JP2006508047A - Methods and formulations for treating conditions associated with neovascularization - Google Patents

Abstract

The present invention provides a method of inhibiting neovascularization in tissue by delivering an effective amount of sulforaphane or a pharmaceutically acceptable derivative, salt or prodrug thereof to a cell or tissue. Also provided herein is a method of treating a disease associated with endothelial hyperproliferation and / or neovascularization by administering to a subject an effective amount of sulforaphane or a pharmaceutically acceptable derivative, salt or prodrug thereof. provide. Kits for treating patients are also provided.

Description

(Cross-reference with related applications)
This application claims the benefit under 35 USC 119 (e) of US Provisional Application No. 60 / 401,130 filed on August 5, 2002, the contents of which are hereby incorporated by reference Incorporated into the disclosure.

  The present invention is in the field of medicine. In particular, it relates to the field of anti-angiogenic drugs for disease prevention and treatment.

  Angiogenesis is the process by which new vasculature is created by growth from existing capillaries. In this process, endothelial cells separate from the basement membrane as the support degrades by proteolytic enzymes. These cells then migrate from the parent vessel, separate and form newly differentiated vascular tissue (Risau, (1997) Nature 386: 671-674; Wilting et al. (1995) Cell. Mol. Biol. Res. 41 (4): 219-232). A variety of different biological factors have been shown to function in the control of angiogenesis (Bussolino et al. (1997) Trends IN Biochem Sci 22 (7): 251-256; Folkman and d'Amore, (1996). ) Cell 87: 1153-1155). These include proteins with diverse functions such as growth factors, cell surface receptors, proteases, protease inhibitors and extracellular matrix proteins (Achen and Stacker, (1998) Int. J. Exp. Pathol. 79: 255-265; Devararaja and Richmond, (1999) Trends in Pharmacol. Sci. 20 (4): 151-156; Hanahan, (1997) Science 277: 48-50; Maisonpierre et al. -60; Sun et al. (1996) Cell 87: 1171-1180; Sato et al. (1995) Nature 376: 70-74; d Rifkin, (1996) Enzyme Protein 49: 117-137; Pintucci et al., (1996) Semin Thromb Hemost 22 (6) 517-524; Vernon and Sage, (1995) Am. j. Pathol. Brooks et al. (1994) Science 264: 569-571; Koch et al. (1995) Nature 376: 517-519). The complexity of the angiogenic process and the diversity of factors that control its progression provide a range of useful points for medical procedures that control angiogenesis in vivo.

  Angiogenesis usually occurs in a carefully controlled manner during embryonic development, during development, and in special cases such as wound healing and the female reproductive cycle (Wilting and Christ, (1996) Naturewissenschaffen 83: 153). Goodger and Rogers, (1995) Microcirculation 2: 329-343; Augustin et al. (1995) Am.j. Pathol.147 (2): 339-351). Some of the important steps in the process of pathological angiogenesis are those in many humans, including tumor growth and metastatic cancer, diabetic retinopathy, rheumatoid arthritis, and other inflammatory diseases such as psoriasis. Play a central role in disease (Folkman, (1995) Nature Med. 1 (1): 27-31; Polverini, (1995) Rheumatology 38 (2): 103-112; Healy et al., (1998) Hum. Reprod.Update 4 (5): 736-396). In these cases, disease progression is driven by permanent and unregulated angiogenesis. For example, in rheumatoid arthritis, new capillaries erode joints and destroy cartilage. In diabetic retinopathy, retinal capillaries erode the vitreous and bleed, causing blindness. Tumor growth and metastasis are markedly angiogenic. Most major solid tumors experience a persistent avascular condition with growth limited to approximately 1-2 mm in diameter. Up to this size, tumor cells can obtain the necessary oxygen and nutrient supply by passive diffusion. These microscopic tumor masses can finally switch on angiogenesis and begin to generate capillaries that replenish the surrounding blood vessels and pass the blood vessels through the tumor mass, which continues to expand and malignant Provides the potential for metastasis of cells to distant parts. Although significant progress has been made in understanding the biological events that occur during pathological angiogenesis, there are currently no effective pharmaceutical compositions that help control angiogenesis in vivo. Thus, effective therapies that can control angiogenesis have the potential to alleviate a significant number of human diseases.

  Traditionally, pharmaceutical compounds have been developed by screening synthetic compounds and subsequent in vivo toxicity and efficacy tests for the desired pharmaceutical properties. Compounds selected in this way often have in vivo side effects, and this approach has not succeeded in developing effective angiogenesis inhibitors for disease treatment. More recently, molecular biology techniques have been applied to the development of angiogenesis inhibitors. Protein inhibitors of angiogenesis, such as angiostatin, have been discovered that control angiogenesis in experimental models. Nevertheless, such protein therapies are expensive to produce and have proven difficult to formulate and deliver to subjects. At present, protein angiogenesis inhibitors have not yet been developed as therapeutic drugs for patients. Thus, there is a need for therapeutic agents that can be safely administered to patients and effective in inhibiting the pathological growth of vascular endothelial cells. The present invention provides formulations and methods useful for this purpose and provides related advantages.

  Applicants have found that sulforaphane and its derivatives inhibit the process of endothelial cell growth and proliferation and vascularization. Thus, the present invention provides a method of inhibiting endothelial cell proliferation in specific cells or in tissues that are dividing to a pathological degree. The present invention also provides a method of inhibiting neovascularization in tissue. Each method entails delivering an effective amount of sulforaphane or a pharmaceutically acceptable derivative, salt or prodrug thereof to the cell or tissue. In one aspect, the method is combined with other compounds, formulations or therapies known to inhibit angiogenesis, neovascularization or excessive cell proliferation as occurs in cancer. Such therapies include, but are not limited to, chemotherapy, radiation therapy and administration of anti-angiogenic compounds.

  Also provided herein is a method of treating a disease associated with endothelial cell hyperproliferation and / or neovascularization by administering to a subject an effective amount of sulforaphane or a pharmaceutically acceptable derivative, salt or prodrug thereof. Is done. In one aspect, the method is combined with other compounds, formulations or therapies known to inhibit angiogenesis, neovascularization or excessive cell proliferation as occurs in cancer. Such therapies include, but are not limited to, chemotherapy, radiation therapy and administration of anti-angiogenic compounds. Kits for treating patients are also provided.

  In addition, screening methods are provided for poisoning new therapeutic agents that have the same, similar or better therapeutic effects as sulforaphane or pharmaceutically acceptable derivatives, salts or prodrugs thereof. Said screening method involves comparing the antiproliferative effect of sulforaphane or a pharmaceutically acceptable derivative, salt or prodrug thereof with said drug.

  Furthermore, methods for enhancing the therapeutic or therapeutic benefit of a compound, formulation or therapy that treats or prevents neovascularization, angiogenesis, neoplasia or cancer are also provided. Such treatments include, but are not limited to, chemotherapy, radiation therapy and administration of anti-angiogenic compounds.

  Throughout this disclosure, various publications, patents and published patent specifications are referenced by identifying citations. The disclosures of these publications, patents and published patent publications are hereby incorporated by reference into the present disclosure in order to more fully describe the state of the art relating to the present invention.

  The practice of the present invention uses conventional techniques of molecular biology, microbiology, cell biology, biochemistry and immunology that are within the skill of the art unless otherwise specified (including recombinant techniques). . Such techniques are explained fully in the literature.

Definitions As used herein, certain terms may have the meanings defined below.

  As used in the specification and claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “cell” includes a plurality of cells and includes mixtures thereof.

  As used herein, the term “comprising” is intended to mean that the formulations and methods include the elements described, but do not exclude others. “Consisting essentially of”, when used to define a formulation and method, means that the combination does not include any other essential elements. Thus, a formulation consisting essentially of the elements as defined herein contains trace amounts of foreign matter from pharmaceutical separation and purification methods, such as phosphate buffered saline and preservatives, and pharmaceutically. Will not exclude acceptable carriers. “Consisting of” means excluding more than trace steps of other ingredients and substantial method steps of administering the formulations of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.

  The term “isolated” means that the component, cell, etc., in which the compound is usually separated from substantially accompanying substances.

  A “subject” or “host” is a vertebrate, preferably an animal or mammal, and more preferably a human patient. Mammals include, but are not limited to, mice, monkeys, human patients, farm animals, sport animals, and pets.

  The terms “cancer”, “neoplasm” and “tumor” used interchangeably in the singular or plural refer to cells that have undergone malignant transformation that renders the cell pathological to the host organism. Primary cancer cells (ie, cells obtained near the location of malignant transformation) can be easily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of cancer cell, as used herein, includes not only primary cancer cells, but any cells derived from the original species of cancer cells. This includes metastasized cancer cells, and test tube cultures and cell lines derived from cancer cells. Normally, when referring to a type of cancer that appears as a solid tumor, a “clinically detectable” tumor is a tumor that has been treated by procedures such as CAT scan, nuclear magnetic resonance imaging (MRI), X-ray, ultrasound, or palpation. It can be detected based on the lump. Biochemical or immunological findings alone may not be sufficient to meet this definition.

  As used herein, “inhibit” means stopping, delaying or slowing growth, endothelial cell proliferation, cell division or the formation of blood vessels in a tissue. Methods for monitoring inhibition include, but are not limited to, endothelial cell proliferation analysis, measurement of vascular bed volume by determining blood volume, and quantitative determination of vascular structure density. When the culture is a mixture of cells, neovascularization is monitored by quantitative measurement of cells that represent specific markers of endothelial cells, such as angiogenic factors, proteolytic enzymes and endothelial cell specific cell adhesion molecules .

  “Formulation” means a combination of an active agent with other inactive (eg, detectable agents or labels) or active compounds such as immunostimulants.

  A “pharmaceutical formulation” is intended to include an inert or active carrier and active agent combination that renders the formulation suitable for in vitro, in vivo or in vitro diagnostic or therapeutic use. .

As used herein, the term “pharmaceutically acceptable carrier” refers to standard pharmaceuticals such as phosphate buffered saline solution, water, and emulsions such as oil / water or water / oil emulsions. Includes any of a carrier and various types of wetting agents.
The formulation can further include stabilizers and preservatives.
For examples of carriers, stabilizers and immune aids, see Martin, REMINGTON'S PHARM. SD. , 15TH ED. (Mack Publ. Co., Easton (1975)).

  An “effective amount” is an amount sufficient to effect beneficial or desired results. For example, the amount of treatment is that which achieves the desired therapeutic effect. This amount may be the same or different from the prophylactically effective amount necessary to prevent the onset or symptom of the disease. An effective amount can be administered by one or more administrations, applications or dosages.

As used herein, unless otherwise specified, “sulforaphane” is intended to mean an aglycon product made by destroying sulforaphane glucosinate (SGS), which is primarily a cruciferous vegetable (cabbage). , Broccoli, broccoli sprouts, cabbage, cauliflower, cauliflower sprouts, pak choi, green leaf kanran, corrado leaves, yellow scallop, kohlrabi, mustard, turnip, red box radish, and Dutch pepper. Unless otherwise specified, the term includes derivatives of sulforaphane, pharmaceutically acceptable salts and prodrugs. Young broccoli sprouts and cauliflower sprouts are particularly rich in sulforaphane glycosinate derivatives. Sulforaphane glycosinate is also known as glycosinate glucoraphanin. Sulforaphane has a molecular formula of C ₆ H ₁₁ NOS ₂ and a molecular weight of 177.29 daltons. It is also known as 4-methylsulfinylbutyl isothiocyanate and (−)-1-isothiocyanato-4 (R)-(methylsulfinyl) butane. Sulforaphane has anti-carcinogenic properties due to its ability to generate process II detoxification enzymes (providing protection from certain carcinogens and other harmful electrophiles) such as glutathione S-transferase and quinone reductase It may be possible.

  The inventor has discovered that sulforaphane inhibits endothelial cell growth and has anti-angiogenic properties. In accordance with these discoveries, the present invention provides a method of inhibiting endothelial cell growth by delivering a growth-inhibiting amount of sulforaphane or a pharmaceutically acceptable derivative, salt or prodrug thereof to the cell. The invention further provides a method of inhibiting vascular conduction in tissue by delivering to the tissue an anti-vascular conduction amount of sulforaphane or a pharmaceutically acceptable derivative, salt or prodrug thereof.

  This method can be performed in vitro or in vivo. When performed in vitro, endothelial cells or vascularly conductive tissue are cultured under conditions well known to those skilled in the art, for example, as illustrated. The cells and / or tissues can be from established cell lines or can be cultured from a biopsy sample obtained from a subject. Sulforaphane or a pharmaceutically acceptable derivative, salt or prodrug thereof is then added to the medium and delivered as a component of the pharmaceutical formulation.

  The purification of the racemic sulforaphane is described below. In one aspect, the purified sulforaphane is only D-form. In another aspect, it is L-only. Racemates, D or L form are each commercially available, and methods for isolating each are known in the art. For example, the use of HPLC chiral columns such as CHIRAL-AGP, CHIRAL-CBH and CHIRAL-HAS is possible from Chrom Tech International (AB). Perkins-Elmer also sells chiral separation kits under the Sulfadex chiral separation kit.

  Examples of salts include: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, Camphor sulfonate, cyclopentane propionate, digluconate, dodecyl sulfate, ethane sulfonate, fumarate, flucoheptanoic acid, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride , Odorate, iodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectate Sulfate, phenylpropionate, picrate, pivalate, propionate, oxalate, tartrate, thiocyanate, tosylate and unde Down salt.

Other examples of salts include the anions of the compounds of the present invention in combination with a suitable cation such as Na ⁺ , NH ₄ ⁺ and NW ₄ ⁺ (where W is a C _1-4 alkyl group). included. For therapeutic use, the salts of the compounds of the invention will be pharmaceutically acceptable. However, salts of acids and bases that are not pharmaceutically acceptable may find use, for example, in the preparation or purification of pharmaceutically acceptable compounds.

  Not all treatments are effective for each individual, so an in vitro analysis that measures efficacy for each patient would be advantageous. The method provides these means for determining whether sulforaphane therapy treats an individual subject-specific disease associated with pathological proliferation of endothelial cells. Examples of such are provided here. For example, a tissue biopsy is separated from a patient and contacted with an effective amount of a pharmaceutical formulation or treatment as defined herein under conditions effective for cell growth and proliferation. Inhibition of pathological cell growth as determined by conventional treatments, such as the CPAE analysis described herein, indicates that the inventive formulations and / or therapies can effectively treat patients.

  Angiogenesis, or the formation of new blood vessels, is the fundamental process by which new blood vessels are formed. It is involved in essential physiological phenomena such as reproductive development and wound healing. Under normal conditions, angiogenesis is highly regulated. However, many diseases are activated by permanent and unregulated angiogenesis. In rheumatoid arthritis, new capillaries erode joints and destroy cartilage. In diabetic retinopathy, retinal capillaries erode the vitreous and bleed, causing blindness. Tumor growth and metastasis are angiogenesis dependent. Most major solid tumors experience an avascular, seemingly quiescent growth with a maximum reachable size of 1-2 mm in diameter. Up to this size, tumor cells can obtain the necessary oxygen and nutrients by simple passive diffusion. These microscopic tumor masses begin to generate new capillaries that grow by switching on angiogenesis and eventually penetrate the tumor mass by the replacement of surrounding mature host blood vessels. And thereby promote the relentless expansion of the tumor mass and the potential for hematogenous metastasis as well. The angiogenic switch was initially hypothesized to be triggered by ectopic production and synthesis by tumor cells of a growth factor called “tumor angiogenic factor” (TAF).

  The invention further provides in a subject by administering to a subject a therapeutically effective amount of sulforaphane (racemic, D- or L-) or a pharmaceutical formulation comprising one or more thereof. Methods of treating diseases associated with pathological neovascularization are provided. As used in this context, “treating” means relieving the pathology associated with pathological neovascularization as well as reducing neovascularization. Examples of such conditions include, but are not limited to, arthritic conditions, neovascular-based dermatologic conditions, diabetic retinopathy, Kaposi's sarcoma, age-related macular degeneration, peripheral defect dilatation, glaucoma, keloid, Conical rejection, wound granularization, angiofibroma, Osler-Weber syndrome, myocardial angiogenesis, psoriasis and scleroderma. Typical arthritic conditions are selected from the group consisting of rheumatoid arthritis and osteoarthritis. For the treatment of cancer and solid tumors, “treating” includes inhibition of blood vessel growth, resulting in a lack of nutrients needed for the tumor and / or for growth by the tumor. Tumors and masses will decrease in size and will probably disappear. Medications for the treatment of arthritic conditions will result in decreased angiogenesis in cartilage, particularly joints, and will increase mobility and flexibility in these areas. For the treatment of psoriasis, medication will reduce dermatological symptoms such as scab formation, flaking, and blood vessels visible below the surface of the skin. In diabetic retinopathy, administration of the active fraction will reduce the formation of extraneous blood vessels in the retina, resulting in unshielded vision. In the treatment of Kaposi's sarcoma, administration of the active fraction will inhibit blood vessel growth and / or further formation, thereby inhibiting the resulting lesion and / or tumor formation.

  When the active fraction is administered to a subject such as a mouse, rat or human patient, the drug can be administered systemically, orally, transdermally or locally to the subject in addition to a pharmaceutically acceptable carrier. It is. The amount for treatment can be determined empirically and will vary depending on the lesion being treated, the subject being treated, and the toxicity of the fractional format used in the therapy. The active fraction can be delivered orally, intravenously, intraperitoneally or transdermally. Once delivered to an animal, the method is further useful to confirm the efficacy of the active fraction.

As an example of an animal model, a group of nude mice (Balb / c NCR nu / nu female, Simonsen, Gilroy, Calif.), Each of which is subcutaneously about 10 ⁵ to about 10 ⁹ as defined herein. Inoculate individual hyperproliferative cells.
Once the graft is established, the compound is administered, for example, by subcutaneous injection around the graft.
Measurements to determine graft size reduction are made in two dimensions using a caliper twice a week.

MRL / lpr mice (MRL / MpJ-Fas ^lpr ) from Jackson Laboratories (Maine) are useful for testing or monitoring efficacy in arthritic conditions. The benefits of aggressive treatment include reduction of animal joint and hind limb swellings and reduction of cartilage degradation that can be monitored by x-ray.

  A group of Lewis rats (age 8 weeks, 130-150 g. Jackson Laboratory, Maine, USA) is immune to bovine type II (BII) collagen and causes an arthritic condition. BII is dissolved in 0.1 M acetic acid at 400 μg / ml. It is injected transdermally at the base of the tail with an equal volume of BII and ICFA (incomplete Freund's adjuvant) at a concentration of 20 μg (100 μl). Once the arthritic condition is established, a 4-point observation is made for various physical illnesses for a period of 28 days. Other animal models may be used as appropriate.

  In vivo administration can be accomplished in a single dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and amount of administration are well known to those of skill in the art and will vary depending on the formulation used for treatment, the purpose of the treatment, the target cell being treated and the subject being treated. there will be. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Appropriate formulation dosages and methods of drug administration can be found below.

  The formulations and pharmaceutical formulations of the present invention can be used in the manufacture of pharmaceuticals and dietary supplements to treat humans and other animals by administration according to conventional treatments, such as active ingredients in pharmaceutical formulations. Is possible.

  The pharmaceutical formulation can be administered orally, nasally, parenterally or by inhalation therapy and takes the form of a tablet, electuary, granule, capsule, pill, ampoule, suppository or aerosol type be able to. In addition, they may take the form of suspensions, solutions and emulsions of the active ingredient in aqueous or non-aqueous diluents, syrups, granules, powders. In addition to the compositions of the present invention, the pharmaceutical formulation can also include other pharmaceutically active ingredients.

  In particular, the active fraction mentioned here as an active ingredient is applied to the mouth, rectum, nose, topical (including transdermal, aerosol, buccal and sublingual glands), vagina, parenteral (subcutaneous, intramuscular, intravenous and skin). And may be administered for treatment by any suitable route, including the lung). It would also be good that the preferred route will vary depending on the condition and age of the recipient and the disease being treated.

  When the method is performed in vivo, pathological endothelial cell growth or neovascularization is inhibited in the subject. A therapeutically effective amount of sulforaphane (racemate, D- or L-), or a pharmaceutically acceptable derivative, salt or prodrug thereof, delivered to the subject in an amount effective to elicit its intended result Is done. The invention further comprises administering to a subject a therapeutically effective or growth-inhibiting amount of sulforaphane (racemic compound, D- or L-) or a pharmaceutically acceptable derivative, salt or prodrug thereof, Methods of treating a disease associated with pathological neovascularization in a subject are provided. Such conditions include, but are not limited to, arthritic conditions, neovascular dermatologic conditions, diabetic retinopathy, restenosis, Kaposi's sarcoma, age-related macular degeneration, telangiectasia, glaucoma, keloid, Examples include corneal transplant rejection, wound granulation, angiofibroma, Osler-Weber syndrome, myocardial angiogenesis, psoriasis and scleroderma. A typical arthritic condition is selected from the group consisting of rheumatoid arthritis and osteoarthritis.

  When sulforaphane is administered to a subject, such as a mouse, rat or human patient, the drug is added to the subject systemically, orally, transdermally or locally in addition to a pharmaceutically acceptable carrier. It is possible. The amount to treat can be determined empirically and will vary depending on the lesion being treated, the subject being treated and the condition being treated.

  While the drug component can be administered alone, it is provided with at least one active ingredient as defined above, one or more pharmaceutically acceptable carriers for it and optionally other therapeutic agents. It is preferable to present it as a pharmaceutical formulation containing together. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

  Formulations include those suitable for oral, rectal, nasal, topical (including transdermal, buccal, sublingual gland), vaginal, parenteral (subcutaneous, intramuscular, intravenous and intradermal) and pulmonary administration. The formulations may be conveniently presented in unit dosage form and may be prepared by any method well known in the pharmaceutical art. Such methods include the step of bringing into association the active ingredient with the carrier, which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

  The preparation of the present invention suitable for oral administration is an aqueous or non-aqueous liquid solution or suspension as individual units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient, as a powder or granules. Or as an oil-in-water emulsion or a water-in-oil emulsion. The active ingredient may also be provided as a large pill, electuary or paste.

  A tablet may be compressed or molded, optionally with one or more accessory ingredients. Optional binders (eg povidone, gelatin, hydroxypropyl methylcellulose), lubricating inert diluents, preservatives, tablet disintegrating substances (eg sodium starch glycolate, crosslinked povidone, crosslinked sodium carboxymethylcellulose), surfactants or dispersants Compressed tablets may be prepared by compressing free-flowing active ingredients such as powders and granules mixed with in a suitable machine. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert chemical diluent. Tablets may be optionally coated or scored, for example, using hydroxypropyl methylcellulose at various ratios to provide the desired release profile, to slow or control the release rate of the active ingredient in the tablet. Well, you may mix. Tablets may optionally be provided with an enteric coating for release in the intestinal portion other than the stomach.

  Formulations suitable for topical administration in the mouth usually have the active ingredient in an inactive base such as sucrose and acacia or tragacanth gum and other flavoring bases, gelatino and glycerin or sucrose and acacia Including lozenges and mouthwashes that contain the active ingredients in a suitable liquid carrier.

  Pharmaceutical formulations for topical administration according to the invention may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. Alternatively, the formulation may include a wound dressing or dressing, such as a bandage or bandage soaked with the active ingredient and optionally one or more additives or diluents.

  For diseases of the eyes or other outer tissues such as mouth and skin, the formulations are preferably applied as topical ointments or creams containing the active ingredient. When formulated in an ointment, the drug may be used with a paraffinic or water-soluble ointment base. Alternatively, the medicinal component may be formulated in a cream having an oil-in-water cream base.

  If desired, the aqueous phase of the cream base can contain, for example, about 30% w / w polyhydric alcohols such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerin and polyethylene glycol. Alcohols having one or more hydroxyl groups and mixtures thereof may be included. Desirably, the topical formulation may contain a compound that enhances absorption or penetration of the drug ingredient through the skin or other affected area. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogs.

The oil phase of the emulsions of this invention may be composed of known ingredients in any known manner.
This phase may simply comprise an emulsifier (otherwise known as an emulgent), but desirably comprises a fat or oil or a mixture of both fat and oil and at least one emulsifier. Preferably, the aqueous emulsifier is included with the oil emulsifier and serves as a stabilizer.
It is also preferred that it contains both oil and fat.
Together, emulsifiers with or without stabilizers build so-called emulsifying waxes, and waxes with oils and / or fats build so-called emulsifying ointment bases that form the oil-dispersed phase of cream formulations. .

  Emulsions and emulsion stabilizers suitable for use in the formulations of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glycerol monostearate and sodium lauryl sulfate.

  The choice of oil or fat suitable for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils that will be used in pharmaceutical emulsions is very low. . Preferably, therefore, the cream should be a non-greasy, non-staining and washable product with a consistency suitable to avoid leakage from tubes or other containers. Known as diisoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acid, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or Crodamol CAP Linear or branched mono- or dibasic alkyl ether dibasic alkyl esters such as mixtures of branched esters may be used, the last three being the preferred esters. These may be used alone or in a combination depending on required characteristics. Alternatively, high melting point lipids such as white soft paraffin, liquid paraffin or other mineral oils can be used.

  Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredients are dissolved or suspended in suitable carriers, especially aqueous solvents for the active ingredients. Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.

  Formulations suitable for vaginal administration are provided as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing carriers in addition to the prodrug ingredients as known in the art. Good.

  Formulations suitable for nasal administration in which the carrier is a solid include, for example, a coarse powder having a particle size in the range of about 20 to about 500 microns and nasal passages from a container of powder to snuff, ie close to the nose. Is administered by rapid aspiration through Suitable formulations in which the carrier is a liquid for administration such as aerosol administration by nasal spray, nasal spray or nebulizer include aqueous solutions or oily solutions of the prodrug component.

  Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile infusion solutions that may contain antioxidants, buffers, bactericides and solutes that render the formulation isotonic with the blood of the intended recipient; And aqueous and non-aqueous sterile suspensions that may include suspending and thickening agents and liposomes or other microparticle systems designed to target blood components or compounds to one or more organs Contains liquid. The formulation may be provided in unit dose or multi-dose enclosures, such as ampoules and glass bottles, or freeze-dried immediately before use, requiring only the addition of a sterile liquid carrier, such as water for injection. It may be stored in a (freeze-dried) state. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

  Preferred unit dosage formulations are those containing a daily dose or unit, a daily sub-dose as described herein above, or an appropriate fraction thereof of the drug ingredient.

  In addition to the ingredients specifically mentioned above, the formulations of this invention may contain other conventional agents in the art relating to the type of formulation in question, eg those suitable for oral administration are sweeteners, thickeners It should be understood that additional agents such as and perfumes may be included.

  Sulforaphane (racemic or optically pure composition), pre-drugs, salts or derivatives thereof and formulations thereof may also be provided for use in the form of veterinary preparations, for example, conventional methods in the art May be prepared by

The present invention further provides methods for screening for therapeutic agents that inhibit neovascularization or endothelial cell growth. This selection is
(A) contacting the drug with a suitable cell or tissue sample;
(B) contacting another sample of the appropriate cell or tissue sample with a therapeutically effective amount of sulforaphane or a pharmaceutically acceptable derivative, salt or prodrug thereof;
(C) a step of comparing the growth of the sample of the step (a) with the growth of the sample of the step (b), the step of inhibiting the growth to the same or similar degree as the sample of the step (b) Any drug of a) requires a step that is a therapeutic agent that inhibits neovascularization or endothelial cell growth.

  The present invention also provides a kit for treating a pathology associated with pathological neovascularization in a subject. The kit includes a therapeutically effective amount of sulforaphane (racemic, (D) or (L)) or a pharmaceutically acceptable derivative, salt or prodrug thereof and instructions for use. This kit includes cancer and solid tumors, arthritic conditions, neovascular dermatologic conditions, diabetic retinopathy, Kaposi's sarcoma, age-related macular degeneration, restenosis, telangiectasia, glaucoma, keloids, cornea It is useful for treating a medical condition selected from the group consisting of transplant rejection, wound granulation, angiofibroma, Osler-Weber syndrome, myocardial angiogenesis, scleroderma, psoriasis, rheumatoid arthritis and osteoarthritis.

  The following examples are intended to illustrate but not limit the invention.

Example 1
Isolation and purification of sulforaphane Sulforaphane and sulforaphane nitrile were prepared according to the method of Matusheski et al. Agric. Food Chem. 49: 1867-1872 isolated and purified from Brassicaceae seeds. (D, L) -sulforaphane is described in Kim, s. and Yi x. (1986) 3. Org. Chem 51: 2613-2615. D-sulforaphane is purified from the racemic mixture (D, L) -sulforaphane using a chiral chromatography column. All sulforaphanes are commercially available from LKT Laboratories.

Example 2
Endothelial cell inhibition analysis by sulforaphane Endothelial cell analysis:
Conallyly et al. (1986) Anal. Biochem. 152: 136-4 (Liang and Wong (2000) Angiogenesis: From molecular to integrative pharmacology, by Maraudodakis, Kluwer Academic / Plenum. Publishers, New York). T. T. et al. Connolly et al. (1986) Anal. Biochem. 152: 136-140 The analysis was performed according to the procedure. CPAE (Bovine Cardiopulmonary Endothelial Cells) obtained from the American Type Culture Collection (ATCC) grew to approximately 95% confluence in minimal essential medium 10E. Cells were released from tissue culture flasks with 0.25% trypsin solution and plated in 24-well tissue culture dishes in the same medium at a density of 10,000 cells / well. After the petri dish was cultured at 37 ° C. for 8 hours in a 5.0% CO ₂ incubator. An analytical sample group and a control group were added. To ensure reproducibility, each sample was loaded into two different wells at 100 μL / well. After incubation for 60 hours with the sample, the medium was aspirated and the number of cells was determined based on a colorimetric measurement of cellular acid phosphatase.

Results and discussion:

  Each enantiomer of sulforaphane inhibits endothelial cell growth. The results of the endothelial cell analysis show that the addition of sulforaphane to the endothelial cell analysis demonstrates that it significantly inhibits endothelial cell growth. In the analysis, L-sulforaphane, D-sulforaphane and D, L-sulforaphan all expressed greater than 92% inhibition of endothelial cell proliferation at a concentration of 6.25 μg / mL. These results indicate that sulforaphane is a very effective endothelial cell growth inhibitor.

  In addition, a concentration-dependent test for angiogenesis inhibition was performed on L-sulforaphane, D-sulforaphane and D, L-sulforaphane and is shown in FIG. It is clear that sulforaphane is a very strong inhibitor of endothelial cell proliferation even at very low concentrations.

Example 3
Inhibition of various cancer cell lines by sulforaphane Cancer cell lines Analysis was performed as follows. The cancer cell lines used, Lncap (prostate), SW480 (large intestine) and HTB72 (melanoma) were obtained commercially from the American Type Culture Collection (ATCC). Cancer cells grow to 90% confluence in their respective media in 25 cm ² flasks. Cells are trypsinized, counted and diluted to 10,000 cells per ml. 1 ml of cells is added to each 24-well culture dish and the cells are allowed to attach overnight. Test samples are then added at various concentrations to either 50 ul H ₂ O, PBS (phosphate buffered saline) or 1X medium. Wells containing control samples receive the same volume of solution. Cells are grown for 60 hours at 37 ° C. and 5% CO ₂ . Remove media well from each and rinse cells once with 1 ml PBS to begin analysis. Then 500 μl of base solution (100 mM sodium acetate, 0.2% Triton X-100, pH 5.5, 1 mg / ml p-nitrophenyl phosphate) is added. Incubate the petri dish at 37 ° C. for 2 hours. Thereafter, 1 ml of 0.1 M sodium hydroxide is added and the optical density is read at 410 nm with a microplate reader.

Results and Discussion The results of inhibition analysis for various cancer cell lines with L-sulforaphane are shown below in Tables 4-6.

  We have also found that L-sulforaphane will inhibit the growth of many cancer cell lines including Lncap (prostate cancer cell line), SW480 (colon cancer cell line) and HTB72 (melanoma cell line). did. L-sulforaphane has been shown to potently inhibit the growth of these cell lines at very low concentrations (6.25 μg / ml). It appeared to be particularly effective against Lncap (prostate cancer cell line).

  Although the foregoing invention has been described in considerable detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be made. For example, as will be apparent to those skilled in the art, the inventive methods can be combined with one or more known anti-tumor anti-angiogenic or immune enhancing treatments and formulations (eg, shark cartilage, tyrosphingosine, sphingosine). Accordingly, the scope of the invention should not be construed as limiting the specification and examples, but is defined by the appended claims.

FIG. 1 is a graph showing the concentration-dependent inhibition of endothelial cell proliferation by L-sulforaphane, D-sulforaphane and D, L-sulforaphane in endothelial cell analysis.

Claims (36)

  A method of inhibiting endothelial cell growth, comprising the step of delivering a growth inhibitory amount of sulforaphane or a pharmaceutically acceptable derivative, salt or prodrug thereof to said cells.   The method of claim 1, wherein the sulforaphane is selected from the group consisting of D-sulforaphane, L-sulforaphane and racemic D, L-sulforaphane.   The method of claim 1, wherein the derivative of sulforaphane is sulforaphane nitrile.   A method of inhibiting angiogenesis in a tissue comprising delivering an anti-angiogenic amount of sulforaphane or a pharmaceutically acceptable derivative, salt or prodrug thereof to said tissue.   5. The method of claim 4, wherein the sulforaphane is selected from the group consisting of D-sulforaphane, L-sulforaphane and racemic D, L-sulforaphane.   The method according to claim 4, wherein the derivative of sulforaphane is sulforaphane nitrile.   A method of inhibiting angiogenesis in a major organ comprising the step of delivering an anti-angiogenic amount of D-, L- or racemic sulforaphane or a pharmaceutically acceptable derivative, salt or prodrug thereof to said tissue. How to have.   8. The method of any one of claims 1, 4 or 7, wherein the delivering step is in vitro, in vivo or ex vivo.   A method of treating a condition associated with pathological neovascularization in a host, comprising administering to a subject a therapeutically effective amount of an anti-angiogenic amount of sulforaphane or a pharmaceutically acceptable derivative, salt or prodrug thereof. The method which has the process to do.   10. The method of claim 9, wherein the sulforaphane is selected from the group consisting of D-sulforaphane, L-sulforaphane and racemic D, L-sulforaphane.   The pathologies include cancer and solid tumors, arthritic conditions, neovascular dermatologic conditions, diabetic retinopathy, Kaposi's sarcoma, age-related macular degeneration, restenosis, telangiectasia, glaucoma, keloid, cornea 10. The method of claim 9, selected from the group consisting of transplant rejection, wound granulation, hemangiofibroma, Osler-Weber syndrome, myocardial angiogenesis, psoriasis and scleroderma.   The method according to claim 9, wherein the medical condition is selected from the group consisting of prostate cancer, colon cancer and melanoma.   10. The method of claim 9, wherein the condition is an arthritic condition selected from the group consisting of rheumatoid arthritis and osteoarthritis.   The method according to claim 9, wherein the derivative of sulforaphane is sulforaphane nitrile.   A method of treating a pathology associated with pathological neovascularization in a subject's major organs, comprising administering to the subject a therapeutically effective amount of sulforaphane or a pharmaceutically acceptable derivative, salt or prodrug thereof. A method having steps.   16. The method according to any one of claims 9 to 15, wherein the delivering step is by oral, intravenous, intraperitoneal or transdermal administration.   16. The method according to any one of claims 9 to 15, further comprising administering an effective amount of an anti-angiogenic agent.   The method of claim 16, wherein the host or subject is an animal.   18. A method according to claim 17, wherein the animal is selected from the group consisting of pets, livestock or human patients.   A method for improving the general health and welfare of a subject, comprising the step of administering an effective amount of sulforaphane to the subject.   21. The method of claim 20, wherein the amount is provided as a health supplement.   21. The method of claim 20, wherein the host or subject is an animal.   24. The method of claim 21, wherein the animal is selected from the group consisting of pets, livestock or human patients.   21. The method of claim 20, wherein the sulforaphane is selected from the group consisting of D-sulforaphane, L-sulforaphane and racemic D, L-sulforaphane.   21. The method of claim 20, wherein the sulforaphane derivative is sulforaphane nitrile. A method of screening for therapeutic agents that inhibit neovascularization or endothelial cell growth comprising:
a. Contacting the drug with a suitable cell or tissue sample;
b. Contacting another sample of the appropriate cell or tissue sample with a therapeutically effective amount of sulforaphane or a pharmaceutically acceptable derivative, salt or prodrug thereof;
c. Comparing the growth of the sample of step (a) with the growth of the sample of step (b), wherein the growth is inhibited to the same or similar degree as the sample of step (b). Any drug is a therapeutic agent that inhibits neovascularization or endothelial cell growth; and
Having a method.   27. The method of claim 26, wherein the contacting step is in a test tube or in vivo.   27. The method of claim 26, wherein the sulforaphane is selected from the group consisting of D-sulforaphane, L-sulforaphane and racemic D, L-sulforaphane.   25. The method of claim 24, wherein the derivative of sulforaphane is sulforaphane nitrile.   26. The method of claim 25, further comprising contacting the samples of steps a and b with an effective amount of an anti-angiogenic agent.   A kit for treating a pathology associated with pathological neovascularization in a subject comprising a therapeutically effective amount of sulforaphane or a pharmaceutically acceptable derivative, salt or prodrug thereof and instructions for use .   The pathologies include cancer and solid tumors, arthritic conditions, neovascular dermatologic conditions, diabetic retinopathy, Kaposi's sarcoma, age-related macular degeneration, restenosis, telangiectasia, glaucoma, keloid, cornea 32. The kit of claim 31, selected from the group consisting of transplant rejection, wound granulation, angiofibroma, Osler-Weber syndrome, myocardial angiogenesis, psoriasis and scleroderma.   32. The kit of claim 31, wherein the medical condition is selected from the group consisting of prostate cancer, colon cancer and melanoma.   32. The kit of claim 31, wherein the condition is an arthritic condition selected from the group consisting of rheumatoid arthritis and osteoarthritis.   32. The kit of claim 31, wherein the sulforaphane is selected from the group consisting of D-sulforaphane, L-sulforaphane and racemic D, L-sulforaphane.   32. The kit of claim 31, wherein the derivative of sulforaphane is sulforaphane nitrile.

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