EP0984971A1 - Complexes de vanadium, derives de ces complexes et procedes connexes - Google Patents

Complexes de vanadium, derives de ces complexes et procedes connexes

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Publication number
EP0984971A1
EP0984971A1 EP98916725A EP98916725A EP0984971A1 EP 0984971 A1 EP0984971 A1 EP 0984971A1 EP 98916725 A EP98916725 A EP 98916725A EP 98916725 A EP98916725 A EP 98916725A EP 0984971 A1 EP0984971 A1 EP 0984971A1
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EP
European Patent Office
Prior art keywords
treatment
complex
solution
vanadium
beov
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98916725A
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German (de)
English (en)
Inventor
Zaihui Zhang
Philip Toleikis
Pierre Lemieux
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Angiotech Pharmaceuticals Inc
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Angiotech Pharmaceuticals Inc
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Publication date
Application filed by Angiotech Pharmaceuticals Inc filed Critical Angiotech Pharmaceuticals Inc
Publication of EP0984971A1 publication Critical patent/EP0984971A1/fr
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/34—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D309/36—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms
    • C07D309/38—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms one oxygen atom in position 2 or 4, e.g. pyrones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/005—Compounds of elements of Group 5 of the Periodic Table without metal-carbon linkages

Definitions

  • This invention relates to vanadium(V) complexes, more specifically to hydroxyoxovanadium(V), ⁇ -oxo dimeric oxovanadium(V) and cis-dioxovanadium(V) complexes, to methods for synthesizing such complexes and to the use of such complexes as therapeutic agents.
  • Vanadium complexes have been reported to have therapeutic properties. See, e.g., C. E. Heylinger et al., Science 227:1474, 1985; Y. Shechter, Diabetes 39:1,
  • the present invention is directed to vanadium(V) complexes, including pharmaceutically acceptable salts thereof, of the formula:
  • Z is independently selected from O and NR 4 ;
  • Z 2 is independently selected from O and NR 5 ;
  • Z 3 is independently selected from O, NR 6 and C(R 7 ) 2 ;
  • Ri, R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are each independently selected from H, C,-C
  • compositions containing the above-listed vanadium complexes are a pharmaceutical composition containing one or more vanadium(V) complex as set forth above in admixture with a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention is directed to a method of providing therapeutic treatment to an animal subject in need thereof.
  • a therapeutically effective amount of one or more of the vanadium (V) complexs as identified above is administered to a subject in need thereof.
  • Examples of such therapeutic treatments include treatment of proliferative disorders, bone destruction, metastases, drug resistant tumors, arthritis, psoriasis, multiple sclerosis, diseases involving a passageway of the subject's body, diabetes, diseases of the eye, diabetes-related metabolic complications, such as retinopathy, nephropathy and vasculopathy, hypertension, obesity, chronic inflammatory autoimmune disease, cardiovascular disease, lupus, bacterial infections, joint prostheses failure, periodontal disease, Inflammatory Bowel Disease (IBD), and treatment or prevention of surgical adhesions.
  • proliferative disorders such as proliferative disorders, bone destruction, metastases, drug resistant tumors, arthritis, psoriasis, multiple sclerosis
  • diseases involving a passageway of the subject's body diabetes, diseases of the eye, diabetes-related metabolic complications, such as retinopathy, nephropathy and vasculopathy, hypertension, obesity, chronic inflammatory autoimmune disease, cardiovascular disease, lupus, bacterial infections, joint
  • Figure 1 is a graph showing gross toxicity of various doses (200-600 ⁇ g/dose) of OHBEOV as indicated by body weight changes in DBA-2 mice administered the complex through s.c. injection twice daily for 9 days followed by an observation period of 14 days.
  • Figure 2 is a graph showing gross toxicity of various doses (150-400 ⁇ g/dose) of OHBEOV as indicated by body weight changes in DBA-2 mice administered the complex through intravenous injection twice daily for 9 days followed by an observation period of 14 days.
  • Figure 3 is a graph showing tumor weights at the termination of a tumor efficacy study using a xenograft model of human lung cancer (H460) in immuno- compromised mice (SCID-RAG-2) treated by subcutaneous injection twice daily with various doses of BEOV or OHBEOV for nine days.
  • Figure 4 is a graph relating tumor weights to concentration of vanadium measured at the end of a tumor efficacy study of OHBEOV against MDAY-D2 solid tumors.
  • Figure 5 is a graph depicting plasma leukocyte count following 9 day treatment of a xenograft model of lung cancer (H460) in immuno-compromised mice (SCID-RAG-2) with OHBEOV administered through s.c. injection twice daily at a total dose of 600-1000 ⁇ g/day.
  • Figure 6 is a graph showing tumor volumes derived from caliper measurements taken on a daily basis during a tumor efficacy study using a murine solid tumor model of erythroleukemia (MDAY-D2) in DBA-2 mice treated by s.c. injection twice daily with 500 ⁇ g/dose of OHBEOV for nine days.
  • MDAY-D2 murine solid tumor model of erythroleukemia
  • Figure 7 is a graph showing tumor weights taken at the termination of a tumor efficacy study using a murine solid tumor model of murine lymphoma (MDAY- D2) in DBA-2 mice treated by s.c. injection twice daily with 500 ug/dose of OHBEOV for nine days.
  • Figure 8 is a graph showing tumor volumes derived from caliper measurements taken on a daily basis during a tumor efficacy study using a xenograft model of human lung cancer in immunocompromised (SCID-RAG-2) mice treated with OHBEOV or 0[BEOV] 2 administered by s.c. injection twice daily for nine days.
  • Figure 9 is a graph showing tumor weights taken at the termination of a tumor efficacy study using a xenograft model of human lung cancer in immunocompromised mice (SCID-RAG-2) treated with 500 ⁇ g/dose of OHBEOV or 0[BEOV] 2 administered by s.c. injection twice daily for nine days.
  • the present invention is directed to vanadium(V) complexes, including pharmaceutically acceptable salts thereof, of the formula:
  • Z is independently selected from O and NR 4 ;
  • Z 2 is independently selected from O and NR 5 ;
  • Z is independently selected from O, NR 6 and C(R 7 ) 2 ;
  • R,, R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are each independently selected from H, C,-C l0 alkyl, substituted C,-C 10 alkyl, C 7 -C l5 aralkyl, substituted C 7 -C 15 aralkyl, C 7 -C 15 alkylaryl, substituted C 7 -C 15 alkylaryl, C 6 -C 10 aryl, and substituted C 6 -C, 0 aryl, such that independently R, and R 2 , and R, and R 4 , may together form a C 7 -C ]5 alkylaryl, substituted C 7 -C 15 alkylaryl, C 6 -C ⁇ 0 aryl, and substituted C 6 -C 10 aryl, wherein a substituted alkyl, aral
  • Zdon Z 2 , Z 3 , R quarantine R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are each defined as above; and a ring which includes Z 3 also contains two normalized bonds.
  • R 3 is C 2 -C 10 alkyl, more preferably C 2 -C 5 alkyl. R, is preferably not methyl.
  • the present invention is directed to ⁇ -oxo dimeric oxovanadium(V) complexes, including pharmaceutically acceptable salts thereof, of the formula (I):
  • the invention is directed to hydroxyoxovanadium(V) complexes, including pharmaceutically acceptable salts thereof, of the formula (II):
  • the present invention is directed to cis- dioxovanadium(V) complexes, and pharmaceutically acceptable salts thereof, of the formula (III):
  • Z is independently selected from O and NR 4 ;
  • Z 2 is independently selected from O and NR 5 ;
  • Z 3 is independently selected from O, NRg and C(R 7 ) 2 ;
  • R,, R 2 , R 3 , R 4 , R 5 , R ⁇ and R 7 are each independently selected from H, C,-C 10 alkyl, substituted C r Cj 0 alkyl, C 7 -C ⁇ 5 aralkyl, substituted C 7 -C, 5 aralkyl, C 7 -C, 5 alkylaryl, substituted C 7 -C 15 alkylaryl, C 6 -C l0 aryl, and substituted C 6 -C, 0 aryl, such that independently R, and R 2 , and R, and R 4 , may together form a C 7 -C, 5 alkylaryl, substituted C 7 -C 15 alkylaryl, C 6 -C 10 aryl, and substituted C 6 -C 10 aryl, wherein a substituted alkyl, aralkyl, alkylaryl or aryl contains at least one substituent selected from hydroxyl, fluoro, bromo, chloro, and i
  • Exemplary groups which may be chelants of the vanadium centers in a ⁇ -oxo dimeric oxovanadium(V) complex or a hydroxyoxovanadium(V) complex or a cis-dioxovanadium(V) complex include, without limitation, maltol (i.e., 2-methyl-3-hydroxy-4-pyrone), 3- hydroxyflavone, morin, quercetin, fisetin, and myricetin.
  • Preferred complexes of formulas (I), (II) and (III) have an ⁇ - hydroxypyrone chelant.
  • An -hydroxypyrone chelant has the formula
  • An especially preferred chelant is 2-ethyl-3-hydroxy-4-pyrone (BEOV) which, in a vanadium (V) complex of the invention of formulas (I), (II) and (III), provides a specific complex represented by the following formulas (la), (Ila) and (Ilia), respectively:
  • V vanadium
  • Many of the chelants in the vanadium (V) complexes of the present invention are commercially available.
  • 2-ethyl-3-hydroxy-4-pyrone is commercially available from Aldrich Chemical Co., Milwaukee, WI, among other chemical suppliers.
  • V compounds of the invention include, without limitation: ethoxybis(2-ethyl-3-hydroxy-4-pyronato)oxovanadium(V),
  • the synthesis of the ⁇ -oxo dimeric complexes of formula (I) can be achieved by conventional metallation or transmetallation techniques, e.g., by mixing in solution a soluble vanadium salt with the chelant or a salt or a weaker complex thereof, or by an oxidation process with an oxidant, e.g., hydrogen peroxide, from the corresponding vanadium(IV) complex.
  • the synthesis of the hydroxyoxovanadium(V) complexes of formula (II) can be achieved by metallation techniques under acidic conditions, e.g., by a mixture in solution of a soluble vanadium salt with the chelant or a salt, or by an oxidation process with an oxidant, e.g., hydrogen peroxide, from the corresponding vanadium(IV) complex.
  • metallation techniques under acidic conditions, e.g., by a mixture in solution of a soluble vanadium salt with the chelant or a salt, or by an oxidation process with an oxidant, e.g., hydrogen peroxide, from the corresponding vanadium(IV) complex.
  • the cis-dioxovanadium(V) complexes of formula (III) are negatively charged, and thus are associated with a counterion.
  • Suitable counterions are metal ions, e.g., alkaline and alkaline earth metal ions.
  • Preferred metal ions are sodium and potassium.
  • the counterion may be an organic cation, where a suitable organic counterion is an ammonium ion.
  • the counterion is preferably a pharmaceutically acceptable inorganic counterion.
  • Sodium and potassium are suitable pharmaceutically acceptable inorganic counterions.
  • compositions comprising at least one vanadium (V) complex of the present invention (including a vanadium complex of any of formulas (I), (II) or (III)) in admixture with a carrier, adjuvant or vehicle.
  • the composition is preferably formulated as a pharmaceutical or veterinary composition comprising a pharmaceutically or veterinarily acceptable carrier, excipient or diluent, and optionally, one or more other biologically active ingredients.
  • the present invention provides compositions which include a vanadium(V) complex of the invention in admixture or otherwise in association with one or more inert carriers, as well as optional ingredients if desired.
  • compositions are useful as, for example, assay standards, convenient means of making bulk shipments, or pharmaceutical compositions.
  • An assayable amount of a complex of the invention is an amount which is readily measurable by standard assay procedures and techniques as are well known and appreciated by those skilled in the art. Assayable amounts of a complex of the invention will generally vary from about 0.001 wt% to about 75 wt% of the entire weight of the composition.
  • Inert carriers include any material which does not degrade or otherwise covalently react with a complex of the invention.
  • suitable inert carriers are water; aqueous buffers, such as those which are generally useful in High Performance Liquid Chromatography (HPLC) analysis; organic solvents, such as acetonitrile, ethyl acetate, hexane and the like; and pharmaceutically acceptable carriers.
  • HPLC High Performance Liquid Chromatography
  • the present invention provides a pharmaceutical or veterinary composition (hereinafter, simply referred to as a pharmaceutical composition) containing a vanadium(V) complex as described above, in admixture with a pharmaceutically acceptable carrier, diluent or excipient.
  • a pharmaceutical composition containing an effective amount of a vanadium(V) complex as described above, in association with a pharmaceutically acceptable carrier, diluent or excipient.
  • compositions of the present invention may be in any form which allows for the composition to be administered to a patient.
  • the composition may be in the form of a solid, liquid or gas (aerosol).
  • routes of administration include, without limitation, oral, topical, parenteral, sublingual, rectal, vaginal, and intranasal.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
  • Pharmaceutical composition of the invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient.
  • Compositions that will be administered to a patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of vanadium(V) complex in aerosol form may hold a plurality of dosage units.
  • compositions should be pharmaceutically pure and non-toxic in the amounts used. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of subject (e.g., human), the particular form of the active ingredient, the manner of administration and the composition employed.
  • the pharmaceutical composition includes a vanadium(V) complex as described herein, in admixture with one or more carriers.
  • the carrier(s) may be particulate, so that the compositions are, for example, in tablet or powder form.
  • the carrier(s) may be liquid, with the compositions being, for example, an oral syrup or injectable liquid.
  • the carrier(s) may be gaseous, so as to provide an aerosol composition useful in, e.g., inhalatory administration.
  • the composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
  • the composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form.
  • a solid composition will typically contain one or more inert diluents or edible carriers.
  • binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin
  • excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like
  • lubricants such as magnesium stearate or Sterotex
  • glidants such as colloidal silicon dioxide
  • sweetening agents such as sucrose or saccharin, a flavoring agent such as peppermint, methyl salicylate or orange flavoring, and a coloring agent.
  • composition when in the form of a capsule, e.g., a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or a fatty oil.
  • a liquid carrier such as polyethylene glycol or a fatty oil.
  • the composition may be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension.
  • the liquid may be for oral administration or for delivery by injection, as two examples
  • preferred compositions contain, in addition to at least one complex of the present invention, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer.
  • a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
  • the liquid pharmaceutical compositions of the invention may include one or more adjuvants.
  • Suitable adjuvants include sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, as well as fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium.
  • sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, as well as fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium.
  • Polyethylene glycols, glycerin, propylene glycol or other hydric or non-hydric solvents may also be present.
  • antibacterial agents such as benzyl alcohol or methyl paraben may be included.
  • the compositions preferably do not contain hydric organic compounds, i.e., organic alcohols such as polyethyleneglycols, benzyl alcohol, etc. because organic alcohols may react with the van
  • Antioxidants may be included as an adjuvant in a composition of the invention, however their presence is not preferred because antioxidants may discourage the formation or maintenance of the vanadium(V) state of the complexes of the invention.
  • Antioxidants that could, but are preferably not present include ascorbic acid and sodium bisulfite. Buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose are preferred adjuvants. While the compositions of the invention may contain chelating agents such as ethylenediaminetetraacetic acid, the inclusion of chelating agents in the compositions of the invention is not preferred because the chelating agents may react with the vanadium complexes of the invention.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant.
  • An injectable pharmaceutical composition is preferably sterile.
  • a liquid compositions intended for either parenteral or oral administration should contain an amount of an inventive complex such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of a complex of the invention in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition.
  • Preferred oral compositions contain between about 4% and about 50% of the active vanadium(V) complex.
  • Preferred compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01 to 1% by weight of active complex.
  • the pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base.
  • the base for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.
  • Thickening agents may be present in a pharmaceutical composition for topical administration.
  • the composition may include a transdermal patch or iontophoresis device.
  • Topical formulations may contain a concentration of the inventive complex of from about 0.1 to about 10% w/v (weight per unit volume).
  • the composition may be intended for rectal administration, in the form, e.g., of a suppository which will melt in the rectum and release the drug.
  • the composition for rectal administration may contain an oleaginous base as a suitable non- irritating excipient.
  • bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
  • the composition may include various materials which modify the physical form of a solid or liquid dosage unit.
  • the composition may include materials that form a coating shell around the active ingredients.
  • the materials which form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents.
  • the active ingredients may be encased in a gelatin capsule.
  • composition in solid or liquid form may include an agent which binds to the vanadium(V) complex and thereby assists in the delivery of the active components.
  • agents which may act in this capacity include a monoclonal or polyclonal antibody, a protein or a liposome.
  • the pharmaceutical composition of the present invention may consist of gaseous dosage units, e.g., it may be in the form of an aerosol.
  • aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system which dispenses the active ingredients. Aerosols of complexes of the invention may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. Preferred aerosols may be determined by one skilled in the art, without undue experimentation.
  • the pharmaceutical composition of the present invention may contain one or more known pharmacological agents used in the treatment of asthma, allergy, inflammation (including arthritis) or thrombosis.
  • the pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art.
  • a composition intended to be administered by injection can be prepared by combining the vanadium(V) complex with water so as to form a solution.
  • a surfactant may be added to facilitate the formation of a homogeneous solution or suspension.
  • Surfactants are compounds that non-covalently interact with a vanadium(V) complex of the invention so as to facilitate dissolution or homogeneous suspension of the vanadium(V) complex in the aqueous delivery system.
  • the invention also provides a method of treating an animal subject in need thereof.
  • the animal is preferably warm-blooded.
  • Preferred warm-blooded animals are mammals. Preferred mammals are humans. Livestock, including horses, cows, pigs and fowl are other preferred warm-blooded animal subjects.
  • the method includes the step of administering to the subject a therapeutically effective amount of at least one of the complexes or compositions of the present invention.
  • the treatment may be directed at any one or more of the following conditions: proliferative disorders; bone destruction; metastases; drug resistant tumors; arthritis; psoriasis; multiple sclerosis; a disease involving a passageway of the subject's body; diabetes; a disease of the eye; a diabetes-related metabolic complication selected from retinopathy, nephropathy and vasculopathy; hypertension; obesity; chronic inflammatory autoimmune disease; cardiovascular disease; and lupus.
  • the invention provides a method of treating a proliferative disorder, wherein a patient (subject) in need thereof is administered an effective amount of a vanadium(V) complex of the invention, optionally in combination with a pharmaceutically acceptable carrier, excipient or diluent.
  • the anti-proliferative potential of this complex has been evaluated in two separate solid tumor models in mice.
  • the complex was shown to exhibit significant dose-dependent anti-tumor activity against a xenograft model of human lung cancer with a final tumor size reduction of approximately 70 %.
  • the experiment was repeated using a murine solid tumor model of erythroleukemia and a xenograft model of human lung cancer at the maximum tolerated dose (500 ug/day b.i.d., s.c. for 9 days) and significant tumor efficacy was demonstrated in both models through measurement of tumor sizes on a daily basis using calipers and through tumor weights taken at the termination of the experiment.
  • vanadium levels were measured in tumors following a tumor efficacy protocol and the dose of the drug strongly correlated with the tumor size and vanadium concentration confirming a dose-dependent effect of this complex in tumors.
  • the following examples are offered by way of illustration, not limitation.
  • BKOV bis(kojato)oxovanadium(IV)
  • a solution of sodium orthovanadate (1 mole) in deionized water is added to a solution of kojic acid (2 moles) in 0.005 M HCl with stirring and the pH of the solution is maintained between 2-3 at all times by the addition of HCl.
  • the solid precipitated is collected immediately by filtration, washed with deionized water and dried in vacuo overnight.
  • a suspension of ammonium metavanadate (1 mole) in deionized water is added to a hot solution (60 - 70°C) of kojic acid (2 moles) in 1 M acetic acid with stirring. This mixture is kept stirring at 70°C for 3 hours. The solid obtained is collected by filtration, washed with hot water and dried in vacuo overnight.
  • a suspension of 0(BKOV) 2 in absolute ethanol is heated to 50 - 60°C with stirring for about 1 hour.
  • the volume of the solvent is reduced under reduced pressure and the residue is cooled in a -20°C freezer overnight.
  • the dark red solid precipitated upon cooling is collected by filtration, washed with cooled ethanol and water, and dried in vacuo overnight.
  • EXAMPLE 12 SYNTHESIS OF ⁇ -OXOBIS[BIS(2-METHYL-3-HYDROXY-4- PYRIDINONATO)OXOVANADIUM(V)], [0(VO(MPP) 2 ) 2 ]
  • a suspension of 0(VO(Dpp) 2 ) 2 in absolute ethanol is heated to 50 - 60°C with stirring for about 1 hour.
  • the volume of the solvent is reduced under reduced pressure and the residue is cooled in a -20°C freezer overnight.
  • the dark red solid precipitated upon cooling is collected by filtration, washed with cooled ethanol and water, and dried in vacuo overnight.
  • ammonium metavanadate (1 mole) dissolved in a weak NaOH solution is added slowly to a solution of 8-quinolinol (2 moles) in 1 M acetic acid with stirring. A black precipitate is formed and this mixture is digested on a hot plate at low heat for 5 hours.
  • the black solid is collected by filtration and dried at 120°C.
  • the filtrate is separated from the suspension by vacuum filtration.
  • the volume of the filtrate is reduced under vacuum and the remaining solution is evaporated resulting in the desired product.
  • M.T.D. A Maximum Tolerated Dose (M.T.D.) study was undertaken to determine a safe tolerable dose for administration of OHBEOV through a parenteral (s.c.) route of administration delivered twice daily (b.i.d.) for nine days in female mice (DBA-2).
  • the protocol was as follows. 14 DBA-2 mice were randomly divided into groups of 2 for a total of 7 groups. Groups were treated as follows. Vehicle (sterile isotonic phosphate buffer, pH 7.2) was filtered through 0.22 ⁇ m sterile filter (Costar 140666). OHBEOV was dissolved with gentle heating and stirring for about 20 min. To a concentration of 2 mg/mL in vehicle and filtered through 0.22 ⁇ m sterile filter (Costar 140666).
  • Vehicle control 300 ⁇ l/dose for a total of 600 ⁇ l/day.
  • Injections were s.c. in the neck region and were delivered twice daily between 8 - 9 a.m. and between 4 - 5 p.m. Treatment continued for a total of nine days. Groups were observed and weighed daily for 2 weeks following the final treatment and observed for behavioral signs of toxicity i.e. reduced preening, and mobility and organ failure, i.e., weight loss, death. Following this period, mice were terminated with carbon dioxide.
  • MAXIMUM TOLERATED DOSE STUDY (I.V.) OF OHBEOV A Maximum Tolerated Dose (M.T.D.) study was undertaken to determine a safe tolerable dose for administration of OHBEOV through an intravenous route of administration delivered twice daily for nine days in female mice (DBA-2).
  • the protocol was as follows. 8 DBA-2 female mice were randomly divided into groups of 2 for a total of 4 groups. Groups were treated as follows. Vehicle (sterile isotonic phosphate buffer, pH 7.2) was filtered through 0.22 ⁇ m sterile filter (Costar 140666). OHBEOV was dissolved with gentle heating and stirring to a concentration of 2 mg/mL in vehicle and filtered through 0.22 ⁇ m sterile filter (Costar 140666). Groups were as follows:
  • Vehicle control 300 ⁇ l/dose for a total of 600 ⁇ l/day.
  • CANCER A dose-response study was undertaken to determine the efficacy of OHBEOV using Scid/Rag-2 mice with implanted human H460 tumors (lung cancer). The purpose was to establish safe efficacious levels of s.c, b.i.d. administration of OHBEOV for 9 days.
  • the protocol used is as follows. 20 female Scid/Rag-2 mice were randomly divided into 4 groups. Groups were treated as follows. H460 tumor cells were inoculated (50 ⁇ l 10 6 cells bilaterally in the posterior dorsal region) as discussed earlier. On day 1 1 following tumor inoculation, the 9 day treatment began. Vehicle (isotonic phosphate buffer) filtered through 0.22 ⁇ m sterile filter (Costar 14066).
  • OHBEOV was dissolved with gentle heating and stirring to a concentration of 2 mg/mL in vehicle and filtered through 0.22 ⁇ m sterile filter (Costar 140666).
  • the groups were as follows: 1) Vehicle control: isotonic phosphate buffer. 300 ⁇ l/dose, total of
  • mice were weighed as a group prior to injection in the morning. Injections were s.c. in the neck region distal to the tumor sight and were delivered b.i.d. between 8-9 a.m. and between 4 - 5 p.m. Tumor volumes were estimated on a daily basis from measurements taken with calipers.
  • mice Treatment continued for a total of nine days. Mice were terminated on day 10 with carbon dioxide. Tumors were removed and weighed following blotting. Results and discussion:
  • EXAMPLE 36 TUMOR EFFICACY OF OHBEOV ON MDAY-D2 TUMORS
  • the purpose was to confirm efficacious levels of s.c, b.i.d. administration of OHBEOV for 9 days at 500 ⁇ g/dose.
  • the protocol used is as follows. 10 DBA-2 mice were randomly divided into 2 groups. Groups were treated as follows. MDAY-D2 tumor cells were inoculated (50 ⁇ l 10 6 cells bilaterally in the posterior dorsal region) as discussed earlier. On day 11 following tumor inoculation, the 9 day treatment began.
  • Vehicle isotonic phosphate buffer
  • OHBEOV was dissolved with gentle heating and stirring to a concentration of 2 mg/mL in vehicle and filtered through 0.22 ⁇ m sterile filter (Costar 140666).
  • the groups were as follows: 1) Vehicle control: isotonic phosphate buffer. 300 ⁇ l/dose, total of
  • mice 300 ⁇ l to be injected for a total of 1000 ⁇ g/day.
  • Mice were weighed as a group prior to injection in the morning. Injections were subcutaneous in the neck region distal to the tumor sight and were delivered b.i.d. between 8 - 9 a.m. and between 4 - 5 p.m. Tumor volumes were estimated on a daily basis from measurements taken with calipers. Treatment continued for a total of nine days. Mice were terminated on day 10 with carbon dioxide. Tumors were removed and weighed following blotting.
  • 0[BEOV] 2 using SCID-RAG-2 female mice with implanted human H460 tumors (lung cancer). The purpose was to establish safe efficacious levels of s.c, b.i.d. administration of OHBEOV and 0[BEOV] 2 for 9 days at 500 ⁇ g/dose.
  • Vehicle control isotonic phosphate buffer. 300 ⁇ l/dose, total of 600 ⁇ l/day. 2) 250 ⁇ l of 2 mg/mL OHBEOV mixed with 50 ⁇ l vehicle to make 300 ⁇ l to be injected for a total of 1000 ⁇ g/day.
  • Injections were subcutaneous in the neck region distal to the tumor sight and were delivered b.i.d. between 8 - 9 a.m. and between 4 - 5 p.m. Tumor volumes were estimated on a daily basis from measurements taken with calipers. Treatment continued for a total of nine days. Mice were terminated on day 10 with carbon dioxide. Tumors were removed and weighed following blotting.
  • mice (day 0) and subsequently were kept in an animal facility under standard conditions. Survival of mice was determined only in protocol 2.
  • Protocol 1 Effects of a period of treatment with vanadium complexes on established bone metastases. Animals are inoculated with subconfluent MDA-231 breast cancer cells
  • % oj increase X ⁇ 00 osteolytic metastasis # at day 17
  • Protocol 2 Effects of continuous treatment with vanadium complexes on the development of new bone metastases.
  • vanadium complexes dose 1, 2, and 3 in ⁇ g/mouse/day
  • the control group receives PBS.
  • Radiographs are taken at day 28 to assess the presence of osteolytic bone metastases. Mice are then kept untreated until they die. Survival of each animal is determined by the duration between day of cell inoculation and death.
  • Protocol 3 Effects of a short period of prophylactic treatment with vanadium complexes on the development of new bone metastases.
  • Vanadium complexes are administered s.c. into female nude mice (3 week-old) once a day for 7 days before cell inoculation (day -7) and 7 days after inoculation. At this point, the administration of vanadium complexes is discontinued, and nude mice are then inoculated with MDA-231 breast cancer cells into the left ventricle (day 0). Radiographs of these animals are taken, and the mice are sacrificed for histological examination at day 28. Control mice receive PBS. Radiographs and measurement of osteolytic lesion area:
  • Video images are captured using a frame grabber board (Targa+; Truevision, USA) with an IBM compatible 486/33 MHz computer. Quantitation of lesion area is performed using image analysis software (Jandel Video Analysis, Jandel Scientific, Corte Madera, CA).
  • Fore- and hindlimb long bones are removed from mice at the time of sacrifice, fixed in 10%> buffered formalin, decalcified in 14% EDTA, and embedded in paraffin wax. Sections are cut using a standard microtome, placed on poly-L-lysine- coated glass slides and stained with hematoxylin, eosin, orange G, and phloxine. The following variables are measured in midsections of tibiae and femora, without knowledge of treatment groups, to assess tumor involvement: total bone area, total tumor area, and osteoclasts number expressed per millimeter of tumor/bone interface. Histomorphometric analysis is performed on an OsteoMeasure System (Osteometrics, Atlanta, GA) using an IBM compatible computer.
  • OsteoMeasure System OsteoMeasure System
  • one or several animals of each experiment are sacrificed.
  • the presence of metastases is determined by gross autopsy and examination of H&E stained sections of any suspicious organ and step sections of the lungs and draining lymph nodes.
  • animals suspected of being in distress are sacrificed.
  • VANADIUM COMPLEXES Mammalian cells carrying AP-1 or NF-KB reporter luciferase gene constructs are simultaneously submitted to growth factors and cytokines (EGF, TNF, IL-1B, PDGF, VEGF, IGF-1, etc.) and to various vanadium complexes. This protocol allows the identification of the signal transduction pathways that are affected by the vanadium complexes. Once a signal transduction pathway is identified as being a target, the vanadium complexes are screened to determine which one has the highest activity in that specific transduction pathway. Also, specific enzymatic assays (JNK, MAPK, p38) are carried out in parallel to validate the screening process. The enzymes chosen reflect indirectly AP-1 or NF-KB activities.
  • KB8 Kb8-5 and KB85-11
  • KB3-1 KB3-1
  • DMEM media containing 0 to 50 ⁇ M 0[BEOV] 2 and after 24-72 hours the number of viable cells is determined.
  • EXAMPLE 42 CYTOTOXICITY ASSAYS The relative cytotoxicity of vanadium complexes on tumor cell lines was measured using the MTT microculture tetrazolium colorimetric assay. The relative cytotoxicity of a variety of vanadium complexes (Na 3 V0 4 ,
  • VOS0 4 BMOV, BEOV and Naglivan was determined using the following tumor cell lines: P388 (WT) (murine leukemia), P388 (ADR) (murine leukemia), Lewis Lung (murine lung), MCF7 (WT) (human breast), MCF7 (ADR) (human breast), H460 (human non-small lung), K562 (human erythroleukemia), A431 (human epidermal), LSI 80 (human colon) and SK-OV-3 (human ovarian).
  • P388 (WT) murine leukemia
  • P388 (ADR) murine leukemia
  • Lewis Lung murine lung
  • MCF7 (WT) human breast
  • MCF7 (ADR) human breast
  • H460 human non-small lung
  • K562 human erythroleukemia
  • A431 human epidermal
  • LSI 80 human colon
  • SK-OV-3 human ovarian
  • Chondrocytes are plated and maintained for 48 hours at both high cell density (2 x IO 6 to 4 x IO 6 cells/per well on a six well plate) (non-proliferating) and low cell density (5 x IO 5 to 1 x IO 6 cells/per well on a six well plate) (proliferating). For an additional 48 hours, the cells are incubated in media (HAMS F12) containing 0 to 50 ⁇ M 0[BEOV] 2 . The number of viable cells is then determined.
  • media HAMS F12
  • orthovanadate did not affect the non-proliferating cells although it was toxic to proliferating cells.
  • 0[BEOV] 2 can be administered through several routes of administration.
  • 0[BEOV] 2 is given as a suspension in 1% methyl cellulose at a range of doses (approximately 15 mg/kg).
  • Administration of 0[BEOV] 2 can also given in separate experiments by oral administration in drinking water given ad libitum to rats at a concentration of between 0.2-2.0 mg/mL.
  • 0[BEOV] 2 can also be given through oral gavage at a dose of 100-200 ⁇ mol/kg. Blood glucose levels are measured at intervals using a test strip and glucometer obtained commercially.
  • Male (Wistar) rats are made diabetic by i.v. injection of streptozotocin (STZ) as a single dose of 60 mg/kg dissolved in 0.9%) saline via the tail vein.
  • Control groups are injected with saline vehicle only.
  • 0[BEOV] 2 is administered to normal and STZ -treated rats via drinking water.
  • Four groups are established as follows: control, diabetic, control-treated, diabetic-treated. The diabetic state is confirmed with a "Testape"TM at 3 days post- injection and later confirmed with a glucometer test. Blood glucose and insulin levels are measured through the course of the study. Treatment is started 1 week following STZ injection.
  • Treated animals receive 0.1-1.0 mmol/kg of the complex/day in drinking water over a 3 month period.
  • Parameters measured at various time-points through the study include animal weight, blood glucose levels, food consumption, fluid consumption, glycosylated hemoglobin, plasma triglycerides and cholesterol levels.
  • SHR spontaneously hypertensive rats
  • WKY Wistar Kyoto
  • rats are obtained at 4 weeks of age and divided into 4 groups as follows: SHR (untreated), SHRVan (vanadium- treated), WKY (untreated), WKYVan (vanadium-treated).
  • 0[BEOV] 2 is administered (0.1 - 2.0 mg/mL) in drinking water given ad libitum to the SHRVan and WKYVan groups at 5 weeks of age on a constant basis. Following week 8, when hypertension is fully manifest in the SHR, weekly measurements of plasma insulin and systolic blood pressure (tail cuff method, or implanted arterial catheters) are taken in all groups. Weekly measurements of blood pressure are taken and blood samples collected for subsequent glucose and insulin analysis. As a part of this experiment, the effects of 0[BEOV] 2 are assessed for its appetite suppression effects by measuring weight gain.
  • EXAMPLE 48 THE EFFECT OF 0[BEOV], ON SYNOVIOCYTE PROLIFERATION Incubating plated synoviocytes with 0[BEOV] 2 for 24 hours assesses the effect of 0[BEOV] 2 on synoviocyte proliferation. The number of viable cells is determined by the dye exclusion method.
  • vanadium complexes such as orthovanadate and vanadyl sulphate, inhibited synoviocyte proliferation and were cytotoxic to the cells.
  • CIA in rats is a model of chronic inflammatory synovitis with pannus formation, neovascularization, and joint destruction similar to rheumatoid arthritis (RA).
  • RA rheumatoid arthritis
  • Control rats receive only an aqueous vehicle at a dose of 100 mg / kg / day s.c
  • the experimental group receives an aqueous vehicle at 100 mg / kg / day s.c, as well as 0[BEOV] 2 subcutaneously at the appropriate dose.
  • the severity of clinical arthritis in each limb is scored daily based on an objective integer scale of 0 - 4 (D.E. Trentham et al., J. Exp. Med. 146:851-868, 1977).
  • a score of 0 indicates an unaffected limb, while a score of 4 represents fulminant erythema and edema involving distal digits.
  • the arthritic index is defined as the sum of its four limb scores. Since CIA typically involves only the hind limbs, an arthritic index of 6 to 8 is considered to represent severe arthritis.
  • hind limb radiographs are obtained.
  • the radiographic index is defined as the sum of the limbs.
  • the humoral immunity is evaluated by collecting rat serum on Day 18 post arthritis onset for measuring anti-CII IgG by an enzyme linked immunosorbent assay (ELISA) (E. Brahn and D.E. Trentham, Cell Immunol. 86:421-428, 1984; E. Brahn and D.E. Trentham, Cell Immunol. 118:491-503, 1989).
  • Antibody titers are normalized against a previously standardised curve and absorbance read at 490 nm at a serum dilution of 1 :2500.
  • Synovium is selected from rats on Day 5 and Day 18-post arthritis onset to study joint morphology using electron microscopy.
  • Conventional transmission electron microscopy is also performed on the articular cartilage of the trochlear surfaces of native, arthritic control, and 0[BEOV] 2 -treated animals.
  • EXAMPLE 50 PREVENTION OF ARTHRITIS ONSET BY 0[BEOV] 2 IN THE CIA RAT MODEL
  • CIA is a T cell-dependent animal model of the disease that is induced by immunization of the animals with type II collagen.
  • Syngeneic female Louvain rats weighing 120 to 150 grams are injected intradermally with 0.5 mg of native chick collagen II (Genzyme, Boston, MA) solubilized in 0.1 M acetic acid and emulsified in FIA (Difco, Detroit, MI).
  • FIA FIA
  • animals develop a polyarthritis with histologic changes of pannus formation and bone/cartilage erosions.
  • 0[BEOV] 2 is administered i.p. or s.c.
  • the control and experimental animals are evaluated for disease severity both clinically and radiographically by individuals blinded to treatment groups.
  • Delayed-type hypersensitivity (DTH) to CII is determined by a radiometric ear assay completed on day 28. Radiometric ear indices >1.4 represent a significant response to CII.
  • the presence of anti-CII IgG antibodies is determined by enzyme-linked immunosorbent assay (ELISA). Serum samples obtained on day 26 are diluted to 1 :2,560, and the results are expressed as the mean optical density at 490 nm, in quadruplicate aliquots. Background levels in normal rat serum at this dilution are 0 and are readily distinguishable from collagen-immunized rat serum.
  • Diarrhoea was not observed when BMOV was given at a dose of 10 mg / kg / day. However, when the dose was increased to 15 mg / kg / day on Day 1 1 post arthritis onset, a several experimental rats manifested minor diarrhoea. The mean anti-CII IgG titer of the control group was significantly higher than that of the experimental group (p ⁇ 0.004). The biological significance of this difference, however, remained unclear since the magnitude of the difference was minimal and previous experiments have shown that arthritic rats often produce higher titers of anti-CII IgC than non-arthritic rats. X-rays of control and experimental rat limbs illustrated a typical arthritic control limb as having soft tissue swelling and bone erosion. These features are absent in the vanadate treated experimental limb.
  • the articular cartilage of control rats was characteristically scabrous with an excessive number of erosion sites, pits and adhering cells.
  • the BMOV- treated rats exhibited a normal trochlear surface characterized by scant adhering elements and a smooth articular surface with orderly arranged collagen fibrils. Articular surface was mechanically damaged during dissection.
  • the scanning and transmission electron micrographs demonstrated dramatic cartilage destruction in the control joints with exposed or absent chondrocytes in the denuded cartilage. Joints from BMOV-treated rats demonstrated little cartilage damage and intact cartilage.
  • the results show that the combination of BMOV and NAC significantly regressed established CIA, compared to the control using NAC alone, by both clinical and radiologic criteria.
  • the results indicate that the combination of vanadate and NAC regressed established CIA via decreasing collagenase expression.
  • Collagenase mRNA expression in control arthritic rats were significantly higher than that in combination treated non-arthritic rats.
  • the scanning electron micrographs showed much erosion in the synovium of control joints, with chondrocytes exposed to the synovial surface. In contrast, the surface of combination treated synovium had a smooth appearance without chondrocytes exposed.
  • the single agent NAC had no appreciable effect on the clinical severity of CIA.
  • the combination of vanadate and NAC demonstrated efficacy at regressing established CIA due to at least two molecular mechanisms: decreased collagenase gene expression and decreased hydrogen peroxide concentration.
  • the four-month-old animals are injected with 0[BEOV] 2 after the initial signs of MS reach a score of 1+ in the symptoms described above.
  • the course of treatment is spread over 24 days by treating the animals with 0[BEOV], every three days (or alternate dosing schedule based on maximum tolerated dose studies). The body weight and clinical signs described above are determined on each injection day.
  • tissues are formalin fixed and paraffin embedded. Sections of 5 microns are stained with anti-GFAP antibody (DACO), washed and then reacted with secondary antibody conjugated with HPP. The sections are stained for HPP and counter-stained with haematoxylin.
  • DACO anti-GFAP antibody
  • keratinocytes The effect of OfBEOV], on keratinocytes is assessed by determining the cell number and ⁇ -thymidine incorporation by the cells.
  • thymidine incorporation keratinocytes plated at low density (in DMEM, supplemented with 10% FCS, glutamine, antibiotics) are treated with OfBEOV], concentrations of 0 to IO "4 M for 6 hours during logarithmic growth.
  • ⁇ -thymidine is added to the cells and incubated for a further 6 hours. The cells are harvested and radioactivity determined.
  • To determine the total cell numbers keratinocytes are plated as described and incubated in the presence and absence of OfBEOV], for 4 days. Following incubation, cells are collected and counted by the trypan blue exclusion assay.
  • 0[BEOV] 2 FOR THE TREATMENT OF PSORIASIS Keratinocyte mitosis occurs almost exclusively in the basal layer of the epidermis and therefore to combat hyperproliferation 0[BEOV] 2 must reach the cells of this layer.
  • In vitro testing using an animal skin model will determine the most likely formulation candidates.
  • Mini-pig skin will be used as it most closely approximates the human skin permeation barrier.
  • the topical formulation comprises the following: Labrafil® M2130CS (25%>), Labrasol® (25%), Transcutol® (25%), Arlacel® 165 (12%), isopropyl myristate (10%)) and Compritol (3%>).
  • OfBEOV is dissolved in the formulation concentration of 0.1 %>w/w.
  • a specific weight of formulation is applied to excised, dermis removed mini -pig skin mounted on Franz diffusion cells maintained at 37°C. After 24 hours, the skin is washed clean and removed from the diffusion cells. All fluids are kept and assayed for vanadium via atomic absorbance spectroscopy. The skin is microtomed into sections, and the concentration and distribution of vanadium is determined in each section via atomic absorbance spectroscopy. The sectioned skin is also used to determine where vanadium resides in each section. Both treated and untreated skin samples are examined through light and electron microscopy.
  • One type of systemic formulation for OfBEOV] 2 is comprised of amphiphilic diblock copolymers of micelles consisting of a hydrophobic core and a hydrophilic shell in water.
  • Diblock copolymers of poly(DL-lactide)-block-methoxy polyethylene glycol (PDLLA-MePEG), polycaprolactone-block-me hoxy polyethylene glycol (PCL-MePEG) and poly(DL-lactide-co-caprolactone)-block-methoxy polyethylene glycol (PDLLACL-MePEG) can be synthesized using a bulk melt polymerization procedure, or similar methods.
  • the strong association within the internal core of the polymeric micelles presents a high capacity environment for carrying drugs such as 0[BEOV] 2 .
  • the agents may be coupled to block copolymers to form a micellar structure or can be physically incorporated within the hydrophobic cores of the micelles.
  • the mechanisms of drug release from the micelles include diffusion from the core and the exchange between the single polymer chains and the micelles.
  • the small size of the micelles (normally less than 100 nm) will eliminate the difficulties associated with injecting larger particles.
  • Immunodeficient SCID mice are used as recipients for surface transplants of human keratinocyte lines transfected with vascular endothelial growth factor (VEGF) in sense or antisense orientation. Keratinocytes are transplanted via use of modified silicon transplantation chamber assay onto the skin of recipient mice. Keratinocytes are allowed to differentiate and to induce skin angiogenesis. OfBEOV], is then given either systemically or topically (cream, ointment, solution suspension, lotion, gel), and mo ⁇ hometric measurements of vessel numbers and sizes are performed in untreated and treated groups.
  • VEGF vascular endothelial growth factor
  • mice are sensitized to oxazolone by topical application of the compound onto the skin. Five days later, mice are challenged with oxazolone by topical application onto the ear skin (left ear: oxazolone, right ear: vehicle alone), resulting in a cutaneous inflammatory, "delayed-type hypersensitivity" reaction. The extent of inflammation can be quantified by measurements of the resulting ear swelling over a period of 48 hours.
  • Epon- embedded, Giemsa-stained, 1 ⁇ m-tissue sections are evaluated for the presence of inflammatory cells, for the presence of tissue mast cells and their state of activation, and for the degree of epidermal hype ⁇ lasia. OfBEOV], is given either systemically or topically to quantitate its effect on the cutaneous inflammatory reaction in this in vivo model.
  • New Zealand white rabbits are placed on a powdered chow supplemented by 0.25%> cholesterol by weight.
  • Total plasma cholesterol is measured on a weekly basis by taking samples from a marginal ear vein after an injection of Innovar (0.1 ml/kg) to dilate blood vessels. Samples are mixed with EDTA to achieve a 0.15% concentration in the sample and placed on ice until separation of plasma by low speed centrifugation.
  • the animals are randomized into 3 groups of 10. After anaesthetic induction with Ketamine 35 mg/kg and Xylazine 7 mg/kg, and then general anesthesia via intubation, the fur is shaved and the skin sterilized over the abdomen. A laparotomy is performed and the abdominal aorta isolated. Using a 22g needle, ethylene vinyl acetate paste, ethylene vinyl acetate paste containing 5% OfBEOV], complex, or ethylene vinyl acetate paste containing 33%o OfBEOV] 2 complex is placed in a circumferential manner around the proximal half of the infrarenal abdominal aorta. The distal half of the aorta extending to the aortic bifurcation is not treated. In 10 control rabbits, the infrarenal abdominal aorta is isolated, but nothing is injected around it.
  • the atherogenic chow is continued for 24 weeks.
  • the animals are anesthetized with an injection of Ketamine (350 mg/kg) and Xylazine (7 mg/kg) intramuscularly and then sacrificed with an intravenous overdose of Euthanol (240 mg/ml; 2 ml/4.5 kg).
  • the animals are then perfusion fixed at 100 mm mercury via the left ventricle by perfusing Hanks' balanced salt solution with 0.15 mmol/litre N-2- hydroxyethylpaparazine-N'-2-ethanesulfonic acid (pH 7.4) containing Heparin (1 IU/mL) for ten minutes followed by dilute Karnovsky's fixative for 15 minutes.
  • the thoracic and abdominal aorta and iliac arteries are removed en bloc and are placed in a similar solution for a further 30 minutes.
  • Wistar rats weighing 250 - 300 g are anesthetized by the intramuscular injection of innovar (0.33 ml/kg). Once they are sedated they are placed under Halothane anesthesia. After general anesthesia is established, the fur over the neck region is shaved and the skin cleansed with Betadine. A vertical incision is made over the left carotid artery and the external carotid artery exposed. Two ligatures are placed around the external carotid artery and a transverse arteriotomy is made between them. A 2 Fr Fogarty balloon catheter is introduced into the external carotid artery and passed into the left common carotid artery and the balloon is inflated with saline. The catheter is passed up and down the carotid artery three times to denude the endothelium. The catheter is removed and the ligatures tied off on the left external carotid artery.
  • the animals are randomized into groups of 5. Subgroups of 5 rats are control, carrier polymer alone, carrier polymer plus 1, 5, 10, 20, and 33%) OfBEOV] 2 complex is delivered. There are two carrier polymers to be investigated; EVA and EVA/PLA blend. The polymer mixture is placed in a circumferential manner around the carotid artery. The wound is then closed. Rats in each group are sacrificed at 14 and 28 days. In the interim, the rats are observed for weight loss or other signs of systemic. After 14 or 28 days, the animals are sacrificed by initial sedation with intramuscular Innovar (0.33 ml/kg). The arteries are then examined for histology. EXAMPLE 59
  • the animals end to side anastomoses, the proximal anastomosis on the common carotid artery and the distal anastomosis on the internal carotid artery bilaterally.
  • the intervening bypassed artery is ligated.
  • the animals are randomized into groups of 10 pigs receiving carrier polymer alone, 10 pigs receiving carrier polymer plus 5%> w/w 0[BEOV] 2 complex, and 10 pigs receiving carrier polymer plus 33% w/w OfBEOV], complex adjacent to each surgical created anastamosis on the left side only.
  • the right sided grafts will serve as a control in each pig.
  • the wounds are closed and the pigs recovered.
  • a second group of pigs are studied.
  • the grafts are created in a similar manner. No vasoactive agent is placed next to the anastamotic sites at the time of operation. The animals are recovered. Two weeks after the graft has been performed, a second general anaesthetic is administered and the left carotid artery is reexplored.
  • mice in each group Five pigs in each group are sacrificed. The animals are then perfusion fixed at 100 mm mercury via the left ventricle by perfusing Hanks' balanced salt solution with 0.15mmol/litre N-2-hydroxyethylpaparazine-N'-2-ethanesulfonic acid (pH 7.4) containing Heparin (1 IU/mL) for ten minutes followed be dilute Karnovsky's fixative for 15 minutes. The thoracic and abdominal aorta and carotid arteries are removed en bloc and are placed in a similar solution for a further 30 minutes.
  • Hanks' balanced salt solution with 0.15mmol/litre N-2-hydroxyethylpaparazine-N'-2-ethanesulfonic acid (pH 7.4) containing Heparin (1 IU/mL) for ten minutes followed be dilute Karnovsky's fixative for 15 minutes.
  • the thoracic and abdominal aorta and carotid arteries are removed en bloc
  • Histological sections through the carotid artery immediately proximal to the proximal anastamosis, at the proximal anastamosis, at the distal anastamosis and the carotid artery immediately distal to the distal anastamosis are made.
  • the sections are stained with Movat and H&E and Masson stains. Histologic analysis of intimal and advantitial reaction as well as perivascular reaction are noted. Mo ⁇ hometric analysis with degree of luminal narrowing is calculated.
  • the remaining pigs are studied at 6 months and a similar angiography sacrifice procedures is performed.
  • compositions may be produced: (1) as a "thermopaste” that is applied to a desired site as a fluid, and hardens to a solid of the desired shape at a specified temperature (e.g., body temperature); (2) as a spray (i.e., "nanospray") which may delivered to a desired site either directly or through a specialized apparatus (e.g., endoscopy), and which subsequently hardens to a solid which adheres to the tissue to which it is applied; (3) as an adherent, pliable, resilient, angiogenesis inhibitor-polymer film applied to a desired site either directly or through a specialized apparatus, and which preferably adheres to the site to which it is applied; and (4) as a fluid composed of a suspension of microspheres in an appropriate carrier medium, which is applied to a desired site either directly or via a specialized apparatus, and which leaves
  • Reagents and equipment which are utilized within the following experiments include a sterile glass syringe (1 mL), Corning hot plate/stirrer, 20 mL glass scintillation vial, molds (e.g., 50 ⁇ l DSC pan or 50 mL centrifuge tube cap inner portion), scalpel and tweezers, Polycaprolactone ("PCL" - mol wt 10,000 to 20,000;
  • the syringe may be reheated to 60°C and administered as a liquid which solidifies when cooled to body temperature.
  • Nanospray is a suspension of small microspheres in saline. If the microspheres are very small (i.e., under 1 ⁇ m in diameter) they form a colloid so that the suspension will not sediment under the force of gravity. As is described in more detail below, a suspension of 0.1 ⁇ m to 1 ⁇ m microparticles may be created suitable for deposition onto tissue through a finger pumped aerosol.
  • Equipment and materials which may be utilized to produce nanospray include 200 mL water jacketed beaker (Kimax or Pyrex), Haake circulating water bath, overhead stirrer and controller with 2 inch diameter (4 blade, propeller type stainless steel stirrer; Fisher brand), 500 mL glass beaker, hot plate/stirrer (Corning brand), 4 X 50 mL polypropylene centrifuge tubes (Nalgene), glass scintillation vials with plastic insert caps, table top centrifuge (Beckman), high speed centrifuge - floor model (JS 21 Beckman), Mettler analytical balance (AJ 100, 0.1 mg), Mettler digital top loading balance (AE 163, 0.01 mg), automatic pipetter (Gilson), sterile pipette tips, pump action aerosol (Pfeiffer pharmaceuticals) 20 ml, laminar flow hood, polycaprolactone ("PCL” - mol wt 10,000 to 20,000; Polysciences, Warrington, Pennsylvania USA), "washed”
  • the solution can be prepared by following the procedure given below, or by diluting the 5% (w/v) PVA stock solution prepared for production of microspheres. Briefly, 17.5 g of PVA is weighed directly into a 600 mL glass beaker, and 500 mL of distilled water is added. Place a 3 inch Teflon coated stir bar in the beaker. Cover the beaker with a cover glass to reduce evaporation losses. Place the beaker in a 2000 mL glass beaker containing 300 mL of water. This will act as a water bath. Stir the PVA at 300 ⁇ m at 85°C (Corning hot plate/stirrer) for 2 hours or until fully dissolved.
  • Dissolving of the PVA can be determined by a visual check; the solution should be clear. Use a pipette to transfer the solution to a glass screw top storage container and store at 4°C for a maximum of two months. This solution should be warmed to room temperature before use or dilution.
  • nanospray containing OfBEOV To manufacture nanospray containing OfBEOV], prepare the polymer drug stock solution, weigh the appropriate amount of OfBEOV], directly into a 20 mL glass scintillation vial. The appropriate amount is determined based on the percentage of OfBEOV], to be in the nanospray. For example, if nanospray containing 5% 0[BEOV] 2 was required, then the amount of OfBEOV] 2 weighed would be 25 mg since the amount of polymer added is 10 mL of a 5% polymer in DCM solution (see next step).
  • the term film refers to a polymer formed into one of many geometric shapes.
  • the film may be a thin, elastic sheet of polymer or a 2 mm thick disc of polymer. This film is designed to be placed on exposed tissue so that any encapsulated drug is released from the polymer over a long period of time at the tissue site. Films may be made by several processes, including for example, by casting, and by spraying.
  • polymer In the casting technique, polymer is either melted and poured into a shape or dissolved in DCM and poured into a shape. The polymer then either solidifies as it cools or solidifies as the solvent evaporates, respectively.
  • the spraying technique the polymer is dissolved in solvent and sprayed onto glass, as the solvent evaporates the polymer solidifies on the glass. Repeated spraying enables a build up of polymer into a film that can be peeled from the glass.
  • Reagents and equipment which were utilized within these experiments include a small beaker, Corning hot plate stirrer, casting molds (e.g., 50 mL centrifuge tube caps) and mold holding apparatus, 20 mL glass scintillation vial with cap (Plastic insert type), TLC atomizer, nitrogen gas tank, polycaprolactone ("PCL” - mol wt 10,000 to 20,000; Polysciences), OfBEOV],, ethanol, "washed” (see previous) ethylene vinyl acetate (“EVA”), poly(DL)lactic acid (“PLA” - mol wt 15,000 to 25,000; Polysciences), dichloromethane (HPLC grade Fisher Scientific).
  • Nanopaste is a suspension of microspheres suspended in a hydrophilic gel.
  • the gel or paste can be smeared over tissue as a method of locating drug-loaded microspheres close to the target tissue. Being water based, the paste will soon become diluted with bodily fluids causing a decrease in the stickiness of the paste and a tendency of the microspheres to be deposited on nearby tissue. A pool of microsphere encapsulated drug is thereby located close to the target tissue.
  • Reagents and equipment which were utilized within these experiments include glass beakers, Carbopol 925 (pharmaceutical grade, Goodyear Chemical Co.), distilled water, sodium hydroxide (1 M) in water solution, sodium hydroxide solution (5 M) in water solution, microspheres in the 0.1 lm to 3 lm size range suspended in water at 20% w/v.
  • MANUFACTURE OF MICROPHERES Equipment which is preferred for the manufacture of microspheres described below include: 200 mL water jacketed beaker (Kimax or Pyrex), Haake circulating water bath, overhead stirrer and controller with 2 inch diameter (4 blade, propeller type stainless steel stirrer - Fisher brand), 500 mL glass beaker, hot plate/stirrer (Corning brand), 4 x 50 mL polypropylene centrifuge tubes (Nalgene), glass scintillation vials with plastic insert caps, table top centrifuge (GPR Beckman), high speed centrifuge- floor model (JS 21 Beckman), Mettler analytical balance (AJ 100, 0.1 mg), Mettler digital top loading balance (AE 163, 0.01 mg), automatic pipetter (Gilson).
  • Reagents include polycaprolactone ("PCL” - mol wt 10,000 to 20,000; Polysciences, Warrington Pennsylvania, USA), “washed” (see later method of “washing") ethylene vinyl acetate (“EVA”), poly(DL)lactic acid (“PLA” - mol wt 15,000 to 25,000; Polysciences), polyvinyl alcohol (“PVA” - mol wt 124,000 to 186,000; 99% hydrolyzed; Aldrich Chemical Co., Milwaukee WI, USA), dichloromethane (“DCM” or “methylene chloride”; HPLC grade Fisher scientific), and distilled water.
  • PCL polycaprolactone
  • EVA ethylene vinyl acetate
  • PVA poly(DL)lactic acid
  • PVA polyvinyl alcohol
  • DCM dichloromethane
  • HPLC grade Fisher scientific distilled water
  • the stirrer is then started, and 10 mL of polymer solution (polymer solution used based on type of microspheres being produced) is then dripped into the stirring PVA over a period of 2 minutes using a 5 mL automatic pipetter. After 3 minutes the stir speed is adjusted (see Table I), and the solution stirred for an additional 2.5 hours.
  • the stirring blade is then removed from the microsphere preparation, and rinsed with 10 mL of distilled water so that the rinse solution drains into the microsphere preparation.
  • the microsphere preparation is then poured into a 500 mL beaker, and the jacketed water bath washed with 70 mL of distilled water, which is also allowed to drain into the microsphere preparation.
  • the 180 mL microsphere preparation is then stirred with a glass rod, and equal amounts are poured into four polypropylene 50 mL centrifuge tubes. The tubes are then capped, and centrifuged for 10 minutes (force given in Table I). A 5 mL automatic pipetter or vacuum suction is then utilised to draw 45 mL of the PVA solution off of each microsphere pellet.
  • microsphere preparation is transferred into a pre-weighed glass scintillation vial. The vial is capped, and left overnight at room temperature (25°C) in order to allow the microspheres to sediment out under gravity. Microspheres, which fall in the size range of 0.1 ⁇ m to 3 ⁇ m, do not sediment out under gravity, so they are left in the 10 mL suspension.
  • microspheres After the microspheres have sat at room temperature overnight, a 5 mL automatic pipetter or vacuum suction is used to draw the supernatant off of the sedimented microspheres.
  • the microspheres are allowed to dry in the uncapped vial in a drawer for a period of one week or until they are fully dry (vial at constant weight). Faster drying may be accomplished by leaving the uncapped vial under a slow stream of nitrogen gas (flow approx. 10 ml/min.) in the fume hood. When fully dry (vial at constant weight), the vial is weighed and capped. The labelled, capped vial is stored at room temperature. Microspheres are normally stored no longer than 3 months.
  • microspheres will not sediment out, so they are left in suspension at 4°C for a maximum of four weeks.
  • concentration of microspheres in the 10 mL suspension a 200 ⁇ l sample of the suspension is pipetted into a 1.5 mL preweighed microfuge tube. The tube is then centrifuged at 10,000 g (Eppendorf table top microfuge), the supernatant removed, and the tube allowed to dry at 50°C overnight. The tube is then re-weighed in order to determine the weight of dried microspheres within the tube.
  • Microspheres containing 0[BEOV] 2 may then be produced essentially as described above in steps (C) through (E).
  • Microspheres were manufactured from poly (DL) lactic acid (PLA), poly methylmethacrylate (PMMA), polycaprolactone (PCL) and 50:50 ethylene vinyl acetate
  • EVA EVA:PLA essentially as described in Example 2. Size ranged from 10 to 100 ⁇ m with a mean diameter 45um.
  • HBSS Hanks Buffered Salt Solution
  • Neutrophil activation levels were determined by the generation of reactive oxygen species as determined by chemiluminescence.
  • chemiluminescence was determined by using an LKB luminometer with 1 uM luminol enhancer.
  • Plasma pre-coating (or opsonization) of microspheres was performed by suspending 10 mg of microspheres in 0.5 mL of plasma and tumbling at 37°C for 30 min.
  • Microspheres were then washed in 1 mL of HBSS and the centrifuged microsphere pellet added to the neutrophil suspension at 37°C.
  • Microsphere surfaces were modified using a surfactant called Pluronic F127 (BASF) by suspending 10 mg of microspheres in 0.5 mL of 2% w/w solution of F 127 in HBSS for 30 min at 37°C.
  • BASF Pluronic F127
  • Untreated microspheres give chemiluminescence values less than 50 mV. These values represent low levels of neutrophil activation. By way of comparison, inflammatory microcrystals might give values close to 1000 mV, soluble chemical activators might give values close to 5000 mV. However, when the microspheres are pre-coated with plasma, all chemiluminescence values are amplified to the 100 to 300 mV range. These levels of neutrophil response or activation can be considered mildly inflammatory. PMMA gave the biggest response and could be regarded as the most inflammatory. PLA and PCL both become three to four times more potent in activating neutrophils after plasma pre-treatment (or opsonization) but there is little difference between the two polymers in this regard.
  • Plasma precoating of PCL, PMMA, PLA and EVA:PLA as well as the effect of pluronic F127 pre-coating prior to plasma precoating of microspheres all show the same effect: (1) plasma pre-coating amplifies the response; (2) pluronic F127 precoating has no effect on its own; (3) the amplified neutrophil response caused by plasma pre-coating can be strongly inhibited by pre-treating the microsphere surface with 2% pluronic F127.
  • ELVAX poly-l-lactic acid mixture in DCM.
  • Microspheres are then prepared in a dissolution machine (Six-spindle dissolution tester, VanderKanp, Van Kell Industries Inc., U.S.A.) in triplicate at 200 ⁇ m, 42°C, for 3 hours. Microspheres so prepared are washed twice in water and sized on the microscope.
  • encapsulation is undertaken in a uv/vis assay (uv/vis lambda max. at 237 nm, fluorescence assay at excitation 237, emission at 325 nm. After 18 hours of tumbling in an oven at 37° C, the total of OfBEOV] 2 released from the microspheres is determined.
  • EXAMPLE 64 THERAPEUTIC AGENT-LOADED POLYMERIC FILMS COMPOSED OF ETHYLENE VINYL ACETATE AND A SURFACTANT Two types of films are investigated within this example: pure EVA films loaded with OfBEOV] 2 and EV A surfactant blend films loaded with OfBEOV] 2 .
  • the surfactants being examined are two hydrophobic surfactants (Span 80 and Pluronic LI 01) and one hydrophilic surfactant (Pluronic F127).
  • the pluronic surfactants are themselves polymers, which is an attractive property since they can be blended with EVA to optimize various drug delivery properties.
  • Span 80 is a smaller molecule which is in some manner dispersed in the polymer matrix, and does not form a blend.
  • Surfactants are useful in modulating the release rates of OfBEOV] 2 from films and optimising certain physical parameters of the films.
  • One aspect of the surfactant blend films which indicates that drug release rates can be controlled, is the ability to vary the rate and extent to which the compound will swell in water. Diffusion of water into a polymer-drug matrix is critical to the release of drug from the carrier. Pure EVA films do not swell to any significant extent in over 2 months. However, by increasing the level of surfactant added to the EVA it is possible to increase the degree of swelling of the compound, and by increasing hydrophilicity swelling can also be increased.
  • MePEG methoxypolyethylene glycol 350
  • MePEG/PCL paste is prepared by first dissolving a quantity of OfBEOV], into MePEG, and then inco ⁇ orating this into melted PCL.
  • One advantage with this method is that no DCM is required.
  • the melting point of PCL/MePEG polymer blends may be determined by differential scanning calorimetry from 30°C to 70°C at a heating rate of 2.5°C per minute. Briefly, MePEG (as determined by thermal analysis) decreases the melting point of the polymer blend in a concentration dependent manner. This lower melting point also translates into an increased time for the polymer blends to solidify from melt. A 30:70 blend of MePEG:PCL takes more than twice as long to solidify from the fluid melt than does PCL alone.
  • a CT-40 mechanical strength tester is used to measure the strength of solid polymer "tablets" of diameter 0.88 cm and an average thickness of 0.560 cm.
  • the polymer tablets are blends of MePEG at concentrations of 0%, 5%, 10% or 20%> in PCL.
  • MOLECULAR WEIGHT POLY(D,L-LACTIC ACID) As discussed above, depending on the desired therapeutic effect, either quick release or slow release polymeric carriers may be desired. For example, polycaprolactone (PCL) and mixtures of PCL with poly(ethylene glycol) (PEG) produce compositions which release agents over a period of several months.
  • PCL polycaprolactone
  • PEG poly(ethylene glycol)
  • low molecular weight poly(DL-lactic acid) gives fast degradation, ranging from one day to a few months depending on its initial molecular weight. The release of drug, in this case, is dominated by polymer degradation.
  • Another feature of low mol. wt.PDLLA is its low melting temperature, (i.e., 40°C-60°C), which makes it suitable material for making thermopaste.
  • several different methods can be utilized in order to control the polymer degradation rate, including, for example, by changing mol. wt. of the PDLLA, and/or by mixing it with high mol. wt. PCL, PDLLA, or poly(lactide-co- glyocide) (PLGA).
  • D,L-lactic acid was purchased from Sigma Chemical Co., St. Louis, MO., mol. wt.10-20,000, was obtained from Polysciences, Warrington, PA., mol. wt.PDLLA (intrinsic viscosity 0.60 dl/g) and PLGA (50:50 composition, viscosity 0.58 dl/g) were from Birmingham Polymers.
  • Low mol. wt.PDLLA was synthesized from DL-lactic acid through polycondensation. Briefly, DL-lactic acid was heated in a glass beaker at 200°C with nitrogen purge and magnetic stirring for a desired time. The viscosity increased during the polymerization, due to the increase of mol. wt.. Three batches were obtained with different polymerization times, i.e., 40 min (mol. wt.800), 120 min, 160 min. C. Formulation of OfBEOV]2 Thermopastes
  • thermopastes it is anticipated that 0[BEOV] 2 will be loaded into the following materials by hand mixing at a temperature about 60°C. 1) low mol. wt.PDLLA with polymerization time of 40 min. 2) low mol. wt.PDLLA with polymerization time of 120 min.
  • Mixtures of high mol. wt.PDLLA or PLGA with low mol. wt.. PDLLA obtained by dissolving the materials in acetone or other solvent followed by drying.
  • Low mol. wt.PDLLA 40 min was a soft material with light yellow color. The color is perhaps due to the oxidation during the polycondensation.
  • Low mol. wt.PDLLA 120 min (yellow) and 160 min (brown) were brittle solids at room temperature. They all melt at 60°C.
  • Mixtures of 50:50 high molecular weight PDLLA or PLGA with low molecular weight PDLLA 40 min also melt about 60°C.
  • low molecular weight PDLLA 40 min and 120 min broke up into fragments within one day.
  • polymeric carriers may be manufactured, including for example, (1) low mol. wt. (500- 10,000) poly(D,L-lactic acid), poly(L-lactic acid), poly(glycolic acid), poly(6- hydroxycaproic acid), poly (5 -hydroxy val eric acid), poly(4-hydroxybutyric acid), and their copolymers; (2) blends of above (#1) above; (3) blends of (#1) above with high mol.
  • microparticles of co-precipitates of 0[BEOV]Jadditive can be prepared and subsequently added to PCL to form pastes.
  • the samples are coated with 60% Au and 40% Pd (thickness 10 - 15 nm) using a Hummer Instrument (Technics, USA).
  • Osmotic or swellable, hydrophilic agents embedded as discrete particles in the hydrophobic polymer result in drug release by a combination of the erosion of the matrix, diffusion of drug through the polymer matrix, and/or diffusion and/or convective flow through pores created in the matrix by the dissolution of the water soluble additives.
  • Osmotic agents and swellable polymers dispersed in a hydrophobic polymer would imbibe water (acting as wicking agents), dissolve or swell and exert a turgor pressure which could rupture the septa (the polymer layer) between adjacent particles, creating microchannels and thus facilitate the escape of the drug molecules into the surrounding media by diffusion or convective flow.
  • the swelling and cracking of the paste matrix likely resulted in the formation of microchannels throughout the interior of the matrix.
  • Pastes prepared as described above (using mesh size 140 fractions of the OfBEOV] 2 -gelatin microparticles) are filled into 8 x 1 mL syringes (BD Insulin Syringe, 1/2 cc) each syringe containing 150 mg of the paste.
  • mice are divided into two groups of eight, the tumor site opened under anesthesia and 150 mg of the paste, previously heated to about 60°C is extruded at the tumor site and the wound closed.
  • One group is implanted with the OfBEOV] 2 -loaded paste and the other group with control paste containing gelatin and PCL only.
  • the mice are sacrificed and the weight of the mice and the excised tumor are measured.
  • PDLLA-PEG-PDLLA and low molecular weight PDLLA DL-lactide is purchased from Aldrich.
  • Polyethylene glycol (PEG) with molecular weight 8,000. stannous octoate, and DL-lactic acid are obtained from Sigma.
  • Poly- ⁇ -caprolactone (PCL) with molecular weight 20,000 is obtained from Birmingham Polymers (Birmingham. AL).
  • Polystyrene standards with narrow molecular weight distributions are purchased from Polysciences (Warrington, PA).
  • Acetonitrile and methylene chloride are HPLC grade (Fisher Scientific).
  • the triblock copolymer of PDLLA-PEG-PDLLA is synthesized by a ring opening polymerization. Monomers of DL-lactide and PEG in different ratios are mixed and 0.5 wt% stannous octoate is added. The polymerization is carried out at 150°C for 3.5 hours. Low molecular weight PDLLA is synthesized through polycondensation of DL-lactic acid. The reaction is performed in a glass flask under the conditions of gentle nitrogen purge, mechanical stirring, and heating at 180°C for 1.5 hours. The PDLLA molecular weight was about 800 measured by titrating the carboxylic acid end groups. B. Manufacture of paste formulations
  • the OfBEOV] loaded pastes will be weighed into 1 mL syringes and stored at 4°C.
  • the molecular weights and distributions of the PDLLA-PEG-PDLLA copolymers are determined at ambient temperature by GPC using a Shimadzu LC- 10 AD HPLC pump and a Shimadzu RID-6A refractive index detector (Kyoto, Japan) coupled to a IO 4 A Hewlett Packard Plgel column. (Other suitable equipment may be substituted.)
  • the mobile phase is chloroform with a flow rate of 1 ml/min.
  • the injection volume of the sample is 20 ⁇ l at a polymer concentration of 0.2%> (w/v).
  • the molecular weights of the polymers are determined relative to polystyrene standards.
  • the intrinsic viscosity of PDLLA-PEG-PDLLA in CHCI3 at 25°C is measured with a Cannon-Fenske viscometer.
  • Thermal analysis of the copolymers is carried out by differential scanning calorimetry (DSC) using a TA Instruments 2000 controller and DuPont 91 OS DSC (Newcastle, Delaware). The heating rate is 10 °C/min and the copolymer and 0[BEOV],/copolymer matrix samples are weighed (3 - 5 mg) into crimped open aluminum sample pans.
  • DSC differential scanning calorimetry
  • the water phase is decanted and the DCM phase is dried under a stream of nitrogen at 60°C.
  • the dried residue is reconstituted in a 40:60 water:acetonitrile mixture and centrifuged at 10,000g for about 1 min. The amount of the OfBEOV], in the supernatant is then analyzed.
  • mice Ten week old DBA/2J female mice are acclimatized for 3-4 days after arrival. Each mouse is injected subcutaneously in the posterior lateral flank with lOxlO 5 tumor cells (MDAY-D2 or other suitable cancer tumor cells) in 100 ⁇ l of PBS on day 1. On day 6, the mice are randomly divided into two groups. Group 1 are implanted with paste alone (control), and group 2 are implanted with paste loaded with OfBEOV],. A subcutaneous pocket near the tumor is surgically formed under anesthesia and approximately 100 mg of molten paste (warmed to 50°C-60°C) is placed in the pocket and the wound closed. On day 16, the mice are sacrificed, and the tumors are removed and weighed. Day 16 is selected to allow the tumor growing into a easily measurable size within the ethical limit.
  • MDAY-D2 or other suitable cancer tumor cells lOxlO 5 tumor cells
  • PEG-PDLLA relative to polystyrene standards, were measured by GPC.
  • the intrinsic viscosity of the copolymer in CHC1 3 at 25 °C was determined using a Canon-Fenske viscometer.
  • the molecular weight and intrinsic viscosity decreased with increasing PEG content.
  • the polydispersity of PDLLA-PEG-PDLLA with PEG contents of 10% - 40%) were from 2.4 to 3.5.
  • the copolymer with 70%) PEG had a narrow molecular weight distribution with a polydispersity of 1.21. This might be due to a high PEG content reduced the chance of side reactions such as transesterification that results in a wide distribution of polymer molecular weight.
  • a coiled structure of the hydrophobic-hydrophilic block copolymers may result in an artificial low polydispersity value.
  • PEG might represent the glass transition of the PDLLA region. No thermal changes occurred in the copolymers with PEG contents of 10%o, 20%> and 30%> in a temperature range of 10 - 250°C, indicating that no significant crystallization had occurred.
  • PCL containing MePEG at various concentrations can be tested for tensile strength and time to fail by a CT-40 Mechanical Strength Tester.
  • Microspheres (50g) were prepared using PCL (nominal molecular weight 80,000) using the solvent evaporation method described below.
  • PVA mol. Wt 13,000-23,000; 99% hydrolyzed
  • Microspheres were manufactured in the size ranges 0.5 - 10 ⁇ m, 10 - 20 ⁇ m and 30 - lOO ⁇ m using standard methods (polymer was dissolved in dichloromethane and emulsified in a polyvinyl alcohol solution with stirring as previously described in PCL or PDLLA microspheres manufacture methods).
  • Various ratio's of PLLA to GA were used as the polymers with different molecular weights [given as Intrinsic Viscosity (I .Vis.)]
  • Microspheres were manufactured successfully from the following starting polymers:
  • Diblock copolymers of poly(DL-lactide)-block-methoxy polyethylene glycol PLLA-MePEG
  • PCL- MePEG polycaprolactone-block-methoxy polyethylene glycol
  • PCL- MePEG polycaprolactone-block-methoxy polyethylene glycol
  • PCL- MePEG polycaprolactone-block-methoxy polyethylene glycol
  • PLLACL-MePEG poly(ethylene glycol)
  • monomers DL-lactide, caprolactone, and methoxy polyethylene glycols with different molecular weights were heated (130°C) to melt under the bubbling of nitrogen and stirring.
  • the catalyst stannous octoate (0.2%> w/w) was added to the molten monomers.
  • the polymerization was carried out for 4 hours.
  • the molecular weights, critical micelle concentrations, and the maximum drug loadings are measured with GPC, fluorescence, and solubilization testing, respectively.
  • 0[BEOV] can be encapsulated into nylon microcapsules using the interfacial polymerization techniques. Briefly, a specified amount of 0[BEOV], and 100 mg of Pluronic F-127 will be dissolved in 1 mL of dichloromethane (DCM) or other suitable solvent and 0.4 mL (about 500 mg) of adipoyl chloride (ADC) is added. This solution is homogenized into 2% PVA solution using the Polytron homogenizer (1 setting) for 15 seconds. A solution of 1 ,6-hexane- diamine (HMD) in 5 mL of distilled water is added dropwise while homogenizing. The mixture is homogenized for a further 10 seconds after the addition of HMD solution. The mixture is transferred to a beaker and stirred with a magnetic stirrer for 3 hours. The mixture is centrifuged, collected and resuspended in 1 mL distilled water.
  • DCM dichloromethane
  • ADC adipoyl
  • PDLLA-MePEG and PDLLA-PEG-PDLLA are block copolymers with hydrophobic (PDLLA) and hydrophilic (PEG or MePEG) regions. At appropriate molecular weights and chemical composition, they may form tiny aggregates of hydrophobic PDLLA core and hydrophilic MePEG shell. It is anticipated that 0[BEOV] 2 can be loaded into the hydrophobic core, thereby providing 0[BEOV], with an increased "solubility".
  • D,L-lactide was purchased from Aldrich, Stannous octoate, poly (ethylene glycol) (mol. wt. 8,000), MePEG (mol. wt. 2,000 and 5,000) were from Sigma. MePEG (mol. wt. 750) was from Union Carbide.
  • the copolymers were synthesized by a ring opening polymerization procedure using stannous octoate as a catalyst (Deng et al., J. Polym. Sci. Polym. Lett. 25:41 1-416, 1990; Cohn et al., J. Biomed, Mater. Res. 22:993-1009, 1988).
  • the polymers are dissolved in acetonitrile or other suitable solvent and centrifuged at 10,000 g for 5 minutes to discard any non-dissolvable impurities. It is anticipated that OfBEOV], acetonitrile (or other solvent) solution will then be added to each polymer solution to give a solution with 0[BEOV] 2 . The solvent will then be removed to obtain a clear 0[BEOV],/PDLLA-MePEG matrix, under a stream of nitrogen and 60°C warming. Distilled water, 0.9%> NaCl saline, or 5% dextrose is added at four times weight of the matrix. The matrix is finally "dissolved” with the help of vortex mixing and periodic warming at 60°C.
  • EXAMPLE 75 ANALYSIS OF DRUG RELEASE A known weight of a polymer (typically a 2.5 mg pellet) is added to a 15 mL test tube containing 14 mL of a buffer containing 10 mm Na 2 HP0 4 -NaH 2 P0 4 , 0.145 m NaCl and 0.4 g/1 bovine serum albumin. The tubes are capped and tumbled at 37°C. At specific times all the 14 mL of the liquid buffer are removed and replaced with fresh liquid buffer.
  • a polymer typically a 2.5 mg pellet
  • the liquid buffer is added to 1 milliliter of methylene chloride or other suitable solvent and shaken for 1 minute to extract all the 0[BEOV] 2 into the methylene chloride (or other suitable solvent).
  • the aqueous phase is then removed and the solvent phase is dried under nitrogen.
  • the residue is then dissolved in 60% acetonitrile: 40%> water and the solution is analysed using the appropriate assay.
  • Microspheres were made from 100k g/mol PLLA with a particle diameter range of 10-60 ⁇ m. The microspheres were incubated in a sodium hydroxide solution to produce carboxylic acid groups on the surface by hydrolysis of the polyester. The reaction was characterized with respect to sodium hydroxide concentration and incubation time by measuring surface charge. The reaction reached completion after 45 minutes of incubation in 0.1 M sodium hydroxide. Following base treatment, the microspheres were coated with dimethylaminoproylcarbodiimide (DEC), a cross- linking agent by suspending the microspheres in an alcoholic solution of DEC and allowing the mixture to dry into a dispersible powder.
  • DEC dimethylaminoproylcarbodiimide
  • microspheres The weight ratio of microspheres to DEC was 9:1. After the microspheres ere dried, they were dispersed with stirring into a 2% w/v solution of poly (acrylic acid) and the DEC allowed to react with PAA to produce a water insoluble network of cross-linked PAA on the microspheres surface. Scanning electron microscopy was used to confirm the presence of PAA on the surface of the microspheres. EXAMPLE 77 THE EFFECT OF O[BEOV] 2 IN THE TREATMENT OF BACTERIAL INFECTIONS
  • 0[BEOV] 2 is incubated at various concentrations in vitro with isolated strains of bacteria such as Streptococcus pneumoniae. The minimum inhibitory concentration is determined from these studies of 0[BEOV] 2 to determine the sensitivity of the bacterial strains to the compounds. Examination for inhibition of inco ⁇ oration of thymidine, uridine, leucine various ions and glucose into the cells of the bacteria is determined to understand the effect of OfBEOV], on the transport of substrates and ions through the membrane and thus the mechanism of inhibition of 0[BEOV] 2 .
  • the process of cellular recruitment in aseptic loosening of prosthetic joint implants involves the association of macrophages with particulate debris from the cement mantle consisting of polymethylmethacrylate (PMMA) at the joint-tissue interface. This involvement eventually leads to further cellular recruitment, bone reso ⁇ tion and loosening of the joint. As part of this process, cytokines released by osteoblasts stimulate the recruitment of macrophages and osteoclasts into sites of inflammation at the bone-cement interface.
  • PMMA polymethylmethacrylate
  • experiments designed to test the efficacy of 0[BEOV] 2 in the treatment of joint prostheses failure involve exposure of macrophages to PMMA particles with and without OH[BEOV], followed by measure of tumor necrosis factor (TNF), prostaglandin E2 and interleukin 1 (Perry et al., British Journal of Rheumatology, 34: 1127-1134, 1995), (Horowitz et al., Calcif Tissue Int., 57: 301-305, 1995). Furthermore, the effects of 0[BEOV] 2 on cytokine release of osteoblasts in vitro are then tested.
  • TNF tumor necrosis factor
  • prostaglandin E2 prostaglandin E2
  • interleukin 1 interleukin 1
  • Osteoblasts are incubated with conditioned medium from macrophages exposed to PMMA with and without 0[BEOV] 2 followed by measure of granulocyte macrophage colony stimulating factor (GM-CSF), interleukin 6 (IL-6) and prostaglandin E2 (PGE-2).
  • GM-CSF granulocyte macrophage colony stimulating factor
  • IL-6 interleukin 6
  • PGE-2 prostaglandin E2
  • Periodontitis defined as an inflammation of the supporting tissue of the teeth is a progressively destructive disease leading to loss of bone and periodontal ligament. It is characterized by reso ⁇ tion of the alveolar bone and loss of soft tissue attachment to the tooth and is a major cause of tooth loss in the adult.
  • 0[BEOV] administration on periodontal disease
  • subjects are treated with 0[BEOV], at weekly intervals and the degree of bone density assessed over time and prevalence of subgingival bacteria including Porphyromonas gingivalis, Prevotella intermedia, Bacteroides for sy thus, and Actinobacillus actinomycetemcomitans evaluated.
  • Methods to assess the degree of osteopenia include various measures of bone density through one or more of the following single photon abso ⁇ tiometry, dual photon abso ⁇ tiometry, duel energy X-ray abso ⁇ tiometry, quantitative computerized tomography or, but not limited to, digital subtraction radiography.
  • Methods to assess the degree of periodontitis include oral measurements such as alveolar crest height, clinical attachment loss, and residual ridge reso ⁇ tion as well as clinical outcomes including tooth loss, bleeding and edentulousness. Assessment of these key areas over time will determine the efficaciousness of 0[BEOV], in the treatment of this disease.
  • IBD Inflammatory bowel disease
  • IBD Inflammatory bowel disease
  • TLB 2,4,6- trinitrobenzene sulphonic acid
  • a single administration initiates an acute and chronic inflammation that persists for several weeks.
  • the rabbit colon has been shown to resemble the human colon more so than does the rat (Gastroenterology, 99: 13424-1332, 1990).
  • mice Female New Zealand white rabbits are used in all experiments.
  • the animals are anesthetized intravenously (i.v.) with phenobarbitol.
  • An infants' feeding tube is inserted rectally, so that the tip is 20 cm proximal to the anus, for injection of the TNB (0.6 ml; 40 mg in 25% ethanol in saline).
  • TNB 0.6 ml; 40 mg in 25% ethanol in saline
  • the rabbits are randomized into 3 treatment groups. At this time, the animals receive either no treatment, vehicle alone (i.v.) or OfBEOV], (i.v.). This is repeated every 4 days for a total of 4 treatments.
  • the rabbits are sacrificed with Euthanol at 24 hours and 1, 2, 4 and 6 weeks.
  • the entire colon is isolated, resected and opened along the anti-mesenteric border, washed with saline and placed in Hank's balanced salt solution containing antibiotics.
  • the colon is examined with a stereomicro scope and scored according to the same criteria as at endoscopy.
  • specimens of colon are selected at autopsy, both from obviously inflammed and ulcerated regions and from normal colon throughout the entire length from anus to ascending colon.
  • the tissues are fixed in 10% formaldehyde and processed for embedding in paraffin; 5 mm sections are cut and stained with hemotoxylin and eosin.
  • the slides are examined for the presence or absence of IBD histopathology.
  • the initial experiment can be modified for the use of oral 0[BEOV] 2 following induction of colitis in rabbits by the intracolonic injection of TNB.
  • the animals are randomized into 3 groups receiving no treatment, vehicle alone or orally formulated OH[BEOV] 2 .
  • New Zealand female white rabbits are anesthetized and a laparotomy is performed through a midline incision.
  • the uterine horns are exposed and a 5 cm long segment off each is abraded using a scalpel blade. This abrasion is sufficient to remove the serosa, resulting in punctate bleeding.
  • Rabbits are randomly assigned to the control or paclitaxel treated groups and to post-operative evaluation periods of two, four and eight weeks.
  • each uterine horn is completely wrapped with OfBEOV],- loaded film following abrasion.
  • the musculoperitoneal layer is closed with sutures and the cutaneous layer with skin staples.
  • the total grade is additive, with an adhesion score range of 0 - 4 which represents both extent and severity.

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Abstract

L'invention se rapporte à des complexes d'organovanadium et, plus particulièrement, à des complexes d'hydroxyoxovanadium (V), de ν-oxo dimère oxovanadium (V) et de cis-dioxovanadium. Ces complexes peuvent être formulés sous forme d'une composition pharmaceutique. Ces complexes et/ou compositions peuvent être utilisés dans le traitement d'une pluralité d'états pathologiques, notamment en tant qu'agents anti-prolifératifs et/ou anti-métastatiques, et/ou en vue de traiter des tumeurs résistant aux médicaments. L'invention concerne également des procédés permettant de réduire l'aptitude des tumeurs à métastaser et/ou de traiter du diabète, l'arthrite, la sclérose en plaques, des maladies impliquant des voies du corps humain, et des maladies auto-immunes, notamment le psoriasis ou le lupus, mais pas uniquement.
EP98916725A 1997-04-24 1998-04-24 Complexes de vanadium, derives de ces complexes et procedes connexes Withdrawn EP0984971A1 (fr)

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WO2000024730A1 (fr) * 1998-10-28 2000-05-04 The University Of British Columbia Complexes organiques de vanadium(iii), et utilisation correspondante
US6878388B1 (en) * 2000-11-15 2005-04-12 Parker Hughes Institute Vanadium compounds for treating proliferative cell disorders
EP3988092A4 (fr) * 2019-06-24 2022-08-24 Hunan Fangshengtai Medical Technology Co., Ltd. Nouvelle application d'un complexe métallique

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FR2678622B1 (fr) * 1991-07-03 1994-11-18 Adir Nouveaux complexes de vanadium, leur procede de preparation et les compositions pharmaceutiques qui les contiennent.
US5300496A (en) * 1991-09-30 1994-04-05 The University Of British Columbia Complexed vanadium for the treatment of diabetes mellitus
US5527790A (en) * 1991-09-30 1996-06-18 The University Of British Columbia Bis(maltolato)oxovanadium compositions for the treatment of elevated blood sugar
US5565491A (en) * 1994-01-31 1996-10-15 Bristol-Myers Squibb Company Use of phosphotyrosine phospatase inhibitors for controlling cellular proliferation

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AU7022198A (en) 1998-11-24
WO1998049173A1 (fr) 1998-11-05

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