JP2005510448A - Compositions containing active fractions isolated from Hedyotis Diffusae and methods of use - Google Patents

Abstract

  The present invention provides a process for extracting a pharmaceutically active fraction from Hedyotis Diffusae. The present invention provides a method for inhibiting endothelial cell proliferation by delivering a growth inhibiting amount of a fraction to the cells. The present invention also provides a method for inhibiting angiogenesis in tissue by delivering an anti-angiogenic amount fraction to the tissue. Also provided herein are methods of treating various diseases including cancer. A process for preparing a biologically active fraction AHD04 from a composition of Hedyotis Diffusae comprising the step of extracting a fraction having an optical absorbance between about 210 nm and about 250 nm. Include.

Description

(Cross-reference to related applications)
This application is filed in US Provisional Application 60 / 255,502 (filed December 13, 2000), the contents of which are hereby incorporated by reference into this disclosure. S. C. Claim the benefits under 119 (e).

(Field of Invention)
The present invention is an invention of the pharmaceutical field. In particular, the invention relates to the field of anti-angiogenic medicaments for preventing and treating diseases.

(background)
Angiogenesis is a process caused by growth from capillaries where new vasculature already exists. In this process, endothelial cells are detached from the basement membrane when the basement membrane support is degraded by proteolytic enzymes. These cells then migrate out of the parent vessel, divide and form newly differentiated vasculature (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 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 various functions, such as growth factors, cell surface receptors, proteases, protease inhibitors, and extracellular matrix proteins (Achen and Stacker (1998) Int. J. Exp. Pathol. 79: Devalaraja and Richmond (1999) Trends in Pharmacol. Sci. 20 (4): 151-156; Hanahan (1997) Science 277: 48-50; Maisonpierre et al. (1997) Science 277: 55-60S; (1996) Cell 87: 1171-1180; Sato et al. (1995) Nature 376: 70-74; Mignati and R. fkin (1996) Enzyme Protein 49: 117-137; Pintucci et al. (1996) Semin Thromb Hemost 22 (6) 517-524; Vernon and Sage (1995) Am. J. Pathol. 147 (4): 873-883; (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 development provide many useful points for therapeutic interventions to control angiogenesis in vivo.

  Angiogenesis usually occurs in a carefully controlled manner during embryonic development, during proliferation, and in special cases (eg, wound healing and the female reproductive cycle) (Wilting and Christ (1996) Naturewissenschaffen). 83: 153-164; Goodger and Rogers (1995) Microcirculation 2: 329-343; Augustin et al. (1995) Am. J. Pathol.147 (2): 339-351). Some important steps in the angiogenesis process are: 1) growth factor (ie, vascular endothelial growth factor, VEGF) signaling; 2) matrix metalloproteinase (MMP) and VEGF receptor interaction; 3 ) Endothelial cell migration to the site of growth factor signaling; and 4) Endothelial tubule formation. Pathological angiogenesis plays a central role in many 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; Heary et al. (1998) Hum.Reprod. Update 4 (5): 736-396). In these cases, disease progression is driven by persistent disordered angiogenesis. For example, in rheumatoid arthritis, new capillaries invade indirectly and destroy cartilage. In diabetic retinopathy, the capillaries in the retina invade, bleed, and cause blindness in the vitreous humor. Importantly, tumor growth and metastasis are angiogenesis-dependent. Most primary solid tumors undergo a long avascular state (during this time growth is limited to a diameter of about 1-2 mm). Up to this size, tumor cells can obtain the necessary oxygen and nutrient supply by passive diffusion. These microscopic tumor masses eventually switch to angiogenesis, recruiting the surrounding blood vessels to initiate the sprouting capillaries that vascularize the tumor mass, and Provides sustainability of tumor expansion and malignant cell metastasis. While the understanding of the biological events that occur during pathological angiogenesis has advanced greatly, there are currently no effective pharmaceutical compounds that are useful for controlling angiogenesis in vivo. Thus, effective treatments that can control angiogenesis have the potential to alleviate a number of human diseases.

  Traditionally, pharmaceutical compounds have been developed by screening synthetic chemical compounds for the desired pharmaceutical properties and then testing them for toxicity and effects in vivo. Compounds selected in this way often have toxic side effects in vivo and this approach has not been successful in developing effective angiogenesis inhibitors for disease treatment. More recently, molecular biology techniques have been applied to develop angiogenesis inhibitors. Angiogenic protein inhibitors that control angiogenesis have been discovered in experimental models: for example, Angiostatin (O'Reilly et al. (1994) Cell 79 (2): 315-328) and Endostatin (O ' Reilly et al. (1997) Cell 88 (2): 277-285). Nevertheless, performing such protein therapies is expensive and it has been found that such protein therapies are difficult to formulate and deliver to a subject. Currently, protein angiogenesis inhibitors still need to be developed into therapeutic drugs for disease patients. Accordingly, there is a need for therapeutic compounds that can be safely administered to patients and that can be effective in inhibiting the pathological proliferation of vascular endothelial cells. The present invention provides compositions and methods that are useful for this purpose and provides related advantages as well.

(Disclosure of the Invention)
The present invention provides a process for extracting a pharmaceutically active fraction (also referred to as “extract”, “compound” or “drug”) from Hedyotis Diffusae. In one aspect, this process involves a fraction of Heyotis Diffusae hot (about 30 minutes, about 80-90 ° C.) tea (tea) termed AHD04 (which is an optical between about 210 nm and about 250 nm). Extraction step) having an absorbance and isolated by column chromatography. One means to obtain this fraction is to soak an effective amount of Hedyotis Diffusae in an effective amount of hot water to obtain a liquid extract, and then filter the extract to obtain a clear liquid extract by. This crude extract (referred to as 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.

  The present invention provides a method for inhibiting endothelial cell proliferation by delivering a growth-inhibiting amount of a fraction to cells. The present invention also provides a method of inhibiting angiogenesis in a tissue by delivering an anti-angiogenic amount fraction to the tissue. Methods for treating various diseases including cancer are also provided herein.

(Form for carrying out the invention)
Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identified 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 belongs.

  The practice of the present invention, unless otherwise indicated, includes conventional techniques of molecular biology (including recombinant techniques), microbiology, organic chemistry, cell biology, biochemistry and immunology (these are those of ordinary skill in the art). Is within the range. Such techniques are explained fully in the literature.

(Definition)
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 “a cell” includes a plurality of cells (including mixtures thereof).

  As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but do not exclude others. The “Consisting essentially of”, when used to define compositions and methods, should mean excluding other elements that are inherently significant to the combination. Accordingly, a composition consisting essentially of the elements defined herein contains trace impurities and pharmaceutically acceptable carriers (eg, phosphate buffered saline) derived from isolation and purification methods. Water, preservatives, etc.) are not excluded. “Consisting of” should mean excluding other ingredients and even the finest components of the substantial method steps for administering the compositions of the invention. Embodiments defined by each of these conversion terms are within the scope of this invention.

  All numerical designations (eg, pH, temperature, time, concentration, and molecular weight) (including range) are approximate and change to (+) or (-) with 0.1 increments. It is to be understood that all numerical designations are preceded by the term “about”, although not necessarily explicitly indicated. It is also to be understood that although not necessarily explicitly indicated, the reagents described herein are merely exemplary and their equivalents are well known in the art.

  The term “isolated” means a state in which the compound is separated from the components with which it is normally associated in nature (cellular or other components).

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

  The terms “cancer”, “neoplasm”, and “tumor” are used interchangeably to refer to cells that have undergone malignant transformation and become pathogenic to the host organism, in either one or more forms. Say. Primary cancer cells (ie, cells obtained from near the site of malignant transformation) can be easily distinguished from non-cancerous cells by well-established techniques (particularly histological examination). As used herein, the definition of cancer cell includes not only primary cancer cells, but also any cell derived from a cancer cell ancestor. These include metastasized cancer cells, and in vivo cultures and cell lines derived from cancer cells. When referring to a type of cancer usually expressed as a solid tumor, a “clinically detectable” tumor is based on a tumor mass (eg, CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound, Or a detectable tumor (by a procedure such as palpation). To meet this definition, biochemical or immunological knowledge alone may be insufficient.

  As used herein, “inhibit” means stopping, slowing, or slowing the growth, proliferation, or cell division, or angiogenesis, of endothelial cells in a tissue. Methods for monitoring inhibition include, but are not limited to, endothelial cell proliferation assay, determination of blood content and determination of vascular bed volume by quantitative determination of vascular structure density. When the culture is a mixture of cells, neovascularization is a quantitative measurement of cells that express endothelial cell-specific markers (eg, angiogenic factors, proteolytic enzymes, and endothelial cell-specific cell adhesion molecules). Monitored by

  “Composition” is intended to mean a combination of an active agent with another compound or composition (which may be inactive (eg, a detectable agent or label) or active) (eg, an adjuvant).

  A “pharmaceutical composition” includes a combination of an active agent and a carrier (which may be inert or active) that makes the composition suitable for in vitro, in vivo, or ex vivo diagnostic or therapeutic use. Intended.

  As used herein, the term “pharmaceutically acceptable carrier” refers to any standard pharmaceutical carrier such as phosphate buffered saline, water, and emulsions (oil / water emulsions or Water / oil emulsion), and various types of wetting agents.The composition may also include stabilizers and preservatives.Martin, REMINGTON'S PHARM as examples of carriers, stabilizers, and adjuvants See SCI, 15th Edition (Mack Publ. Co., Easton (1975)).

  An “effective amount” is an amount sufficient to produce a beneficial or desired result. For example, a therapeutic amount is an amount that achieves a desired therapeutic effect. This amount can be the same as or different from the prophylactically effective amount (the amount required to prevent the disease or symptoms of the disease from developing). An effective amount can be administered in one or more administrations, applications, or dosages.

  The inventors have identified a process for extracting pharmaceutically active fractions from Hedyotis Diffusae. In one aspect, the process comprises extracting a fraction having an absorbance between about 210 nm and about 250 nm from a high temperature (about 80-90 ° C. for about 30 minutes) extract (hot tea) of Hedyotis Diffusae. Need. In a further aspect, the active fraction has an absorbance of about 220 nm to about 240 nm. In yet a further aspect, the pharmaceutically active fraction has an absorbance after chromatography of about 230 nm. An effective amount of Hedyotis Diffusae is decocted in an effective amount of hot water to provide a liquid extract, which is then filtered to obtain a clean liquid extract. This extract is lyophilized and then resuspended in a suitable carrier, and the pharmaceutically active fractions are separated by chromatography.

  The inventors have also discovered that this fraction inhibits endothelial cell proliferation and has anti-angiogenic properties. In accordance with these findings, the present invention provides a method of inhibiting the growth of cells by delivering a growth inhibiting amount of the fraction to endothelial cells. The invention also provides a method of inhibiting angiogenesis in tissue by delivering an anti-angiogenic amount of this fraction to the tissue.

  The method can be performed in vitro or in vivo. When performed in vitro, endothelial cells or vascularized tissue are cultured under conditions well known to those skilled in the art, eg, as exemplified below. The cells and / or tissues can be derived from the constructed cell line or can be cultured from a biopsy sample obtained from a subject. This fraction is then added directly to the culture medium or delivered as a component of the pharmaceutical composition.

  Since not all treatments are effective for each individual, in vitro assays that assess the cost for each patient are effective. The present invention provides a means for determining whether the method of the present invention treats a patient. For example, a tissue biopsy is isolated from a patient and contacted with an effective amount of a pharmaceutical composition or treatment as defined herein under conditions effective for cell growth and proliferation. Inhibition of pathological cell proliferation as determined by conventional procedures (eg, the CPAE assay described herein) may allow the compositions and / or therapies of the invention to be effective in treating patients. Indicates.

  Angiogenesis or the formation of new vasculature is the basic process of forming new blood vessels. This is involved in essential physiological phenomena such as reproductive system development and wound healing. Under normal conditions, angiogenesis is highly regulated. However, many diseases are caused by persistent unregulated angiogenesis. In rheumatoid arthritis, new capillaries invade the joint and destroy cartilage tissue. In diabetic retinopathy, new capillaries in the retina invade the vitreous, bleed, and cause blindness. Tumor growth and metastasis are angiogenesis-dependent. The most primary solid tumors undergo a long-term avascular state, apparent dormancy, and growth (the maximum reachable size is about 1-2 mm in diameter). Up to this size, tumor cells can acquire the necessary oxygen and nutrients by simple passive diffusion. These microscopic tumor masses grow towards the tumor mass and eventually recruit surrounding mature host blood vessels to initiate the generation of new capillaries that penetrate the tumor mass. Can eventually initiate angiogenesis. This creates the possibility of unrelenting expansion of the tumor mass and hematogenous metastatic spread. It was initially assumed that the initiation of angiogenesis is induced by assimilation by ectopic products and growth factors called "tumor angiogenesis factor" (TAF) by tumor cells.

  The present 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 an extract or a pharmaceutical composition comprising the extract. . As used for this reason, “treating” means to alleviate symptoms associated with pathological neovascularization and reduced neovascularization. Such conditions include arthritic conditions, skin conditions based on neovascularization, diabetic retinopathy, capage sarcoma, age-related macular degeneration, restenosis, telangectasia, glaucoma, keloids, corneal transplant rejection, wounds Examples include, but are not limited to, granulation, angiofibroma, Osler-Webber syndrome, myocardial angiogenesis, and scleroderma. An example of an arthritic condition is selected from the group consisting of rheumatoid arthritis, psoriatic arthritis, and osteoarthritis. For the treatment of cancer and solid tumors, “treating” includes inhibiting the growth of blood vessels. Inhibition of blood vessel growth depletes tumor nutrients and / or cancer cells required by the tumor for growth. Tumors and growth can diminish in size and disappear. Administration to treat arthritic conditions causes a decrease in angiogenesis in cartilage tissue, particularly joints. This increases the mobility and flexibility in these areas. For the treatment of psoriasis, administration reduces dermatological symptoms such as scabbing, flaking, and visible blood vessels under the skin surface. In diabetic retinopathy, administration of the active fraction reduces the formation of extraneous blood vessels in the retina. Thereby, the field of view is not disturbed. In the treatment of Kaposi's sarcoma, administration of the activated fraction inhibits growth and / or further angiogenesis, thereby inhibiting the formation of lesions and / or tumors.

  When the active fraction is administered to a subject (eg, a mouse, rat, or human patient), the agent can be added to a pharmaceutically important carrier and administered to the subject systemically, orally, transdermally Administration or local administration. The therapeutic amount can be determined empirically and varies with the toxicity of the pathology being treated, the subject being treated, and the fractional form used in the therapeutic method. The active fraction can be delivered orally, intravenously, intraperitoneally, or transdermally. When administered to animals, this method is useful to further confirm the effect of the active fraction.

As an example of an animal model, nude mice (Balb / c NCR nu / nu female, Simonsen, Gilroy, CA) each have about 10 ⁵ to about 10 ⁹ pathological cells subcutaneously as described herein. Vaccinated. When the transplant is complete, this fraction, extract, or compound is administered, for example, by subcutaneous injection near the implant. Measurements to determine the reduction in implant size are performed two-dimensionally with a veneer scissor twice a week.

MRL / lpr mice (MRL / MpJ-Fas ^lpr ) from Jackson Labs (Maine) are useful for testing or monitoring effects in arthritic conditions. Positive therapeutic benefits include decreased swelling of the animal's joints and hind paws and decreased cartilage tissue degradation, which can be monitored by x-ray.

  In vivo administration can be performed in a single dose, multiple doses, continuously, or intermittently throughout the course of treatment. Methods of determining the most effective means of administration and dosage 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 specialist. Methods for administering appropriate dosage formulations and reagents can be found below.

  The compositions and pharmaceutical formulations of the present invention can be used in the manufacture of medicaments and to treat humans and other animals by administering according to conventional procedures (eg, active ingredients in pharmaceutical compositions). .

  The pharmaceutical composition may be administered orally, intranasally, parenterally or by inhalation therapy, in the form of tablets, troches, granules, capsules, pills, ampoules, suppositories, or aerosol forms Can take. They can also take the form of suspensions, solutions, and emulsions of the active ingredient in aqueous or non-aqueous diluents, syrups, granules, or powders. In addition to the compounds of the present invention, the pharmaceutical compositions may also contain other pharmaceutically active ingredients.

  More particularly, the active fraction, referred to herein as the active ingredient, can also be any suitable route (including oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), It can be administered for treatment by the vagina, parental (including subcutaneous, intramuscular, intravenous and intradermal) and lung. It will also be appreciated that the preferred route will vary with the condition and age of the recipient and the disease being treated.

  It will be appreciated that the appropriate dosage of the active fraction of the present invention may depend on the type and severity and stage of the disease and may vary from patient to patient. The determination of the optimal dosage is generally related to a balance in the level of therapeutic benefit for all risks or adverse side effects in the treatment of the present invention.

  Ideally, this fraction, extract or compound (“drug”) or composition comprising it should be administered at the site of the disease so that the concentration of the active compound reaches a peak. This can be accomplished, for example, by intravenous injection of the drug (if desired, in saline) or oral administration (eg, a tablet, capsule or syrup containing the active ingredient). The desired blood level of the drug can be maintained by continuous infusion to provide a therapeutic amount of the active ingredient within the diseased tissue. The use of an effective combination provides a therapeutic combination that requires a lower total dosage of each component drug than can be required when each individual therapeutic compound or drug is used alone, By reducing harmful side effects.

  The drug component may be administered alone, but at least one as described above, together with one or more pharmaceutically acceptable carriers for this drug component and optionally other therapeutic agents. It is preferably presented as a pharmaceutical formulation comprising one active ingredient. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation.

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

  Formulations of the present invention suitable for oral administration are, for example, as discrete units such as capsules, cachets or tablets, each of which contains a predetermined amount of the active ingredient; as a powder or granules; Or as a solution or suspension in a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. These active ingredients can also be presented in boluses, electuaries or pastes.

  A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be in free-flowing form (eg, binders (eg, povidone, gelatin, hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (eg, sodium starch glycolate (optional) soda starch glycolate), cross-linked povidone, cross-linked sodium carboxymethyl cellulose), powders or granules mixed with surfactants or dispersants) may be prepared by pressing with a suitable machine. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Tablets may be coated or scored as required, and provide, for example, sustained or controlled release of the active ingredient therein, using, for example, various proportions of hydroxypropyl methylcellulose. Can be formulated to provide the desired release profile. Tablets may optionally be provided with an enteric coating to provide release in portions of the gastrointestinal tract other than the stomach.

  Formulations suitable for topical administration in the mouth include lozenges (usually containing the active ingredient in a flavored base which is sucrose and gum arabic or tragacanth); pastilles (gelatin and glycerin or sucrose and Active ingredients in an inert base such as gum arabic); and mouthwashes (containing the active ingredient in a suitable liquid carrier).

  A pharmaceutical composition for topical administration according to the invention may be formulated as an ointment, cream, suspension, lotion, powder, solution, paste, gel, spray, aerosol or oil. Alternatively, the formulation may comprise a patch or bandage such as a dressing or adhesive cast soaked in the active ingredient and optionally one or more excipients or diluents.

  For diseases of the eye or other external tissue (eg mouth and skin), the formulation is preferably applied as a topical ointment or cream containing the active ingredient. When formulated in an ointment, the drug can be used with either a paraffinic or water-miscible ointment base. Alternatively, the drug component can be formulated in a cream with an oil-in-water cream base.

  If desired, the aqueous phase of the cream base may comprise, for example, at least about 30% w / w polyhydric alcohol (ie, an alcohol having two or more hydroxyl groups (eg, propylene glycol, butane-1,3-diol). Mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof)). Topical formulations may desirably include compounds that enhance the absorption or penetration of drug components through the skin or other diseased areas. Examples of such dermal penetration enhancers include dimethyl sulfoxide and related analogs.

  The oily phase of the emulsions of the present invention can be composed of known ingredients in any known manner. This phase may contain only emulsifiers (otherwise known as elimination promoters), but desirably contains at least one emulsifier with fat or oil or with fat and oil. Preferably, a hydrophilic emulsifier is included with a lipophilic emulsifier that acts as a stabilizer. It is also preferred that both oil and fat are included. At the same time, emulsifiers with or without stabilizers constitute so-called emulsifying waxes, and the waxes, together with oils and / or fats, are so-called emulsifying ointment bases (which are oily dispersions of cream formulations). Forming a phase).

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

  Since the solubility of the active compound in most oils probably used in pharmaceutical emulsion formulations is very low, the selection of an appropriate oil or fat for the formulation is based on achieving the desired aesthetic properties. Thus, the cream should preferably be a non-greasy, non-colorable and washable product with an appropriate consistency to avoid leakage from tubes or other containers. Linear or branched, basic monoalkyl or dibasic alkyl esters (e.g., di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acid, isopropyl myristate, decyl oleate, isopropyl palmitate, Mixtures of branched chain esters known as butyl stearate, 2-ethylhexyl palmitate, or Crodamol CAP can be used, which can be used alone or in combination depending on the properties required. Alternatively, high melting point lipids (eg, 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 (eg containing cocoa butter or a salicylate).

  Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foaming agents or spray formulations, which are known in the art to be suitable in conjunction with the active ingredient. Including careers.

  Formulations suitable for nasal administration (the carrier is a solid) include, for example, a coarse powder having a particle size in the range of between about 20 to about 500 microns, which is inhaled through the nose ( I.e., rapidly inhaled through a nasal cavity from a powder container, tightly attached to the nose). Suitable formulations include aqueous or oily solutions of the active ingredient, where the carrier is a liquid for administration by, for example, nasal spray, administration as a nasal spray, or aerosol administration through a nebulizer.

  Formulations suitable for parenteral administration can make the formulation isotonic with the blood of the intended recipient and can be prepared as aqueous and non-aqueous sterile injection solutions (including antioxidants, buffers, And aqueous and non-aqueous sterile suspensions (which may include suspensions and thickeners), and liposomes or other particulate systems (where the compound is a blood component) Or designed to target one or more organs). These formulations can be presented in single or multiple dose sealed containers (eg, ampoules and vials) and require only the addition of a sterile liquid carrier (eg, water for injection) immediately prior to use. It can be stored in a freeze-dried (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 comprising a daily dose or unit, daily sub-dose, or an appropriate portion thereof of a drug component, as listed herein above. It is.

  Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, or an appropriate portion thereof, of a drug component, as listed herein above. They can also contain additional active agents such as anti-tumor agents, anti-cancer agents, anti-angiogenesis agents or immune enhancing agents.

  In addition to the ingredients specifically mentioned above, it is to be understood that the formulations of the present invention may include other drugs routine in the art in view of the type of formulation in question. (For example, agents suitable for oral administration may include additional agents such as sweeteners, thickeners and flavoring agents).

  The same extract (“drug”) or composition can also be shown for use in the form of veterinary formulations, which can be prepared, for example, by methods routine in the art.

The present invention further provides a method of screening for therapeutic agents for inhibiting neovascularization or endothelial cell proliferation. This screening includes:
(A) contacting the drug with an appropriate cell or tissue sample;
(B) contacting a separate sample of suitable cells or tissues with a therapeutically effective amount of an extract of the invention or a pharmaceutically acceptable composition containing the extract; and (c) step ( comparing the growth of the sample of a) with the growth of the sample of step (b), wherein the growth of step (a) is inhibited to the same or similar degree as the sample of step (b) Any agent is a therapeutic agent for inhibiting neovascularization or endothelial cell proliferation.

  As used herein, a suitable sample is intended to be any sample containing endothelial cells. The method can be performed in vitro or in vivo as described herein.

  Also provided by the present invention is a kit for disordering a disease associated with pathological neovascularization in a subject. The kit includes a therapeutically effective amount of extract and instructions for use. This kit is useful for treating a disorder selected from the group consisting of: arthritic condition, angiogenic skin condition, diabetic retinitis, Kaposi's sarcoma, age-related muscle degeneration, restenosis , Telangectasia, glaucoma, keloid, corneal transplant rejection, wound granulation, hemangiofibroma, Osler-Webber syndrome, myocardial angiogenesis, scleroderma, chronic Rheumatoid arthritis, psoriatic arthritis and osteoporosis.

  The following examples are for illustrative purposes and do not limit the invention.

(Example)
(material)
The following materials were used in the methods described below.

  Dry plants of Herba Hedyotis Diffusae are homogenized and hot water (80-90 ° C. (Sigma); sodium acetate (Sigma); ammonium sulfate (Sigma); hydrochloric acid (VWR Scientific); phosphatase substrate (Sigma); Sephadex G-25 (Sigma); and Triton X-100 (Sigma)).

(Example 1)
(Isolation and purification)
The present invention provides several embodiments of a process for preparing biologically active fractions from Hedyotis Diffusae. In one aspect, for example, the process shown in FIG. 1 includes extracting a soluble fraction AHD04 having an optical absorbance between about 210 nm and about 250 nm. In a further aspect, this fraction has an absorbance of about 220 nm to about 240 nm. In yet a further aspect, the absorbance of this active fraction is about 230 nm. In a further aspect, plant leaves and / or stems are immersed in hot water (any temperature above about 60 ° C., preferably above about 90 ° C.). The tea is extracted and concentrated to provide EHDa.

(Example 2)
(Determining angiogenesis inhibition by endothelial cell culture (EGG) assay)
The assay used to determine the rate of inhibition of angiogenesis is a modification to determine cell number by the level of acid phosphatase activity (Liang and Wong (1999) ANGIOGENESIS: FROM THE MOLECULAR TO INTEGRATIVE PHARMMACOLOGY (Maraudoudais) A variation of the assay developed by DT Connally et al. (1986) Anal. Biochem. 152: 136-4, with Kluwer Academic / Plenum.Publishers, New York) American Type TissueCC Culture. CPAE (cardiopulmonary artery endothelial cells, bovine) obtained from These cells were grown from tissue culture flasks using 0.25% trypsin solution and 24 wells in the same culture medium at a density of 10,000 cells / well. The tissue culture plates were then planted, and the plates were then cultured in a 5.0% CO ₂ incubator for 8 hours at 37 ° C. Assay samples and controls were added, each sample ensuring reproducibility. At 100 μL / well, two different wells were loaded, after 60 hours incubation with the sample, the medium was aspirated and the cell number was determined based on a colorimetric measurement of the acid phosphatase of the cells.

(Example 3)
(Cytolytic / Cytotoxicity assay)
Bovine pulmonary artery endothelial (CPAE) cells are plated into 24-well culture plates at 10,000 cells per well. After about 60 hours of growth incubation at 37 ° C., 5% CO ₂ , a dose of sample (about 50 μl to about 100 μl) is added to each sample well and reincubated for 30 minutes. After incubation, the cells are assayed visually under an inverted microscope to detect the presence of the cells and assayed through the use of an ECC assay. Both methods are used to detect the presence or absence of endothelial cells in each well. Control cells without sample were used and grew normally.

(Example 4)
(Sample titration assay)
To determine whether anti-angiogenic activity from a fraction (eg, ADH04) is dose related, a titration assay was performed on endothelial cells. Samples were made at titration concentrations from 1.0 mg / mL to 0.00625 mg / mL. Different samples are loaded into the cells, while the blank is the control. The results are shown in Table 1 (below).

  Table 1: Angiogenesis inhibition assay of AHD04 at different concentrations

（実施例５）
（阻害特異性）
この阻害活性が、内皮細胞特異的であるか否かを決定するために、ＦＣＣアッセイを、コントロールとしてＨＥＰ−２細胞（ヒト喉頭組織から単離された）を使用することによって、実行した。結果は、ＨＥＰ−２細胞が、活性な画分に対して感受性でないことを示す。

(Example 5)
(Inhibition specificity)
To determine if this inhibitory activity is endothelial cell specific, FCC assays were performed by using HEP-2 cells (isolated from human laryngeal tissue) as a control. The results show that HEP-2 cells are not sensitive to the active fraction.

  The results are shown in Table 2.

（実施例６）
（ＭＭＰアッセイ）
Ｐ．Ｃ．Ｂｒｏｏｋｓら（１９９６）は、「Ｌｏｃａｌｉｚａｔｉｏｎ ｏｆ ｍａｔｒｉｘ ｍｅｔａｌｌｏｐｒｏｔｅｉｎａｓｅ ＭＭＰ−２ ｔｏ ｔｈｅ ｓｕｒｆａｃｅ ｏｆ ｉｎｖａｓｉｖｅ ｃｅｌｌｓ ｂｙ ｉｎｔｅｒａｃｔｉｏｎ ｗｉｔｈ ｉｎｔｅｇｒｉｎ αｖβ３」、Ｃｅｌｌ ８５：６８３−９３において、マトリックスメタロプロテイナーゼとαｖ／β３インテグリンとの相互作用に対するインビトロアッセイを記載する。ＭＭＰ−２／αｖβ３インテグリン複合体に対する実験サンプルの効果は、このサンプルの作用機構が、脈管形成経路のこの区域の任意の破壊を含むか否かを決定する。これは、この実験サンプルが、αｖβ３インテグリンとのＭＭＰ−２の相互作用を阻害し得るか否かを試験する工程を包含する。最初に、これを、ＭＭＰ−２に対する抗体を使用するＥＬＩＳＡによって行い、そしてサンプルに対するこれらの抗体の結合を試験する。陽性の結果が生じるか否かのさらなる研究を続ける。ＭＭＰ−２の公知の天然のインヒビターであるＴＩＭＰ−２（マトリックスメタロプロテアーゼ−２の組織インヒビター）を、コントロールとして使用する。

(Example 6)
(MMP assay)
P. C. Brooks et al. (1996), “Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with α αββ3”, The assay is described. The effect of the experimental sample on the MMP-2 / αvβ3 integrin complex determines whether this sample's mechanism of action involves any disruption of this area of the angiogenic pathway. This involves testing whether this experimental sample can inhibit the interaction of MMP-2 with αvβ3 integrin. Initially, this is done by ELISA using antibodies against MMP-2 and the binding of these antibodies to the sample is tested. Continue to study further whether a positive result occurs. TIMP-2 (a tissue inhibitor of matrix metalloproteinase-2), a known natural inhibitor of MMP-2, is used as a control.

(Example 7)
(Endothelial tubule / cortex formation assay)
Matrigel (60 μl of 10 mg / ml; Collaborative Lab # 35423) is placed in each well of an ice-cooled 96-well plate. The plate is left at room temperature for 15 minutes and then incubated at 37 ° C. for 30 minutes to allow matrigel polymerization. Meanwhile, HUVEC are prepared in EGM-2 (Clonetic # CC3162) at a concentration of 2 × 10 ⁵ cells / ml. The test compound is prepared at 2 × the desired concentration (5 concentration levels) in the same medium. Cells (500 μl) and 2 × fractions or compounds (500 μl) are mixed and 200 μl of this suspension is placed in duplicate on the polymerized Matrigel. After 24 hours of incubation, photographs are taken in triplicate for each concentration using a Bioquant Image Analysis system. Drug effect (IC ₅₀ ) is assessed relative to untreated controls by measuring the length of cords / tubules formed and the number of connections. TNP-470 (NSC 642492) and paclitaxel (NSC 125973) are used as reference compounds.

(Example 8)
(Endothelial cell migration assay)
Migration is assessed using a 48 well Boyden chamber and 8 μm pore size collagen coated (10 μl / ml rat tail collagen; Collaborative Laboratories) polycarbonate filter (Osmonics, Inc.). Lower chamber wells receive 27-29 μl of DMEM medium alone (reference) or chemoattractant (bFGF, VEGF or Swiss 3T3 cell conditioned medium). The upper chamber receives 45 μl of HUVEC cell suspension (1 × 10 ⁶ cells / ml) prepared in DMEM + 1% BSA with or without fractions or compounds. After 5 hours incubation at 37 ° C., the membranes are rinsed with PBS, fixed and stained in Diff-Quick solution. The filter is placed on a glass slide with the migrated cells facing down and the top cells are removed using a Kimwipe. The test is performed 4-6 replicates and 5 fields are counted from each well. Negative unstimulated control values were subtracted from stimulated controls, and values and data tested with fractions or compounds were expressed as mean migrating cells ± S. D. Plot with IC ₅₀ is calculated from the plotted data. TNP-470 (NSC 642492) and paclitaxel (NSC 125973) are used as reference compounds.

  Although the foregoing invention has been described in some 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 practiced. For example, as will be apparent to those of skill in the art, the methods of the present invention may include one or more known anti-tumor, anti-angiogenic or immune enhancing therapeutic agents and compositions (eg, shark cartilage, tyrosphingosine or sphingosine). ). Accordingly, the description and examples should not be construed as limiting the scope of the invention which is defined by the appended claims.

FIG. 1 illustrates an exemplary process of the present invention. However, although not always explicitly stated, it should be understood that the reagents described herein are merely exemplary and that such equivalents are well known in the art. . The following are examples and their equivalents are within the scope of the invention. FIG. 1 shows a procedure for isolating an active fraction designated EHDa that is useful as a food and dietary supplement, as well as a process for isolating a pharmaceutically active fraction AHD04. Briefly, extraction of the crude extract from Hedyotis Diffusae begins by soaking the dried plant in hot water (temperature range of about 60 ° C. to about 100 ° C.) for about 10 minutes to about 60 minutes. The liquid extract is then filtered using cheesecloth or Miracloth to remove all physical and very large molecules from the liquid extract. The extract is then centrifuged at 1000 rpm for about 10 minutes. The clear liquid supernatant is taken and then lyophilized to concentrate the active fraction isolated from Hedyotis Diffusae. A CPAE assay is performed to determine the effective concentration of this new crude extract (EHDa) in inhibiting angiogenesis. All fractions, pellets and supernatants were assayed for anti-angiogenic activity using the CPAE assay after each step confirming that anti-angiogenic activity was not lost. For example, further isolation by concentration via chromatography provides a substantially purified active fraction AHD04. FIG. 2 is a graph showing the activity of fractions when isolated from G-25 gel filtration chromatography [column = volume 20 cm × 80 cm; run at 4 ° C. and 3.0 ml / mL water flow rate. ]. FIG. 3 is a graph showing the activity of fractions when isolated from a C-18 chromatography column. FIG. 4 is a graph of inhibition (%) (Y axis) versus concentration (X axis) after the second run through a C-18 column.

Claims (21)

A process for preparing a biologically active fraction AHD04 from a composition of Hedyotis Diffusae, the process comprising extracting a fraction having an optical absorbance between about 210 nm and about 250 nm. Inclusive process. A process for preparing a biologically active fraction EHDa from a composition of Hedyotis Diffusae, comprising immersing an effective amount of Hedyotis Diffusae in an effective amount of hot water and submerging the liquid extract. And obtaining the EHDa by filtering the extract. The process of claim 1, wherein the process comprises the following steps:
(A) immersing an effective amount of Hedyotis Diffusae in hot water to obtain a liquid extract;
(B) centrifuging to obtain a clear liquid extract;
(C) drying and resuspending the liquid extract in an acceptable carrier; and (d) a biologically active extract AHD04 having an optical absorbance between about 210 nm and about 250 nm. Obtaining the active fraction by chromatography so as to obtain
Including the process. A biologically active extract obtained by the process according to claim 1. A composition comprising the extract of claim 4 and a pharmaceutically acceptable carrier. 6. The composition of claim 5, further comprising an effective amount of an agent selected from the group consisting of an anti-angiogenic agent, an anti-tumor agent and an immunopotentiator. A method for inhibiting endothelial cell proliferation comprising delivering a growth inhibiting amount of the extract of claim 4 to said cells. A method of inhibiting angiogenesis in a tissue, the method comprising delivering an anti-angiogenic amount of the extract of claim 4 to the tissue. A method of treating a disorder associated with pathological neovascularization in a subject, the method comprising administering to the subject a therapeutically effective amount of the extract of claim 4. 10. The method of claim 9, wherein the disorder is cancer, arthritic condition, neovascular-based dermatological condition, diabetic retinitis, restenosis, Kaposi's sarcoma, age-related macular degeneration, telangiectasia, A method selected from the group consisting of glaucoma, keloid, corneal transplant rejection, wound granulation, angiofibroma, Osler-Weber syndrome, myocardial angiogenesis and scleroderma. 11. The method of claim 10, wherein the disorder is an arthritic condition selected from the group consisting of rheumatoid arthritis, psoriatic arthritis and osteoarthritis. 11. The method of claim 10, wherein the delivering step is by oral administration, intravenous administration, intraperitoneal administration or transdermal administration. The method of claim 9, wherein the subject is an animal. 14. The method according to claim 13, wherein the animal is selected from the group consisting of a pet animal, livestock or a human patient. 10. The method of any one of claims 7, 8 or 9, further comprising administering to the subject an effective amount of an agent selected from the group consisting of an anti-angiogenic agent, an anti-tumor agent or an immunopotentiator. The method according to item. A method for screening for a therapeutic agent to inhibit neovascularization or endothelial cell proliferation comprising the following steps:
(A) contacting the agent with a suitable cell or tissue sample;
(B) contacting a separate sample of a suitable cell sample or tissue sample with a therapeutically effective amount of the extract of claim 4; and (c) expanding the sample of step (a), wherein step (b ), Wherein any agent of step (a) that inhibits the growth to the same or similar degree as the sample of step (b) is neovascularization or endothelium A therapeutic agent for inhibiting cell proliferation,
Including the method. The method of claim 16, wherein the contacting is in vitro or in vivo. 17. The method of claim 16, further comprising contacting the sample of step (b) with an agent selected from the group consisting of an anti-angiogenic agent, an anti-tumor agent and an immunopotentiator. A kit for treating a disorder associated with pathological neovascularization in a host, the kit comprising a therapeutically effective amount of the extract of claim 4 and instructions for use. The kit of claim 19, wherein the disorder is cancer, arthritic condition, neovascular-based dermatological condition, diabetic retinitis, capage sarcoma, age-related macular degeneration, restenosis, telangiectasia, A kit selected from the group consisting of glaucoma, keloid, corneal transplant rejection, wound granulation, angiofibroma, Osler-Weber syndrome, myocardial angiogenesis and scleroderma. 21. The kit of claim 20, wherein the disorder is an arthritic condition selected from the group consisting of rheumatoid arthritis, psoriatic arthritis and osteoarthritis.

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