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WO2002060464A2 - Compositions contenant une fraction active isolee a partir de hedyotis diffusae et procedes d'utilisation - Google Patents

Compositions contenant une fraction active isolee a partir de hedyotis diffusae et procedes d'utilisation Download PDF

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Publication number
WO2002060464A2
WO2002060464A2 PCT/US2001/048144 US0148144W WO02060464A2 WO 2002060464 A2 WO2002060464 A2 WO 2002060464A2 US 0148144 W US0148144 W US 0148144W WO 02060464 A2 WO02060464 A2 WO 02060464A2
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Prior art keywords
extract
growth
effective amount
fraction
group
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WO2002060464A3 (fr
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Kin-Ping Wong
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Wackvom Ltd
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Wackvom Ltd
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Priority to JP2002560655A priority Critical patent/JP2005510448A/ja
Priority to EP01994223A priority patent/EP1357925A2/fr
Publication of WO2002060464A2 publication Critical patent/WO2002060464A2/fr
Anticipated expiration legal-status Critical
Publication of WO2002060464A3 publication Critical patent/WO2002060464A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is in the field of pharmaceuticals.
  • it is related to the field of anti-angiogenic pharmaceuticals for the prevention and treatment of disease.
  • Angiogenesis is the process through which new vascular structures arise by outgrowth from pre-existing capillaries.
  • endothelial cells become detached from the basement membrane as this support is degraded by proteolytic enzymes. These cells then migrate out from the parent vessel, divide, and form into a newly differentiated vascular structure (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 found to function in controlling blood vessel formation (Bussolino et al., (1997) Trends in Biochem Sci 22(7):251-256; Folkman and D'Amore, (1996) Cell 87: 1153-1155).
  • Angiogenesis normally occurs in a carefully controlled manner during embryonic development, during growth, and in special cases such as wound healing and the female reproductive cycle (Wilting and Christ, (1996) Naturwissenschaften 83:153-164; 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 angiogenesis are: 1) growth factor (i.e.
  • vascular endothelial growth factor VEGF
  • MMP matrix metalloproteinases
  • VEGF receptor interaction VEGF receptor interaction
  • endothelial cell migration to site of growth factor signaling
  • endothelial cell tubule formation VEGF signaling
  • endothelial cell tubule formation VEGF signaling
  • MMP matrix metalloproteinases
  • endothelial cell migration to site of growth factor signaling
  • endothelial cell tubule formation endothelial cell tubule formation.
  • Pathological angiogenesis plays a central role in a number of human diseases including tumor growth and metastatic cancer, diabetic retinopathy, rheumatoid arthritis, and other inflammatory diseases such as psoriasis (Folkman, (1995) Nature Med. 1(1):27-31; Polverini, (1995) Rheumatology 38(2): 103-112; Healy et al., (1998) Hum. Reprod. Update 4(5)
  • angiogenesis In these cases, progression of disease is driven by persistent unregulated angiogenesis.
  • new capillary blood vessels invade the joints and destroy the cartilage.
  • diabetic retinopathy capillaries in the retina invade the vitreous, bleed and cause blindness.
  • tumor growth and metastisis are angiogenesis dependent.
  • Most primary solid tumors go through a prolonged avascular state during which growth is 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.
  • protein angiogensis inhibitors have yet to be developed into therapeutic pharmaceuticals for disease patients.
  • therapeutic compounds that can be safely administered to a patient and be effective at inhibiting the pathological growth of vascular endothelial cells.
  • the present invention provides compositions and methods that are useful for this purpose and provides related advantages as well.
  • This invention provides processes for extracting pharmaceutically active fractions (also termed “extract”, “compound” or “drug”) from from Hedyotis Diffusae.
  • the process is extracting from a hot (about 80-90°C for about 30 minutes) tea of Hedyotis Diffusae a fraction, designated AHD04, isolated by column chromatography that has an optical absorbance between about 210 nm and about 250 nm.
  • One means to obtain this fraction is by steeping an effective amount of Hedyotis Diffusae in an effective amount of hot water to obtain a liquid extract and then filtering the extract to obtain a clear liquid extract.
  • This crude extract, designated EHDa can be processed for use as a food or health supplement.
  • EHDa is lyophilized, resuspended and the pharmaceutically active fraction ADH04 is separated by chromatography.
  • This invention provides a method for inhibiting the growth of endothelial cells by delivering to the cells a growth inhibitory amount of a fraction.
  • This invention also provides a method of inhibiting vascularization in a tissue by delivering to the tissue an anti-vascularization amount of a fraction.
  • Methods of treating various diseases, including cancer, are also provided herein.
  • Figure 1 depicts exemplary processes of this invention. However, it is to be understood, although not always explicitly stated that the reagents described herein are merely exemplary and that equivalents of such are well known in the art. The following are examples and equivalents thereof are within the scope of this invention:
  • Figure 1 depicts procedures for isolating active fraction designated EHDa that is useful as food and health supplements as well as the process for isolating the pharmaceutically active fraction, AHD04. Briefly, the extraction of the crude extract from Hedyotis Diffusae starts with the steeping of the dry plant in hot water ranging in temperature from about 60°C to about 100°C for a time period of about 10 minutes to about 60 minutes. This liquid extract is then filtered using cheesecloth or Miracloth to remove all the physical items and extremely large molecules from the liquid extract. This extract is then centrifuged at 1000 rpm for approximately 10 min. The clear liquid supernatant is saved and then lyophilized to concentrate the active fraction that has been isolated from Hedyotis Diffusae.
  • a CPAE assay is run to determine the effective concentrations of this new crude extract, EHDa, in inhibiting angiogenesis. All fractions, pellets, supernatants are assayed for anti-angiogenic activity using the CPAE assay after each step to insure that no anti-angiogenic activity is lost. Further isolation by concentration through chromatography, for example, yields the substantially purified active fraction AHD04.
  • Figure 3 is a graph showing activity of the fractions as they are isolated off the C- 18 chromatography column.
  • Figure 4 is a graph where Y axis is % inhibition versus concentration (X axis) after second run through C-18 column. MODES FOR CARRYING OUT THE INVENTION [013] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.
  • a cell includes a plurality of cells, including mixtures thereof.
  • compositions and methods include the recited elements, but not excluding others.
  • Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination.
  • a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
  • a "subject” or “host” is a vertebrate, preferably an animal or mammal, more preferably a human patient. Mammals include, but are not limited to, murines, simians, human patients, farm animals, sport animals, and pets.
  • cancer refers to cells that have undergone a malignant transformation that makes them pathological to the host organism.
  • Primary cancer cells that is, cells obtained from near the site of malignant transformation
  • the definition of a cancer cell includes not only a primary cancer cell, but also any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.
  • a "clinically detectable" tumor is one that is detectable on the basis of tumor mass; e.g., by such procedures as CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation. Biochemical or immunologic findings alone may be insufficient to meet this definition.
  • inhibit means to stop, delay or slow the growth, proliferation or cell division of endothelial cells or the formation of blood vessels in tissue.
  • Methods to monitor inhibition include, but are not limited to endothelial cell proliferation assays, measurement of the volume of a vascular bed by determination of blood content and quantitative determination of the density of vascular structures.
  • endothelial cell proliferation assays measurement of the volume of a vascular bed by determination of blood content
  • quantitative determination of the density of vascular structures When the culture is a mixture of cells, neovascularization is monitored by quantitative measurement of cells expressing endothelial cell specific markers such as angiogenic factors, proteolytic enzymes and endothelial cell specific cell adhesion molecules.
  • composition is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.
  • a "pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • the term "pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin, REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975)).
  • an "effective amount” is an amount sufficient to effect beneficial or desired results.
  • a therapeutic amount is one that achieves the desired therapeutic effect. This amount may be the same or different from a prophylatically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • Applicant has identified a process for extracting pharmaceutically active fractions from Hedyotis Diffusae.
  • the process requires extracting from a hot (about 80-90°C for about 30 minutes) tea of Hedyotis Diffusae a fraction wherein said fraction has an optical absorbance between about 210 nm and about 250 nm.
  • the active fraction has an optical absorbance of about 220 to about 240 nm.
  • the pharmaceutically active fraction has an optical absorbance after chromatography of about 230 nm.
  • Hedyotis Diffusae is steeped in an effective amount of hot water to obtain a liquid extract and then filtering the extract to obtain a clear liquid extract.
  • the extracted is lyophilized and then resuspended in a suitable carrier and the pharmaceutically active fraction is separated by chromatography.
  • this invention provides a method for inhibiting the growth of endothelial cells by delivering to the cells a growth inhibitory amount of a fraction.
  • This invention also provides a method of inhibiting vascularization in a tissue by delivering to the tissue an anti-vascularization amount of the fraction.
  • This method can be practiced in vitro or in vivo.
  • endothelial cells or vascularized tissue are cultured under conditions well known to those of skill in the art, e.g., as exemplified below.
  • the cells and/or tissue can be from an established cell line or cultured from a biopsy sample obtained from a subject.
  • the fraction is then directly added to the culture medium or delivered as a component of a pharmaceutical composition.
  • an in vitro assay to gauge efficacy for each patient would be advantageous. The present method provides these means to determine whether the methods of this invention will treat the patient.
  • a tissue biopsy is isolated from the patient and contacted with an effective amount of a pharmaceutical composition or therapy as defined herein and under conditions effective for growth and proliferation of the cells.
  • a pharmaceutical composition or therapy as defined herein and under conditions effective for growth and proliferation of the cells.
  • Angiogenesis or the formation of new vasculature is a fundamental process by which new blood vessels are formed. It participates in essential physiological events, such as reproduction development and wound healing. Under normal conditions, angiogenesis is highly regulated. However, many diseases are driven by persistent unregulated angiogenesis. In rheumatoid arthritis, new capillary blood vessels invade the joints and destroy the cartilage. In diabetic retinopathy, new capillaries in the retina invade the vitreous, bleed, and cause blindness. Tumor growth and metastasis are angiogenesis-dependent. Most primary solid tumors go through a prolonged state of avascular, and apparently dormant, growth in which the maximum size attainable is ⁇ l-2mm in diameter.
  • tumor cells can obtain the necessary oxygen and nutrient by simple passive diffusion.
  • These microscopic tumor masses can eventually switch on angiogenesis by recruiting surrounding mature host blood vessels to begin sprouting new blood vessel capillaries which grow toward, and eventually infiltrate the tumor mass, thus setting in motion the potential for relentless expansion of tumor mass and hematogenous metastatic spread as well.
  • the angiogenic switch was initially hypothesized to be triggered by the ectopic production and elaboration by tumor cells of a growth factor called "tumor angiogenesis factor" (TAF).
  • TAF tumor angiogenesis factor
  • This invention also provides a method of treating a disorder associated with pathological neovascularization in a subject by administering to the subject a therapeutically effective amount of the extract or a pharmaceutical composition containing the extract.
  • to "treat” means to alleviate the symptoms associated with pathological neovascularization as well as the reduction of neovascularization.
  • Such conditions include, but are not limited to arthritic conditions, neovascular-based dermatological conditions, diabetic retinopathy, Karposi's Sarcoma, age-related macular degeneration, restenosis, telangectasia, glaucoma, keloids, comeal graft rejection, wound granularization, angiofibroma, Osier- Webber Syndrome, myocardial angiogenesis, and scleroderma.
  • exemplary arthritic conditions are selected from the group consisting of rheumatoid arthritis, psoriatic arthritis and osteoarthritis.
  • to "treat” includes inhibition of the growth of blood vessels resulting in a lack of nutrients for the tumors and/or cancer cells needed by the tumor for its growth. Tumors and growths will decrease in size and possibly disappear.
  • Administration for the treatment of arthritic conditions will result in decreased blood vessel formation in cartilage, specifically joints, resulting in increased mobility and flexibility in these regions.
  • administration for the treatment of psoriasis will reduce dermatological symptoms such as scabbing, flaking and visible blood vessels under the surface of the skin.
  • administration of the active fraction In diabetic retinopathy, administration of the active fraction will reduce the formation of extraneous blood vessels in the retina, resulting in unobstructed vision.
  • administration of the active fraction will inhibit the growth and/or further formation of blood vessels, thereby inhibiting the formation of lesions and/or tumors that arise.
  • the agent when the active fraction is administered to a subject such as a mouse, a rat or a human patient, the agent can be added to a pharmaceutically acceptable carrier and systemically, orally, transdermally or topically administered to the subject.
  • Therapeutic amounts can be empirically determined and will vary with the pathology being treated, the subject being treated and the toxicity of the form of the fraction used in the therapeutic method.
  • the active fraction can be delivered orally, intravenously, intraperitoneally, or transdermally. When delivered to an animal, the method is useful to further confirm efficacy of the active fraction.
  • mice groups of nude mice (Balb/c NCR nu/nu female, Simonsen, Gilroy, CA) are each subcutaneously inoculated with about 10 5 to about 10 9 pathological cells as defined herein.
  • the fraction, extract or compound is administered, for example, by subcutaneous injection around the graft. Measurements to determine reduction of graft size are made in two dimensions using venier calipers twice a week.
  • MRL/lpr mice from Jackson Labs (Maine) are useful to test or monitor efficacy in arthritic conditions.
  • a positive therapeutic benefit includes reduced swelling of the joints and hindlegs of animals and reduced cartilage degradation which can be monitored by X-ray.
  • Administration in vivo can be effected in one dose, multiple doses, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents can be found below.
  • compositions and pharmaceutical formulations of the present invention can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as an active ingredient in pharmaceutical compositions.
  • the pharmaceutical compositions can be administered orally, infranasally, parenterally or by inhalation therapy, and may take the form of tablets, lozenges, granules, capsules, pills, ampoules, suppositories or aerosol form. They may also take the form of suspensions, solutions and emulsions of the active ingredient in aqueous or nonaqueous diluents, syrups, granulates or powders. In addition to a compound of the present invention, the pharmaceutical compositions can also contain other pharmaceutically active ingredients.
  • the active fraction also referred to herein as the active ingredient
  • the fraction, extract, or compound (“drug”) or composition containing it should be administered to achieve peak concentrations of the active compound at sites of disease. This may be achieved, for example, by the intravenous injection of the drug, optionally in saline, or orally administered, for example, as a tablet, capsule or syrup containing the active ingredient. Desirable blood levels of the drug may be maintained by a continuous infusion to provide a therapeutic amount of the active ingredient within disease tissue.
  • operative combinations is contemplated to provide therapeutic combinations requiring a lower total dosage of each component agent than may be required when each individual therapeutic compound or drug is used alone, thereby reducing adverse side effects.
  • the drug ingredient While it is possible for the drug ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation comprising at least one active ingredient, as defined above, together with one or more pharmaceutically acceptable carriers therefor and optionally other therapeutic agents.
  • 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 and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier that 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.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented a bolus, electuary or paste.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g., povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • compositions for topical administration may be formulated as an ointment, cream, suspension, lotion, powder, solution, paste, gel, spray, aerosol or oil.
  • a formulation may comprise a patch or a dressing such as a bandage or adhesive plaster impregnated with active ingredients and optionally one or more excipients or diluents.
  • the formulations are preferably applied as a topical ointment or cream containing the active ingredient.
  • the drug may be employed with either a paraffinic or a water-miscible ointment base.
  • the drug ingredients may be formulated in a cream with an oil-in-water cream base.
  • the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-l,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof.
  • the topical formulations may desirably include a compound that enhances absorption or penetration of the drug ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.
  • the oily phase of the emulsions of this invention may be constituted from known ingredients in any known manner. While this phase may comprise merely an emulsifier (otherwise known as an emulgent) it desirably comprises a mixture of at lease one emulsifier with fat or oil or with fat and oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier that acts as a stabilizer. It is also preferred to include both oil and fat.
  • an emulsifier otherwise known as an emulgent
  • a hydrophilic emulsifier is included together with a lipophilic emulsifier that acts as a stabilizer. It is also preferred to include both oil and fat.
  • the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax
  • the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
  • Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate.
  • the choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is very low.
  • the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers.
  • Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
  • Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient.
  • 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 may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient, such carriers as are known in the art to be appropriate.
  • Formulations suitable for nasal administration include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebulizer include aqueous or oily solutions of the active ingredient.
  • Formulations suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • sterile liquid carrier for example water for injections, immediately prior to use.
  • 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, daily subdose, as herein above recited, or an appropriate fraction thereof, of a drug ingredient.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily subdose, as herein above recited, or an appropriate fraction thereof, of a drug ingredient. They may also contain additional active agents, e.g., anti-tumor, anti-cancer, anti-angiogenic or immune enhancers.
  • the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable of oral administration may include such further agents as sweeteners, thickeners and flavoring agents.
  • suitable of oral administration may include such further agents as sweeteners, thickeners and flavoring agents.
  • the extract ("drug") or compositions of the same may also be presented for the use in the form of veterinary formulations, which may be prepared, for example, by methods that are conventional in the art.
  • This invention further provides a method for screening for a therapeutic agent for inhibiting neovascularization or endothelial cell growth.
  • the screen comprises:
  • step (c) comparing the growth of the sample of step (a) with the growth of the sample of step (b), and wherein any agent of step (a) that inhibits the growth to the same or similar extent as the sample of step (b) is a therapeutic agent for inhibiting neovascularization or the growth of endothelial cells.
  • a suitable sample intends any sample that contains endothelial cells.
  • the method can be practiced in vitro or in vivo as described herein.
  • a kit for treating a disorder associated with pathological neovascularization in a subject also is provided by this invention.
  • the kit includes a therapeutically effective amount of an extract and instructions for use.
  • the kit is useful to treat disorders selected from the group consisting of arthritic conditions, neovascular-based dermatological conditions, diabetic retinopathy, Karposi's Sarcoma, age-related macular degeneration, restenosis, telangectasia, glaucoma, keloids, comeal graft rejection, wound granularization, angiofibroma, Osier- Webber Syndrome, myocardial angiogenesis, scleroderma, rheumatoid arthritis, psoriatic arthritis and osteoarthritis.
  • the process shown for example in Figure 1, comprises extracting a soluble fraction AHD04 that has an optical absorbance of between about 210 nm to about 250 nm.
  • the fraction has an absorbance from about 220 nm to and 240 nm.
  • the active fraction's absorbance is about 230 nm.
  • leaves and/or stems of the plant are steeped in hot water (any temperature at or above about 60°C, and preferably above about 90°C). The tea is extracted and concentrated to provide EHDa.
  • CPAE Cardiopulmonary Artery Endothelial Cells, bovine acquired from American Type Tissue Culture (ATTC) were grown to nearly 95% confluence in MEM-10E.
  • the cells were released from the tissue culture flask with a 0.25% trypsin solution and planted in 24-well tissue culture plates in the same culture medium at a density of 10,000 cell/well. After the plates were cultivated for 8 hours at 37°C in a 5.0% CO 2 incubator. Assay samples and controls were added. Each sample was loaded in two different wells at 100 ⁇ L/well to insure reproducibility. After incubation with the sample for 60 hours, the medium was aspirated, and the number of cells was measured on the basis of the colorimetric measurement of cellular acid phosphatase. [069]
  • Example 3 Example 3
  • Calf Pulmonary Arterial Endothelial (CPAE) cells are plated at 10,000 cells per well in 24 well culture plates. After growth incubation at 37°C, 5% CO 2 for about 60 hours, a dosage of the sample is added (about 50 ⁇ l to about lOO ⁇ l) to each sample well and re- incubated for 30 minutes. After incubation, cells are assayed visually under an inverted microscope to detect the presence of cells and through the use of the ECC assay. Both methods are used to detect the presence or absence of endothelial cells in each well. Control cells containing no sample were used and grew normally.
  • CPAE Calf Pulmonary Arterial Endothelial
  • the titration assay was carried out on endothelial cells. Sample was made in concentration tifrations from l.Omg/mL to 0.00625mg/mL. Different samples were loaded on cell, while blank is the control. Results are shown in Table 1 (below).
  • FCC assay was carried out by using HEP-2 cells (isolated from human laryngeal tissue) as control. The results show that HEP-2 cells are not sensitive to the active fraction. [072] The results are shown in Table 2.
  • Matrigel (60 ⁇ l of lOmg/ml; Collaborative Lab # 35423) is placed in each well of an ice-cold 96-well plate. The plate is allowed to sit at room temperature for 15 minutes then incubated at 37°C for 30 minutes to permit the matrigel to polymerize.
  • HUVEC are prepared in EGM-2 (Clonetic # CC3162) at a concentration of 2X10 5 cells/ml.
  • the test compound is prepared at 2X the desired concentration (5 concentration levels) in the same medium.
  • Cells (500 ⁇ l) and 2X fraction or compound (500 ⁇ l) is mixed and 200 ⁇ l of this suspension are placed in duplicate on the polymerized matrigel.
  • the bottom chamber wells receive 27-29 ⁇ l of DMEM medium alone (baseline) or medium containing chemo-attractant (bFGF, VEGF or Swiss 3T3 cell conditioned medium).
  • the top chambers receive 45 ⁇ l of HUVEC cell suspension (1X10 6 cells/ml) prepared in DMEM+1% BSA with or without the fraction or compound. After a 5 hour incubation at 37°C, the membrane is rinsed in PBS, fixed and stained in Diff-Quick solutions.
  • the filter is placed on a glass slide with the migrated cells facing down and cells on top are removed using a Kimwipe. The testing is performed in 4-6 replicates and five fields are counted from each well. Negative unstimulated control values are subtracted from stimulated control and fraction or compound treated values and data is plotted as mean migrated cell ⁇ S.D. IC 50 is calculated from the plotted data. TNP-470 (NSC 642492) and paclitaxel (NSC 125973) are used as reference compounds.

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Abstract

La présente invention concerne des procédés permettant d'extraire des fractions pharmaceutiquement actives de Hedyotis Diffusae. Cette invention concerne un procédé permettant d'inhiber la croissance des cellules endothéliales par administration à ces cellules d'une quantité d'une fraction pouvant inhiber la croissance. La présente invention concerne également un procédé permettant d'inhiber la vascularisation dans un tissu par administration à ce tissu d'une quantité de fraction anti-vascularisation. L'invention concerne également des méthodes permettant de traiter diverses maladies, parmi lesquelles le cancer.
PCT/US2001/048144 2000-12-13 2001-12-12 Compositions contenant une fraction active isolee a partir de hedyotis diffusae et procedes d'utilisation Ceased WO2002060464A2 (fr)

Priority Applications (2)

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JP2002560655A JP2005510448A (ja) 2000-12-13 2001-12-12 ＨｅｄｙｏｔｉｓＤｉｆｆｕｓａｅから単離された活性な画分を含む組成物および使用方法
EP01994223A EP1357925A2 (fr) 2000-12-13 2001-12-12 Compositions contenant une fraction active isolee a partir de hedyotis diffusae et procedes d'utilisation

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2019201371B2 (en) * 2018-02-27 2025-01-02 Helen Chui Lan Chai Composition of combined plant proteins for treatment of cancer and diabetes

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CN101496845B (zh) * 2008-02-01 2013-02-20 中国科学院大连化学物理研究所 一种白花蛇舌草提取物及其分离制备方法

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AU5213586A (en) * 1985-10-03 1987-04-09 John Ky Extract of plant species hedyotis used as an anti-cancer tea

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2019201371B2 (en) * 2018-02-27 2025-01-02 Helen Chui Lan Chai Composition of combined plant proteins for treatment of cancer and diabetes

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