) type structures and compounds based on these structures in which one or more of the tungsten atoms are removed and, in the majority of cases, exchanged by other metal atoms.
4+
Vacancies in the structures are most often created by the extraction of WO 10 or W306 6+ from the Keggin (XW12O40 n") or Dawson (P2W18O62 6~) species. Isomers of these unsaturated (lacunary) polyanions are possible, a consequence of the location of the vacancy. (R. Massart R. Contant, J. M. Fruchart, J. M. Ciabrini, M.
Foumier, inorg. Chem. 1977, 16, 2916; T. L. Jorris, M. Kozik, N. Casan-Pastor, P.
J. Domaille, R. G. Finke, W. K. Miller and L. C. W. Baker, J. Am. Chem. Soc. 1987, 15 109, 7402; T. J. R. Weakley, Polyhedron 1987, 6, 931 ; R. Contant and J.-P.
Ciabrini, J. Chem. Res. (S), 1977, 222; R. G. Finke, M. W. Droege and P. J.
Domaille, Inorg. Chem., 1987, 26, 3886; M. T. Pope, Ηeteropoly and Isopoly
Oxometalates", Springer- Verlag, Berlin, 1983.)
10
20 The position of the vacancy in P2 17061 is defined by the prefix c - for a belt vacancy or α2- for a cap vacancy. The rotation of W3-oxide triads in the structures leads to a number of isomers. Thus a 60° rotation of a W3 triad cap can convert, for example, an α- isomer to the β- isomer.
25 In the trivacant polyanions of the type XW9034 n~, A- or B- forms are obtained, depending upon whether a corner-linked W3 oxide triad is lost (A- form) or an edge-linked W3 oxide triad has been removed(B- form).
Unsaturated heteropolyanions can behave as ligands by bonding, at their 30 vacant site, with metal ions. These metal ions, when not sterically crowded, can carry ligands such as water, organic coordinating species or organometallic groups.
Organometallic moieties can also react with exposed oxygen atoms on, for example, trisubstituted Keggin or Dawson structures (R. G. Finke and M. W. Droege, J. Am. Chem. Soc, 1984, 106, 7274 and R. G. Finke, B. Rapko and P. J. Domaille, Organometallics 1986, 5, 175).
An oxygen atom on the Keggin structure can also be alkylated with reagents such as trimethyloxonium salts (W. H. Knoth and R. L. Harlow, J. Am. Chem. Soc. 1981 , 103, 4265).
Some of the oxygen atoms on heteropolytungstates can also be exchanged for fluorine atoms (F. Chauveau, P. Doppelt and J. Lefebvre, Inorg. Chem. 1980, 19, 2803; T. L. Jorris, M. Kozik and L. C. W. Baker, Inorg. Chem. 1990, 29, 4584).
Other heteropolyanion species are formed by reaction of two W5018H ions with metal ions such as the lanthanoids (R. D. Peacock and T. J. R. Weakley, J. Chem. Soc. A, 1971 , 1836). Heteropolyanions having PW7 phosphotungstate groups, generally bridged by phosphate group(s), are known (J. Fuchs and R. Palm, Z. Naturforsch. 1988, 43b, 1529 and R. Acerete, J. Server-Carrio, A. Vegas and M. Martinez-Ripoll, J. Am. Chem. Soc, 1990, 112, 9386).
The central atom in the compounds can vary widely, especially in the case of the simpler Keggin type structures. The central atom in the Dawson type structures is most often phosphorus.
Heteropolytungstate species are often more stable in solution than the corresponding heteropoiymolybdates. Heteropoly compounds of other metals, such as niobium and vanadium, have also been made but often are stable only over a more limited pH range.
In work leading to the present invention, the inventors have made the unexpected discovery that heteropolytungstate polyanions containing a "central" species (designated X in the examples of structural types listed below) are active as angiogenesis inhibitors as shown in the tests described below using the inhibition of growth of blood vessels in rat aorta rings.
SUMMARY OF THE INVENTION
According to the present invention, there is provided a method of prophylactic or therapeutic inhibition of angiogenesis in a human or non-human animal patient, which comprises administration to the patient of an effective amount of a polyoxometallate compound selected from the group of compounds of
Formulae 1 to 14 hereinafter, and dimers, isomers, solvates, or oxidised or reduced forms thereof, or a pharmaceutically acceptable derivative thereof.
DETAILED DESCRIPTION OF THE INVENTION
The compounds of Formulae 1 to 14 may be prepared by the literature methods or adaptions thereof, varying reactants and conditions as required to obtain the target compound. General review articles, describing the preparation, structure and properties of many of the compounds, include P. Souchay, "Ions Mineraux Condenses", Masson, Paris, 1969; M. T. Pope, "Heteropoly and Isopoly Oxometalates", Springer-Verlag, Berlin, 1983; T. J. R. Weakley, Structure and Bonding, Springer-Verlag, Berlin, 1974, 18, 131 ; M. T. Pope and A. Mϋller, Angew. Chem. Int. Ed. Engl. 1991 , 30, 34. The compounds of the invention useful as active angiogenesis inhibitors, are listed as Formulae 1-14 below along with appropriate methods of preparation for each sub-type.
A. Compounds based on the Keggin structure.
1.) AnlYM uOss]
Wherein
X = Bm, Pv, Siιv, Geιv, Zn", Co", Co1", Fe1", Ga1", Tiιv, or Zr,v. M = Mn", MnMI, Fe", Fe1", Cu", Co", Co"1, Ga"1, Ni", Zn", Tiιv,
Zrιv, Al1", In"1, Vιv, Vv, Movl, Pb", or Nbv Y = ligand (e.g. H20, OH", O2", NH3, NCS", N02 ", CN", SO3 2", aromatic/aliphatic amines, or cyclopentadienyl group.)
These compounds may be made by following the procedures described in the literature, e.g. C. M. Tourne, G. F. Tourne, S. A. Malik and T. J. R. Weakley, J. inorg. nucl. Chem. 1970, 32, 3875-3890 and references therein; M. Bauchet, C. M.
Tourne and G. Tourne, C. R. Acad. Sci. Paris, 1972, C275, 407; F. Zonnevijlle, C.
M. Tourne and G. F. Tourne, Inorg. Chem. 1982, 21 , 2742-2750, 2751-2757 and references therein; P. J. Domaille, J. Am. Chem. Soc, 1984, 106, 7677; T. J. R.
Weakley, J. Chem. Soc, Dalton Trans. 1973, 341 ; L. C. W. Baker and T. P.
McCutcheon, J. Am. Chem. Soc. 1956, 78, 4503 and subsequent papers; M.
Michelon and G. Herve, C. R. Acad. Sci. Paris, 1972, C274, 209; J. Liu, W. Wag,
Z. Zhu, E. Wang and Z. Wang, Transition Met. Chem. 1991 , 16, 169; F. Ortega and M. T. Pope, Inorg. Chem. 1984, 23, 3292. The cyclopentadienyl compounds were prepared by the methods of R. K. C. Ho and W. G. Klemperer, J. Am. Chem.
Soc. 1978, 100, 6772 or W. H. Knoth, J. Am. Chem. Soc. 1979, 101 , 2211.
2.) An[M(XW11O39)2] Wherein X = Ga1", Pv, Siιv, Geι , or Tilv M = La"1, Ce"1, Ceιv, Pr"1, Sm"1, Nd"1, Eu"1, Gd1", Tb"1, Dy1", Ho1", Tm1", or Yb"1.
These compounds may be made by following procedures given in the literature, e.g. J. Liu, W. Wang, Z. Zhu, E. Wang and Z. Wang, Transition Met. Chem. 1991 , 16, 169 and references therein; Gh. Marcu and M. Rusu, Rev. Roum. Chim. 1977, 22, 227.
3.) An[XM2W10O40]
Wherein
X = Pv, Siιv, or Vv
M = Tiιv, Zrιv, Vv, Zn", Co", Fe", or FeIM.
These compounds may be prepared by following the methods of P. J.
Domaille, Inorg. Synth., 1990, 27, 102-104. (Ed. A. P. Ginsberg) Whiley- lnterscience and references therein; J. Canny, R. Thouvenot, A. Teze, G. Herve, M. Leparulo-Loftus and M. T. Pope, Inorg. Chem. 1991 , 30, 976; P. J. Domaille and W. H. Knoth, Inorg. Chem. 1983, 22, 818; C. M. Tourne and G. F. Tourne, J.Chem. Soc. Dalton Trans. 1988, 2411 ; C. M. Flynn Jr. and M. T. Pope, Inorg. Chem. 1971 , 10, 2745.
4.) An[XM3W9O40]
Wherein X = Pv, Fe1", Silv, or V
M = Vv, Fe1", Nbv, Cr1", Tilv or Zrlv
These compounds may be prepared by following the methods of P. J. Domaille, J. Am. Chem. Soc, 1984, 106, 7677; R. G. Finke, C. A. Green and B. Rapko, Inorg. Synth., 1990,.27, 129 (Ed. A. P. Ginsberg) Whiley-lnterscience and references therein; P. Jun, Q. Lun-yu and C. Ya-guang, Inorg. Chim. Ada 1991 , 183, 157; D. J. Ediund, R. J. Saxton, D. K. Lyons and R. G. Finke, Organometallics 1988, 7, 1692; C. M. Flynn Jr. and M. T. Pope, Inorg. Chem. 1971 , 10, 2745.
5 B. Saturated Compounds based on the Dawson structure.
5.) An[X2MW17O62]
Wherein
X = Pv
10 M = Co", Co1", Ni", Zn", Mn", Mn"1, Fe1", Al1", Ga"1,
In1", Tiιv, Zrιv, Vv, MoVi, or π-RC5H4Ti where R = organic residue.
These compounds may be obtained by the reactions of S. A. Malik and T. J. R. Weakley, J. Chem. Soc, Chem. Commun. 1967, 1094; J. Chem. Soc. (A), 1968, 15 2647; D. K. Lyon, W. K. Miller, T. Novet, P. J. Domaille, E. Evitt, D. C. Johnson and R. G. Finke, J. Am. Chem. Soc. 1991 , 113, 7209 and references therein; J. F. W. Keana and M. D. Ogan, J. Am. Chem. Soc. 1986, 108, 7951.
6.) An[M(X2W17O61)2]
20 Wherein
X = Pv
M = Eu , Cem, Ceιv, Sm'", or other stable lanthanoid metal ion.
These compounds may be made by the reactions described by R. D. 25 Peacock and T. J. R. Weakley, J. Chem. Soc. (A), 1968, 1836; A. V. Botar and Rev. Roum. Chim. 1973, 18, 1155; Gh. Marcu, M. Rusu and A. V. Botar,f?ev. Roum. Chim. 1974, 19, 827. 7.) An[X2M3W15O62]
Wherein
X = Pv
M = Vv, Tiιv, Movl, or Nbv
The compounds where M = V, may be made by following method described by R. G. Finke, B. Rapko, R. J. Saxton and P. J. Domaille, J. Am. Chem. Soc. 1986, 108, 2947. The compounds where M = Nb, monomers and/or dimers, may be formed according to D. J. Ediund, R. J. Saxton, D. K. Lyons and R. G. Finke, Organometallics 1988, 7, 1692. The compounds where M = Mo, may be made according to J. P. Ciabrini, R. Contant and J. M. Fruchart, Polyhedron 1983, 2, 1229.
C. Compounds in which two or three trivacant A- or B-XWg034 n~ or two B- X2W15056 n~ polyanions are connected via transition metal or lanthanoid metal ion(s).
8.) An[M4(H2O)y(XW9O34)2]
Wherein X = Pv, Fe'", Zn", or Co",
M = Mn", Fe", Co", Ni", Cu", Zn", ZrO, [FeCu]1/2, [WZn3]1/4, [WZnMnM 2]1/4, [WZnMn"'2]1/4, [WMn"3]1/4, [WMn"<3]1/4, [ Fe"3]1/4, [WFe'"3]1/4, [WNi"3]1/4, [WCu"3]1/4, [WZnVlv 2]1/4, [WZn"Fe"2]1/4, [WZn"Co"2]1/4, WNi3", [WZnMNi"2]1/4,
[WZn"Vlv 2]1/4, [WZn"Pd"2]1/4, [WCoΛ 14'
[WCo'^n11^, [WCo"Fe"2]1/4, [WCo"Ni"2]1/4, or [WCo"Zn"2]1/4. y is 1-6 These compounds may be made following the methods of H. T. Evans, C. M. Tourne, G. F. Tourne and T. J. R. Weakley, J. Chem. Soc. Dalton Trans., 1986, 2699: R. G. Finke, M. W. Droege and P. J. Domaille, Inorg. Chem.,, 1987, 26, 3886; S. H. Wasfi, A. L. Reingold, G.F.Kokoszka and A. S. Goldstein, Inorg. Chem., 1987, 26, 2934; C. M. Tourne, G. F. Tourne and F. Zonnevijlle, J. Chem. Soc. Dalton Trans. 1991 , 143.
9.) An[(FeOA)4(PW9O34)2
These compounds may be made by reflux of an aqueous mixture of Δ-Na8HPW9O34 with an Fe(lll) acetate species in a 1 :2 molar ratio as described in International Patent Application PCT/AU91/00280 (WO 92/00078). The structure of the polyanion is not known.
10.) An[Ma(XW9O34)2] Wherein
X = Pv, or Siιv.
If a = 1 or 2; M = WO2; if a = 3; M = Zr(OH), CeO,
Cu(N03)1/3, Cu(NO2)1/3, WO2 Zn", Mn", Mn'", Fe", Fe'", Ni", Co", [Fe2(W02)]1/3, [FeCo(W02)]1/3, [Co"Cu"W02]1/3, [Cu"2Co"]1/3, [Cu"2Co"NO3]1/3, [Zn2(WO2)]1/3,
[Ni2(WO2)]1/3, [Co2(W02)]1/3, or [p-C5H5Tilv(OH2)].
These compounds may be made by the methods described by R. G. Finke, B. Rapko and T. J. R. Weakley, Inorg. Chem. 1989, 28, 1573 and references therein; C. Tourne, A. Revel and G. Tourne, Rev. Chim. Minerale 1977, 14, 537 ([Cθ2(W02)]- 3); C. M. Tourne and G. F. Tourne, J .Chem. Soc. Dalton Trans. 1988, 2411. 11 •) An[Co9(OH)3(H2O)6(HPO4)2(PW9O34)3]
These compounds may be prepared following the method of T. J. R. Weakley, J. Chem. Soc, Chem. Commun. 1984, 1406; J. Chem. Soc, Dalton Trans., 1986, 2699.
12.) An[M4(H2O)2(X2W15O56)2] Wherein
X = Pv
M = Mn", Fe", Co", Ni", Cu" or, Zn".
These compounds may be made following the general method of R. G. Finke, M. W. Droege and P. J. Domaille, Inorg. Chem., 1987, 26, 3886. When the cobalt compound (e.g. Na salt) is warmed in aqueous solution to 80-90° for several hours, the red-brown compound that crystallizes from the solution on cooling to room temperature is referred to as the 'high temp.' form of the compound.
13.) AnP2W15O56
These compounds are made by the general method of R.G. Finke, M.W. Droege and P.J. Domaille, Inorg. Chem. 1987, 26, 3886.
14.) An[M4X2W16O65].n H2O
Wherein
X= Pv
M= Co", Co'", Ni", Zn", Mn'^Mn'", Fe'", Al"' or Ga"'.
These compounds can be made by method of M. Cholewa, I.F. Turnbull, G.J.F. Legge, H. Weigold, S.M. Marcuccio, G. Holan, E. Tomlinson and P.J. Wright, Cellular and Molecular Biology, 1996, 42 (1) 69-76.
In each of Formulae 1 to 14 above, A is a cation, and n is the number of such cations necessary for electrical neutrality of the molecule. In the compounds of Formulae 1 to 14, when a transition metal atom(s) replace(s) one or more tungsten atoms in the structure, the oxygen on the transition metal atom(s) may be either doubly protonated (H20), singly protonated (OH), or completely deprotonated (O). The acidity of these protons, and the compounds that are obtained, as is known to one skilled in the art of heteropolytungstate chemistry, depends on the nature of the transition metal atom, its oxidation state, the basicity of the polyanion formed and the basicity of the solution from which the compounds were isolated. In the compounds of the invention not all oxygen atoms are necessarily oxo groups and the charge (and hence the number of counter cations (A)) on the polyanion will depend on the number of protons attached to the oxygen atom(s). Furthermore, compounds containing groups such as, for example, FeOH, may dimerize by an intermolecular condensation reaction. Dimers, where formed, of the compounds listed, are also included in the invention.
Many of the compounds of the invention can occur in a number of isomeric forms. In fact, it is at times difficult to obtain isomerically pure compounds. All isomers or isomer mixtures are included in this invention.
Many of the compounds can undergo one or more electron reductions. The reduced compounds are also included in this invention. Compounds, such as those containing Fe", may undergo oxidation and the oxidised species formed are included in this invention.
The charge on the polyanions can vary, depending upon the extent of protonation of the polyanions, as noted earlier, and upon the oxidation states of the metal atoms. The number of associated counter cations (A) will vary correspondingly. A may be a proton, an alkali metal ion, an alkali earth ion, or ammonium or alkyl ammonium ion of type R4.nHπN+, where R is an alkyl chain of from 1 to 6 carbon atoms. The required cation is generally introduced into the compound either by use of an ion exchange resin or by precipitation with excess of a salt of that cation. It is to be noted that, as one skilled in the art of heteropolyanion chemistry would know, not all combinations of the elements given in Formulae 1 to 14 are stable. This invention does not include such unstable compounds.
Specific polyoxometallate compounds of Formulae 1 to 14 prepared by the methods described above are set out in the Examples herein.
As described above, the polyoxymetallate compounds of Formulae 1 to 14 have been found to exhibit significant angiogenesis inhibitory activity. Accordingly, the method of the present invention includes inhibition of angiogenesis in a patient, treatment of conditions where growth of new blood vessels is involved such as chronic inflammation, diabetic retinopathy, psoriasis and rheumatoid arthritis, as well as treatment of related disorders and conditions including, but not limited to, prevention of restenosis by inhibition of vascular smooth muscle cell proliferation, acceleration of wound healing by activation of the release of active growth factors stored in the extracellular matrix, and inhibition of tumour cell metastasis.
In another aspect the present invention provides a pharmaceutical or veterinary composition for prophylactic or therapeutic anti-angiogenetic treatment of a human or non-human animal, which comprises a polyoxometallate compound as broadly described above, in association with at least one pharmaceutically or veterinarily acceptable carrier or diluent.
The formulation of such compositions is well known to persons skilled in this field. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal 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, and it is described, by way of example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
It is especially advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the human subjects to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and/or diluent. The specifications for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active ingredient for the particular treatment.
In yet another aspect, this invention provides the use of a prophylactic- or therapeutic-effective amount of a polyoxometallate compound as broadly described above in the prophylactic or therapeutic anti-angiogenetic treatment of, or in the manufacture of a medicament for prophylactic or therapeutic anti-angiogenetic treatment of a human or non-human animal.
A variety of administration routes are available. The particular mode selected will depend, of course, upon the particular condition being treated and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, may be practised using any mode of administration that is medically acceptable, meaning any mode that produces therapeutic levels of the active component of the invention without causing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, topical, nasal, inhalation, transdermal or parenteral (e.g. subcutaneous, intramuscular and intravenous) routes. Formulations for oral administration include discrete units such as capsules, tablets, lozenges and the like. Other routes include intrathecal administration directly into spinal fluid, direct introduction such as by various catheter and balloon angioplasty devices well known to those of ordinary skill in the art, and intraparenchymal injection into targeted areas.
The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing the active component into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active component into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active component, in liposomes or as a suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, or an emulsion.
Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active component which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in polyethylene glycol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The active compound may also be formulated for delivery in a system designed to administer the active component intranasally or by inhalation, for example as a finely dispersed aerosol spray containing the active component.
Other delivery systems can include sustained release delivery systems.
Preferred sustained release delivery systems are those which can provide for release of the active component of the invention in sustained release pellets or capsules. Many types of sustained release delivery systems are available. These include, but are not limited to: (a) erosional systems in which the active component is contained within a matrix, and (b) diffusional systems in which the active component permeates at a controlled rate through a polymer. In addition, a pump- based hardware delivery system can be used, some of which are adapted for implantation.
The active component is administered in prophylactically or therapeutically effective amounts. A prophylactically or therapeutically effective amount means that amount necessary at least partly to attain the desired effect, or to delay the onset of, inhibit the progression of, or halt altogether, the onset or progression of the particular condition being treated. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition and individual patient parameters including age, physical condition, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgement. It will be understood by those of ordinary skill in the art, however, that a lower dose or tolerable dose may be administered for medical reasons, psychological reasons or for virtually any other reasons.
Generally, daily oral doses of active component will be from about 0.01 mg/kg per day to 1000 mg/kg per day. Small doses (0.01-1 mg) may be administered initially, followed by increasing doses up to about 1000 mg/kg per day. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localised delivery route) may be employed to the extent patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.
The compounds according to the invention may also be presented for use in the form of veterinary compositions, which may be prepared, for example, by methods that are conventional in the art. Examples of such veterinary compositions include those adapted for: (a) oral administration, external application, for example drenches (e.g. aqueous or non-aqueous solutions or suspensions); tablets or boluses; powders, granules or pellets for admixture with feed stuffs; pastes for application to the tongue;
(b) parenteral administration for example by subcutaneous, intramuscular or intravenous injection, e.g. as a sterile solution or suspension; or (when appropriate) by intramammary injection where a suspension or solution is introduced into the udder via the teat;
(c) topical application, e.g. as a cream, ointment or spray applied to the skin; or
(d) intravaginally, e.g. as a pessary, cream or foam.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
Further features of the present invention will be apparent from the following Examples which are included by way of illustration, not limitation of the invention.
In the accompanying drawings: Figure 1 shows the results of angiogenesis inhibition assays using compounds of the present invention and the standard angiogenesis inhibitor PAMPS.
EXAMPLES
EXAMPLE 1 Preparation of Compounds
(i) Preparation of : α- Na12P2W15O56 (BRI-6177)
Preparation of α- β - K6P2W18 018062
Ref: R.G. Finke, M.W. Droege and P.J. Domaille, Inorganic Chemistry 26 (23) 3889 (1987).
H3PO3 (85% 30ml ) was added dropwise to a boiling solution of Na2WO . 2 H2O (20. Og) in water (70 ml). The solution was refluxed for 13 h. It was then cooled and the product precipitated by adding 20g of solid KCI. The light green solid was collected by filtration and air-dried (15.2g). Recrystallisation from water after leaving the solution to stand overnight at 4° gave a light green solid. A portion of this was purified by washing and after drying (1.376g) of clean material was obtained.
Preparation of α- K6P2W18062
R.G. Finke, M.W. Droege and P.J. Domaille, Inorganic Chemistry 26 (23) 3889) (1987). D.K. Lyon, W.K. Miller, T. Novet, P.J. Domaille, E. Evitt, D.C. Johnson and R.G. Finke, J.Amer.Chem.Soc 113 (19) 7218 (1991).
The α,β-K6P2W18062 (1.3 g) was dissolved of water (5ml) with heating. Bromine, 1 drop, was added to the stirred warm solution causing the light green colour to change to light yellow. The solution was cooled to room temperature and KHCO3 (1 M ) was slowly added with stirring. A white solid precipitated and the mixture was stirred for an additional 30min before HCI (1 ml, 6M) was slowly added to give a slightly cloudy yellowish solution. This was filtered and KCI (2g) added to the filtrate to precipitate a yellowish solid. The suspension was kept overnight at 4° and the solid was then collected by filtration; dried, yielding 0,91 g of product.
Preparation of α-Na12P2W15056
R.G. Finke, M.W. Droege and P.J. Domaille, Inorganic Chemistry, 26 (23) 3889 (1987).
α- K6P2W18O62 (0.9g) was dissolved in water (2.5ml) with gentle heating. The solution was cooled to room temperature and NaCIO4 (0.75g) was added to the stirred solution. A white precipitate was formed and the reaction mixture was stirred for an additional 1 h and then filtered. An solution of Na2CO3 (10ml; 1M) was added dropwise to the filtrate. At pH8.5 a white precipitate began forming. The pH was adjusted to 9.0 and kept there by periodic addition of the sodium carbonate solution. The suspension was then filtered and the white solid washed with sat. NaCI, ethanol, ether; then air-dried to give the product - α-Na12P2W15O56 (0.60g) as a white powder.
(ii) Preparation of Na16 [ Mn4 (H2O)2 (P2W15O56)2] . nH2O (BRI-6175)
R.G. Finke, M. W. Droege and P.J. Domaille, Inorganic Chemistry, 26 (23) 3889 (1987). H. Weigold, G. Holan, S.M. Marcuccio, C. Birch and I. Gust. International Patent Publication WO 92/00078 (1991).
α- Nai2P2W1 5θ56 (0.5g) solid was added to a warm solution of MnCl2.4H2θ (50 mg) and sodium chloride (0.3g) in water (5ml). The reaction mixture became red brown and the polyoxometallate slowly dissolved. The reaction mixture was stirred for 30 min then the solution was filtered and the filtrate kept overnight at 4°. The rust coloured solid formed was filtered off and the solid washed with ethanol and diethyl ether. It was then air-dried to yield the product -Na-|6 [ Mn4 (H2O) 2 (P2W.15O55)] . nH2θ (0.246g ) as a rust coloured powder.
(iii) Preparation of K10 [ Co4 (H2O) 2 (PW9O34) 2 ]■ nH2O (BRI-6174)
R.G. Finke, M. W. Droege and P.J. Domaille, Inorganic Chemistry, 26 (23) 3889
(1987).
Co (NO3)2 (0.61 g) was dissolved in water (11 ml) containing nitric acid (70%; 1.0g) and the solution was heated to boiling. A solution of Na2WO4. 2H2O (3.0g) and Na2HPθ4 (0.16g) in water (11ml) was added slowly. After approximately 1/4 of the solution was added to the refluxing cobalt nitrate solution, a precipitate formed which redissolved on continuing addition. The solution was refluxed for 5h, then solid KCI (5g) was added to the cooled burgundy coloured solution. The precipitated solid was filtered off, washed with water and recrystallised from hot water, then dried, to yield (1.060g) as dark blue/purple crystals.
(iv) Preparation of K12 [ Fe'"4 P2 W16 O65] . nH2O (BRI-6178)
M. Cholewa, I.F. Turnbull, G.J.F. Legge, H. Weigold, S.M. Marcuccio, G. Holan, E. Tomlinson and P.J. Wright, Cellular and Molecular Biology 42 (1) 71 (1996).
A solution of NaWO4 . 2H2O (27.9g) and NaH2P0 (1.56g) in water (125ml) was adjusted to pH 8.2 with acetic acid. The solution was heated to 70-80° and then a solution of Fe(NO3)3 . 9 H2O (8.05g) and sodium acetate (10g) in water (40ml) was slowly added. The brown reaction mixture was refluxed for 8 h and the solution filtered and cooled to room temperature. Solid KCI (16g) was then added to the solution precipitating a brown solid. This material was filtered off and reprecipitated from hot water with KCI and then recrystallised from hot water to yield after drying a brown powder (5.456g) of K12 [Fe'"4 P2 W16 065] .nH20 . (v) Preparation of K7 [(Fe111 (H2O) P2W17O61] . 21H2O (BRI-6176)
Preparation of
Figure imgf000022_0001
D. K. Lyon, W.K. Miller, T. Novet, P.J. Domaille, E. Evitt, D.C. Johnson and R.G. Finke, J.Amer. Chem. Soc. 113 (19) 7209 (1991).
α- β - K6P2W18 018O62 (27g), was dissolved in water (60ml) with gentle warming. KHCO3 (100ml; 1 M) was then added with stirring. A white solid precipitated and the mixture was stirred for an additional 30 min. The solid was collected by filtration and washed with water and then air-dried to yield a white powder (24.43 g) of c 2- K10P2W17O61.
A solution of Fe(NO3)3 .9 H2O (86mg) in water (0.5ml) was added to a hot solution of α2-K10P2W17O61.15H2O (1g) in water (3ml) with stirring to yield a yellow brown solution. A drop of bromine was added to ensure a fully oxidised product. The reaction mixture was boiled for a few minutes and then cooled to room temperature and filtered. Solid KCI (0.5g) was then added which precipitated a yellow-orange solid. The mixture was warmed to achieve solution and then cooled to room temperature. The formed solid was collected by filtration washed with water, ethanol and diethyl ether then dried to give a yellowish powder (0.542g) of K7 [(Feιπ (H2O) P2W17O61] . 21H2O .
EXAMPLE 2 - Preparation of K13[Nd(TiW11O39)2].xH2O
Titanium tetrachloride (0.55ml; O.δmmol) was added to a vigorously stirred solution of Na2W04.2H20 (18.2g; 5.5mmol) in water (100ml). The pH of the solution was adjusted to 4.5 with glacial acetic acid and the mixture then boiled until a clear solution was obtained. A solution of NdCI3 (627mg; 0.25mmol) in water (5ml) was then added dropwise with vigorous stirring to the above solution at 70°C. The solution was cooled to room temperature and a saturated solution of KCI (5ml) was added. Upon standing at 0°C a solid material separated from the solution. The solid was collected by filtration and recrystallised from water (3X) to give a white powder.
5 EXAMPLE 3 - Preparation of K10 [Ni4(H2O)2(PW9O34)2]
(i) Direct preparation.
Ni (NO3)2.6H2O (1.95g; 6.7mmol), NaHPO4 (0.48g; 3.4mmol) and 6N HCI (3.1 ml;
10 18.6mmol) were added sequentially to a solution of Na2WO4.2H2O (10g;
30.3mmol) in water (50ml). A light green suspension resulted and the mixture was heated under reflux for 18 hours, during which time a light green homogenous solution resulted. The solution was cooled, solid KCI (20g) was added, and the greenish yellow solid that precipitated collected by filtration and recrystallised from
15 water. Yield 2.82g.
(ii) Preparation via δ-Na8HPW9034.19H20.
85% H3PO4 (0.75ml) was added to a stirred solution of Na2WO4.2H2O (30.0g) in 20 water (37ml), followed by the addition of glacial acetic acid (5.5ml). A white precipitate of α-Na8HPW9O34 soon formed which was collected by filtration and air dried with suction overnight. Yield 21.86g. δ-Na8HPW9034.19H2O was prepared from this material by drying at 140°C for 2hrs at 1 atm.
25 Solid δ-Na8HPW9O34.19H2O (2.0g) was added to a stirred solution of
Ni(NO3)2.6H20 (407mg; 1.4mmol) in water (15ml) and the mixture heated until a homogenous light yellow solution was obtained. Excess solid KCI (6g) was then added and a fine light yellow precipitate formed. The mixture was cooled to room temperature and the solid collected by filtration. The crude product was
30 recrystallised from a minimum amount of water (5ml) to give K10[Ni4(H2O)2(PW9O34)2] as a pale yellow solid (0.39g). EXAMPLE 4 - Preparation of K6H7 [A-α-SiW9Fe3O40]
6NHC1 (26ml) was added to a stirred solution of Na2W04.2H2O (36.4g) and sodium silicate (2.2g) in water (40ml) and the solution boiled for 1 hr and concentrated to ca. 30ml. The solution was cooled and the precipitated silica residue removed by filtration. A solution of anhydrous sodium carbonate (10g) in water (40ml) was added and Na10[a-SiWgO34] precipitated from the gently stirred solution. The solid was collected by filtration and air dried.
A solution of Fe(NO3)3.9H2O (2.16g; 5.3mmol) in water (20ml) was slowly added to 0.5M sodium acetate solution (pH 6.5, 40ml) to give a deep red solution. Na10[a- SiW9O34] (4.5g; 1.6mmol) was added to this solution in small portions at 70°C. The solution became dark brown and was stirred at 80°C for 1hr, cooled and passed through a cation exchange column (Dowex 50W-X2 [K]). A solution of KCI (1.3g) in water (5ml) was added to the eluate to give a fine brown precipitate. The precipitate was collected by filtration and recrystallised from water to give K6H7[A-α-SiW9Fe3O40].
EXAMPLE 5 - Measurement of Angiogenesis Inhibition
Rat-aorta-ring assay.
The rat-aorta-ring model, initially described by Nicosia and Ottinetti [Cell Dev. Biol. 26 119-128, (1990)] was used. Briefly, a sterile 1.5% solution of agarose was poured into culture dishes and allowed to gel (Agarose, Pharmacia Biotech, Uppsala, Sweden). Rings were obtained by punching two concentric circles, with diameter of 10 and 17 mm, respectively, in the agarose gel. The excess agarose inside and outside the rings was removed. The rings were transferred to a 6 well plate (Nunclon, Roskilde, Denmark), each well containing three rings. Thoracic aortas were obtained from 3-month-old male Wistar rats. The aortas were immediately transferred to a culture dish with serum-free MEM. The fibroadiposic tissue around the aorta was carefully removed in order not to damage the aortic wall. Thin slices (0.5 mm thick) of aortic rings were sectioned and extensively rinsed in 12 consecutive washes of serum free medium. Before transferring the aortic rings to the culture plate, the bottom of each agarose well was coated with 150 μl of clotting fibrinogen. After the fibrin gel had formed, the aortic ring was transferred to the agarose well and positioned in the centre of the agarose well. Then the agarose wells were completely filled with clotting fibrinogen. Partially purified bovine fibrinogen (Sigma) was dissolved in serum-free medium in order to obtain a concentration of 3mg/ml. Clotting was obtained by a adding 20 μl of a 50 U/ml bovine thrombin solution (Sigma) to 1 ml of fibrinogen solution. The fibrin gel formed within 30 seconds at room temperature. After fibrin gelation, 6 ml of medium, supplemented with 20% FCS (Gibco), 10 mM Hepes (Gibco) and 1 mM L-glutamine (Gibco), were added to each well of the 6-well plate and the test compounds were added to the medium at the appropriate concentration.
Quantitation of angiogenesis in the rat-aorta-ring assay.
Cultures were examined daily and scored under an inverted microscope. The growth of microvessels is represented by means of a microvascular growth curve. Formation of more than 200 to 250 microvessels is common due to the three-dimensional complexity of the microvascular network. The margin of error for the observer who is counting the microvessels is high: therefore, the formed microvessels were scored on a scale from 0 (no vessels) to 10 (maximum vessel number). The results are shown in Figure 1.
Standards.
The potent angiogenesis inhibitor PAMPS (Sandra Liekens, Johan Neyts,
Bart Degreve and Erik DeClercq, Oncology Research, in Press, 1997) was used as an active standard while phosphate buffered saline (PBS) was used in the control rings. The results are shown in Figure 1.
Results.
All test compounds inhibited angiogenesis. A complete inhibition of angiogenic growth of microvessels for the 11 day period of the test was shown in aortal rings treated with BRI 6174 at a concentration of 100μg/ml of medium while BRI 6175, BRI 6176 and BRI 6178 have shown no higher score than 1 for the same period of the tests at a concentration of 10Oμg/ml. This compares with the score of 1.75-2.75 after 6 days of the test for the standard angiogenesis inhibitor PAMPS. A noticeable inhibition of angiogenesis was shown by BRI6174 even at concentration of 4μg/ml.

Claims

CLAIMS:
1. A method of prophylactic or therapeutic inhibition of angiogenesis in a human or non-human animal patient, which comprises administration to the patient of an effective amount of a polyoxometallate compound selected from the group of compounds of Formulae 1 to 14 and dimers, isomers, solvates or oxidised or reduced forms thereof:
1.) A^MXW^O^]
Wherein
X = B'", Pv, Siιv, Geιv, Zn", Co", Co'", Fe'", Ga1", Tiιv, or ZrX M = Mn", Mn1", Fe", Fe1", Cu", Co", Co1", Ga"1, Ni", Zn", Tiι ,
Zrι , Al"1, In1", Vιv, Vv, Movl, Pb", or Nbv Y = ligand.
2.) An[M(X\N OZ9)2]
Wherein
X = Ga111, Pv, Siιv, Geιv, or Tilv M = La1", Ce1", Celv, Pr1", Sm1", Nd"1, Eu1", Gd1", Tb"1, Dy"1, Ho^ Tm'", or Yb"1.
3.) An[XM2W10O40]
Wherein
X = Pv, Siιv, or Vv
M = Ti,v, Zrlv, V , Zn", Co", Fe", or Fe"'.
4.) An[XM3W9O40]
Wherein
X = Pv, Fe'", Siιv, or Vv
M = Vv, Fe"', Nbv, CrMI, Tilv or Zrιv
5.) An[X2MW17O62]
Wherein
X = Pv
M = Co", Co"', Ni", Zn", Mn", Mn'", Fe'", Al'", Gam,
In'", Tiιv, Zrιv, Vv, Movl, or π-RC5H4Ti where R = organic residue.
6.) An[M(X2W17O61)2]
Wherein
X = Pv
M = Eu'", Ce'", Ceιv, Srn"', or other stable lanthanoid metal ion.
7.) An[X2M3W15O62]
Wherein
X = Pv
M = Vv, Tiιv, Movl, or Nbv
8.) An[M4(H2O)y(XW9O34)2]
Wherein
X = Pv, Fe"', Zn", or Co",
M = Mn", Fe", Co", Ni", Cu", Zn", ZrO, [FeCu]1/2, [WZn3]1/4,
[WZnMn"2]1/4, [WZnMnMI 2]1/4, [WMn"3]1/4,
Figure imgf000028_0001
[WFe"3]1/4, [WFe'"3]1/4, [WNi"3]1/4,
[WCu"3]1/4, [WZnVlv 2]1/4, [WZn"Fe"2]1/4,
[WZn"CoM 2]1/4, WNi3", [WZn"Ni"2]1/4,
[WZnVv 2]1/4, [WZnMPd"2]1/4, [WCo3"] 1/4.
[WCoMMn"2]1/4, [WCo"Fe"2]1/4, [WCo"Ni"2]1/4, or [WCo"Zn"2]1/4. y is 1-6
9.) An[(FeOA)4(PW9O34)2
10.) An[Ma(XW9O34)2]
Wherein
X = Pv, or Siιv
If a = 1 or 2; M = WO2 if a = 3; M = Zr(OH), CeO,
Cu(NO3)1/3, Cu(NO2)1/3, WO2 Zn", Mn", Mn'", Fe", Fe'", Ni", Co", [Fe2(WO2)]1/3, [FeCo(WO2)]1/3, [Co"Cu"WO2]1/3, [Cu"2Co"]1/3, [Cu"2Co"NO3]1/3, [Zn2(WO2)]1/3, [Ni2(WO2)]1/3, [Co2(W02)]1/3, or [p-C5H5Tilv(OH2)].
11.) An[Co9(OH)3(H2O)6(HPO4)2(PW9O34)3]
12.) An[M4(H2O)2(X2W15O56)2]
Wherein
X = Pv
M = Mn", Fe", Co", Ni", Cu" or, Zn".
13.) AnP2W15O56
14.) An[M4X2W16O65].n H2O
Wherein
X= Pv
M= Co", Co"', Ni", Zn", Mn",Mn"', Fe'", Al'" or Ga"'.
and wherein in each of the above formulae, A is a cation and n is the number of such cations necessary for electrical neutrality of the molecule;
or a pharmaceutically acceptable derivative thereof.
A method as claimed in claim 1 , characterised in that A is a proton, an alkali metal, alkaline earth or ammonium cation, or an alkylammonium cation of the formula R4_mHmN+, where R is an alkyl chain of from 1 to 6 carbon atoms and m is 0, 1 , 2 or 3.
3. A method according to claim 1 or claim 2, wherein said polyoxometallate compound is α-Na12P2W15O56
4. A method according to claim 1 or claim 2, wherein said polyoxometallate compound is Na16[Mn4(H2O)2(P2W15O56)2].nH2O.
5. A method according to claim 1 or claim 2, wherein said polyoxometallate compound is K10[Co4(H2O)2(PW9O34)2].nH2O.
6. A method according to claim 1 or claim 2, wherein said polyoxometallate compound is K12[Fel"4P2W16O65].nH2O.
7. A method according to claim 1 or claim 2, wherein said polyoxometallate compound is K7[(Fe'"(H20)P2W17061].21 H20.
8. A method according to claim 1 or claim 2, wherein said polyoxometallate compound is K^[ d)T\\N„029)2].xH20.
9. A method according to claim 1 or claim 2, wherein said polyoxometallate compound is K10 {Ni4(H2O)2(PW9O34)2].
10. A method according to claim 1 or claim 2, wherein said polyoxometallate compound is K6H7[A-a-SiW9Fe3O40].
11. A method according to any of claims 1 to 10, wherein said treatment comprises inhibition of angiogenesis in a patient, treatment of chronic inflammation, diabetic retinopathy, psoriasis and rheumatoid arthritis and other conditions where growth of new blood vessels is involved, prevention of restenosis by inhibition of vascular smooth muscle cell proliferation, acceleration of wound healing by activation of the release of active growth factors stored in the extracellular matrix, and inhibition of tumour cell metastasis by inhibition of angiogenesis.
12. A pharmaceutical or veterinary composition for prophylactic or therapeutic inhibition of angiogenesis in a human or non-human animal, which comprises a polyoxometallate compound as defined in any of claims 1 to 10, in association with at least one pharmaceutically or veterinarily acceptable carrier or diluent.
13. Use of a polyoxometallate compound as defined in any of claims 1 to 10, in, or in the manufacture of a medicament for, prophylactic or therapeutic inhibition of angiogenesis in a human or non-human animal.
PCT/AU1998/000578 1997-07-24 1998-07-24 Heteropolytungstate angiogenesis inhibitors WO1999004803A1 (en)

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