WO2000057707A1 - Methods of treating alzheimer's disease and other amyloidoses using hypericum perforatum and derivatives thereof - Google Patents

Methods of treating alzheimer's disease and other amyloidoses using hypericum perforatum and derivatives thereof Download PDF

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Publication number
WO2000057707A1
WO2000057707A1 PCT/US2000/006814 US0006814W WO0057707A1 WO 2000057707 A1 WO2000057707 A1 WO 2000057707A1 US 0006814 W US0006814 W US 0006814W WO 0057707 A1 WO0057707 A1 WO 0057707A1
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amyloid
perforatum
disease
mental
pharmacological agent
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PCT/US2000/006814
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French (fr)
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Gerardo Castillo
Alan D. Snow
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Proteotech, Inc.
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Priority to AU38862/00A priority Critical patent/AU3886200A/en
Publication of WO2000057707A1 publication Critical patent/WO2000057707A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/38—Clusiaceae, Hypericaceae or Guttiferae (Hypericum or Mangosteen family), e.g. common St. Johnswort

Definitions

  • the invention relates to compositions and methods for treating Alzheimer's Disease and other amyloidoses, and to methods for isolating pharmaceutical agents from plant matter; more particularly, it relates to compositions and methods for therapeutic intervention in Alzheimer's disease and other amyloidoses using plant matter and de ⁇ vatives thereof
  • Alzheimer's disease is characte ⁇ zed by the accumulation of a 39-43 ammo acid peptide termed the beta-amyloid protein or A ⁇ , in a fib ⁇ llar form, existing as extracellular amyloid plaques and as amyloid within the walls of cerebral blood vessels.
  • a ⁇ beta-amyloid protein
  • Fib ⁇ llar A ⁇ amyloid deposition in Alzheimer's disease is believed to be det ⁇ mental to the patient and eventually leads to toxicity and neuronal cell death, characte ⁇ stic hallmarks of Alzheimer's disease. Accumulating evidence implicates amyloid as a major causative factor of Alzheimer's disease pathogenesis
  • a va ⁇ ety of other human diseases also demonstrate amyloid deposition and usually involve systemic organs (I e oigans or tissues lying outside the central nervous system), with the amyloid accumulation leading to organ dysfunction or failure.
  • systemic organs I e oigans or tissues lying outside the central nervous system
  • amyloid accumulation leading to organ dysfunction or failure.
  • Alzheimer's disease and "systemic" amyloid diseases there is currently no cure or effective treatment, and the patient usually dies within 3 to 10 years from disease onset.
  • New compounds or agents for therapeutic regimes to arrest or reverse amyloid formation, deposition, accumulation and/or persistence that occurs in Alzheimer's disease and other amyloidoses are therefore urgent sought.
  • a p ⁇ mary object of the present invention is to establish new methods for the treatment of the amyloid diseases
  • the amyloid diseases include, but are not limited to, the amyloid associated with Alzheimer's disease, Down's syndrome and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (wherein the specific amyloid is referred to as beta-amyloid protein or A ⁇ ), the amyloid associated with chronic inflammation, va ⁇ ous forms of malignancy and Familial Mediterranean Fever (wherein the specific amyloid is referred to as AA amyloid or inflammation-associated amyloidosis), the amyloid associated with multiple myeloma and other B-cell dyscrasias (wherein the specific amyloid is referred to as AL amyloid), the amyloid associated with type II diabetes (wherein the specific amyloid is referred to as amylin or islet amyloid), the amyloid associated with the p ⁇ on diseases including Creutzfeldt- Jakob disease, Gerstmann
  • Another object of the present invention is to use the plant Hype ⁇ cum perforatum
  • St John's Wort also referred to as St John's Wort
  • its constituents thereof including but not limited to its leaves, buds and flowers
  • amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes and other amyloidoses Hype ⁇ cum perforatum is also referred to as, but not limited to, St John's
  • Another object of the present invention is to use extracts and/or de ⁇ vatives thereof from plant matter related to the family Hype ⁇ caceae, which includes, but is not limited to the genus Hype ⁇ cum, for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes and other amyloidoses
  • Another object of the present invention is to use extracts and/or de ⁇ vatives thereof from plant matter related to the vanous Hype ⁇ cum species, which may include but not limited to, Hype ⁇ cum perforatum, Hype ⁇ cum calycmum, Hype ⁇ cum formosum,
  • Another object of the present invention is to use commercially available pills, tablets, caplets. soft and hard gelatin capsules lozenges, sachets, cachets, vegicaps, liquid drops, elixers, suspensions, emulsions, solutions, syrups, tea bags, aerosols (as a solid or in a liquid medium), supposito ⁇ es, ste ⁇ le injectable solutions, ste ⁇ le packaged powders, d ⁇ ed leaves, d ⁇ ed buds, d ⁇ ed flowers which contain Hype ⁇ cum perforatum to treat patients with Alzheimer's disease, type II diabetes and other amyloidoses
  • Another object of the present invention is to use Hype ⁇ cum perforatum, and/or the hype ⁇ cins contained within Hype ⁇ cum perforatum for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes and other amyloidoses
  • Yet another object of the present invention is to use the flavanoids contained within Hype ⁇ cum perforatum for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes and other amyloidoses.
  • flavanoids include, but are not limited to, hyperoside, biapigenm, rutm, quercetin, quercitin, isoquercit ⁇ n, pseudohype ⁇ cin, hyperfo ⁇ n, procyanidines, amentoflavme, and luteohn
  • Yet another object of the present invention is to use the xanthones contained within Hypencum perforatum for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes and other amyloidoses.
  • Yet another object of the present invention is to use the tannins contained within Hype ⁇ cum perforatum for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes and other amyloidoses.
  • Yet another object of the present invention is to use the carbohydrates contained within Hype ⁇ cum perforatum for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes and other amyloidoses.
  • Such carbohydrates include, but are not limited to, pectin.
  • Yet another object of the present invention is to use the hpids contained within
  • Hype ⁇ cum perforatum for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes and other amyloidoses include, but are not limited to, monoterpenes.
  • Yet another object of the present invention is to use the vitamins contained within Hype ⁇ cum perforatum for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes and other amyloidoses.
  • vitamins include, but are not limited to, vitamin A and vitamin C
  • Yet another object of the present invention is to provide methods to isolate the active ingredients present within Hype ⁇ cum perforatum for use as potent agents which inhibit amyloid formation, amyloid deposition, amyloid accumulation, amyloid persistence, amyloid protein-amyloid protein interactions, and/or cause a dissolution/disruption of preformed or pre-deposited amyloid fibnls in Alzheimer's disease, type II diabetes and other amyloidoses.
  • Methods for isolation of the active ingredients within Hype ⁇ cum perforatum include application of some standard techniques known to those skilled in the art, including, but not limited to, thin layer chromatography using silica-coated plates, and separation and isolation using high pressure liquid chromatography (HPLC).
  • Hype ⁇ cum perforatum found to be potent inhibitors of amyloid formation, amyloid deposition, amyloid accumulation, amyloid persistence, amyloid protein-amyloid protein interactions, and/or cause a dissolution/disruption of pre-formed or pre-deposited amyloid fibnls in Alzheimer's disease, type II diabetes and other amyloidoses, are identified by re- testing of individual bands or fractions (separated by thin layer chromatography, column chromatography and/or HPLC) using specific assay tests as desc ⁇ bed in the examples of the present invention.
  • Yet another object of the present invention is to provide the use of Hype ⁇ cum perforatum and/or its ingredients [(regardless of commercial source and regardless of final form for consumption by humans, i.e. pills, tablets, caplets, soft and hard gelatin capsules, lozenges, sachets, cachets, vegicaps, liquid drops, elixers, suspensions, emulsions, solutions, syrups, tea bags, aerosols (as a solid or m a liquid medium), suppositones, ste ⁇ le injectable solutions, ste ⁇ le packaged powders, d ⁇ ed leaves, d ⁇ ed buds, and d ⁇ ed flowers] for inhibition of amyloid formation, deposition, accumulation, and/or persistence, regardless of its clinical setting.
  • ingredients (regardless of commercial source and regardless of final form for consumption by humans, i.e. pills, tablets, caplets, soft and hard gelatin capsules, lozenges, sachets, cachets, vegicaps
  • Yet another object of the present invention is to provide compositions and methods involving admmiste ⁇ ng to a subject a therapeutic dose of Hype ⁇ cum perforatum (or its active ingredients) which inhibits amyloid deposition. Accordingly, the compositions and methods of the invention are useful for inhibiting amyloidosis in disorders in which amyloid deposition occurs.
  • the compounds of the invention can be used therapeutically to treat amyloidosis or can be used prophylactically in a subject susceptible to amyloidosis.
  • the methods of the invention are based, at least in part, in directly inhibiting amyloid fib ⁇ l formation, inhibiting amyloid fib ⁇ l growth, and/or causing dissolution/disruption of preformed amyloid fibnls.
  • Yet another object of the present invention is to provide pharmaceutical compositions for treating amyloidosis.
  • the pharmaceutical compositions include a therapeutic compound of the invention in an amount effective to inhibit amyloid deposition and a pharmaceutically acceptable vehicle.
  • Yet another object of the present invention is the use of any and all synthetic compounds made similar to Hypencum perforatum and/or its active ingredients for use as potent agents which inhibit amyloid formation, amyloid deposition, amyloid accumulation, amyloid persistence, amyloid protein-amyloid protein interactions, and/or cause a dissolution/ disruption of pre-formed or pre-deposited amyloid fibnls in Alzheimer's disease, type II diabetes and other amyloidoses. It is yet another object of the invention to meet any and all of the needs summanzed above.
  • the invention is the only system that effectively provides for use of extracts from the leaves, flowers and/or buds of Hype ⁇ cum perforatum. and use of the ingredients contained within the vanous commercial preparations of Hype ⁇ cum perforatum, to benefit human patients with Alzheimer's disease and other amyloidoses due to Hypencum perforatum' s newly discovered ability to inhibit amyloid fibnl formation, inhibit amyloid fib ⁇ l growth, inhibit amyloid-proteoglycan interactions, amyloid-glycosaminoglycan interactions, and cause dissolution and/or disruption of preformed amyloid fibnls.
  • the present invention pertains to the identification and surpnsing discovery that an extract from the d ⁇ ed upper plant parts (i.e. leaves, flowers and/or buds) of Hype ⁇ cum perforatum, otherwise known as St. John's Wort, act as an astonishing inhibitor of
  • Hype ⁇ cum perforatum also has the ability to inhibit amyloid protein-amyloid protein interactions, which are believed to be important for the growth of amyloid deposits in tissues.
  • Hypencum perforatum also has the ability to inhibit amyloid protem-proteoglycan (PG)/ glycosaminoglycan (GAG) interactions, which are believed to be important for the formation and persistence of all amyloid deposits in tissues.
  • PG amyloid protem-proteoglycan
  • GAG glycosaminoglycan
  • Hypencum perforatum has the ability to dissolve/disrupt pre-formed amyloid fibnls of the Alzheimer's and type II diabetes types, suggesting that this agent may be useful for patients at latter stages of both Alzheimer's disease, type II diabetes and other amyloidoses.
  • Hype ⁇ cum perforatum extracted from different commercial sources extracts isolated from dned whole plant mate ⁇ als or from powder obtained from gelatin capsules containing a concentrated extract of 0.3% hypencin) were all found to serve as potent inhibitors of Alzheimer's disease amyloid fib ⁇ llogenesis.
  • Hype ⁇ cum perforatum extracts obtained from d ⁇ ed whole plant mate ⁇ als, or from gelatin-capsules containing 0.3% hypencin caused a marked significant inhibition of A ⁇ amyloid fibnl formation as determined using a Thioflavin T fluorometry assay. Extracts of Hype ⁇ cum perforatum obtained from different commercial sources inhibited A ⁇ amyloid fib ⁇ llogenesis in a dose-dependent manner. Hype ⁇ cum perforatum extract also inhibited Alzheimer's A ⁇ -A ⁇ interactions as determined using a solid phase binding assay demonstrating that Hypencum perforatum is additionally an effective inhibitor of Alzheimer's amyloid fibnl growth.
  • Hype ⁇ cum perforatum was effective in the inhibition of A ⁇ -proteoglycan/ glycosaminoglycan (PG/GAG) interactions (an important therapeutic target for all amyloidoses) as determined using a solid phase binding immunoassay.
  • Hype ⁇ cum perforatum extracts de ⁇ ved from different commercial sources was also a potent dissolving/inhibiting agent of pre-formed A ⁇ (1-40) or A ⁇ (1-42) containing amyloid fibnls, and pre-formed amylin fibnls, as determined using a Thioflavin T fluorometry and Congo red staining assays. This latter effect occurred in a dose-dependent
  • Current use of Hypericum perforatum and its ingredients contained within different commercial preparations are anticipated to benefit human patients at all stages of Alzheimer's disease due to Hypericum perforatu 's inherent ability to inhibit A ⁇ amyloid fibril formation (early to mid-stage Alzheimer's disease), inhibit amyloid fibril growth (early to mid-stage Alzheimer's disease), inhibit amyloid-PG/GAG interactions (all stages of Alzheimer's disease) and cause dissolution/disruption of preformed amyloid fibrils (mid to late stages of Alzheimer's disease).
  • Hypericum perforatum is anticipated to benefit patients with different systemic amyloid diseases such as type II diabetes, regardless of the stage of amyloid accumulation and the organ (or tissue) involved.
  • a pharmaceutical agent for treating an amyloid disease in a patient, wherein the pharmacological agent comprises a therapeutically effect amount of plant matter from the genus Hypericum.
  • the pharmaceutical agent is preferably from a plant of the genus Hypericum, species perforatum.
  • the pharmacological agent is preferably an extract obtained from Hypericum perforatum, the extract being derived from the dried leaves, flowers and buds of Hypericum perforatum, and advantageously taken from some commercially available source such as pills, tablets, caplets, soft and hard gelatin capsules, lozenges, sachets, cachets, vegicaps, liquid drops, elixers, suspensions, emulsions, solutions, syrups, tea bags, aerosols (as a solid or in a liquid medium), suppositories, sterile injectable solutions, sterile packaged powders, or plant powder obtained from dried leaves, buds, and/or flowers.
  • some commercially available source such as pills, tablets, caplets, soft and hard gelatin capsules, lozenges, sachets, cachets, vegicaps, liquid drops, elixers, suspensions, emulsions, solutions, syrups, tea bags, aerosols (as a solid or in a liquid medium), s
  • the pharmaceutical agent is an amyloid inhibitory ingredient selected from the group consisting of, but not limited to, flavanoids, xanthones, proanthocyanidins, dianthrones, tannins, monoterpenes, hyperoside, biapigenin, rutin, quercetin, quercitin, isoquercitrin, pseudohypericin, hyperforin, procyanidines, amentoflavine, luteolin, pectin, vitamin A, and vitamin C.
  • an amyloid inhibitory ingredient selected from the group consisting of, but not limited to, flavanoids, xanthones, proanthocyanidins, dianthrones, tannins, monoterpenes, hyperoside, biapigenin, rutin, quercetin, quercitin, isoquercitrin, pseudohypericin, hyperforin, procyanidines, amentoflavine, luteolin, pectin, vitamin A, and vitamin C.
  • the pharmacological agent preferably has a therapeutically effective amount of Hypericum perforatum in a dosage in the range of from about 10 to 1,000 mg/kg of body weight of the patient, and more preferably in the range of about 10 to 100 mg/kg of body weight of the patient.
  • the pharmacological agent preferably has a therapeutically effective amount of hypericin, standardized to contain hypericin at a range of 0.05% to 2%, but more preferably in the range of about 0.1% to 0.5%, per 250mg to 500mg capsule containing Hypericum perforatum.
  • the amyloid disease for treatment with the pharmacological agent is selected from the group consisting of the amyloid associated with Alzheimer's disease, Down's syndrome and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (wherein the specific amyloid is referred to as beta-amyloid protein or A ⁇ ), the amyloid associated with chronic inflammation, various forms of malignancy and Familial Mediterranean Fever (wherein the specific amyloid is referred to as AA amyloid or inflammation-associated amyloidosis), the amyloid associated with multiple myeloma and other B-cell dyscrasias (wherein the specific amyloid is referred to as AL amyloid), the amyloid associated with type II diabetes (wherein the specific amyloid is refened to as amylin or islet amyloid), the amyloid associated with the prion diseases including Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, kuru and animal scrapie (wherein the specific am
  • Preferred pharmaceutical agents have a weight percentage of plant extract in the agent is in the range of from about 70% to about 95%, and may also have a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical agent preferably has an amyloid inhibitory activity or efficacy greater than 50%.
  • Hypencum perforatum has the ability to inhibit the formation of brain amyloid deposits in patients who accumulate brain amyloid deposits that occur dunng normal aging and in a vanety of brain disorders including Alzheimer's disease; it will therefore promote mental alertness in such patients.
  • Hype ⁇ cum perforatum has the ability to reduce, eliminate, prevent or inhibit or disrupt/dissolve amyloid fibnl or protein deposits, brain associated amyloid fibnl deposits or brain associated amyloid protein deposits, as well as amyloid fibnl formation and growth or age associated amyloid fibnl formation and growth, bram associated amyloid fib ⁇ l formation and growth, and interaction of amyloid protein with glycosaminoglycans or proteoglycans; it will therefore promote mental acuity, promote mental alertness, provide nut ⁇ tional support for age or related cognitive or memory decline, promote cognitive well being, support brain function, improve cognitive ability, mental performance or memory, promote concentration and mental sharpness, improve mental vitality, promote greater mental clanty and alertness, improve short term memory, reduce or reverse age associated cognitive or memory decline, support normal bra function, enhance learning or memory; improve concentration, enhance mental performance, reduce mental decline, reduce likelihood of age related bram disorders, and maintain good brain health.
  • Hype ⁇ cum perforatum further has the ability to reduce, eliminate, prevent, inhibit or disrupt/dissolve amyloid fibnl or protein deposits, as well as amyloid fibnl formation and growth, pancreas associated amyloid fib ⁇ l formation and growth, and interaction of amyloid protein with glycosaminoglycans or with proteoglycans; it will therefore support healthy pancreatic function and promote pancreatic function by helping to promote normal insulin function
  • Another aspect of the invention is a method for isolating amyloid inhibitory constituents within Hypencum perforatum plant matter, the method compnsing the following steps: a) extracting the plant matter with an organic solvent or water, b) concentrating the extract, c) removing insoluble mate ⁇ als, d) precipitating amyloid inhibitory constituents with organic solvent or water, e) recove ⁇ ng and redissolvmg the amyloid inhibitory constituents obtained in organic solvent or water, and f) injecting and separation by HPLC.
  • the plant matter is preferably compnsed of commercially obtained pills, tablets, caplets, soft and hard gelatin capsules, lozenges, sachets, cachets, vegicaps, liquid drops, elixers, suspensions, emulsions, solutions, syrups, tea bags, aerosols (as a solid or in a liquid medium), supposito ⁇ es, ste ⁇ le injectable solutions, stenle packaged powders, or plant matter which contain Hypericum perforatum, extracts or derivatives thereof, and may be taken from commercially available gelatin-coated capsules which contain dried-plant material (preferably leaves, buds and flowers) of Hypericum perforatum, extracts or derivatives thereof.
  • a method for treating an amyloid disease in a patient comprising the step of administering to the patient a therapeutically effective amount of plant matter from the plant of the genus Hypericum, species perforatum.
  • the plant matter is preferably administered orally or by aerosal spray or in a parenterally injectable or infusible form.
  • the therapeutically effective amount of plant matter is preferably an amyloid inhibitory ingredient selected from the group consisting of but not limited to, flavanoids, xanthones, proanthocyanidins, dianthrones, tannins, monoterpenes, hyperoside, biapigenin, rutin, quercetin, quercitin, isoquercitrin, pseudohypericin, hyperforin, procyanidines, amentoflavine, luteolin, pectin, vitamin A, and vitamin C.
  • an amyloid inhibitory ingredient selected from the group consisting of but not limited to, flavanoids, xanthones, proanthocyanidins, dianthrones, tannins, monoterpenes, hyperoside, biapigenin, rutin, quercetin, quercitin, isoquercitrin, pseudohypericin, hyperforin, procyanidines, amentoflavine, luteolin, pectin, vitamin A
  • FIGURE 1 is a black and white graph of a 1 week Thioflavin T fluorometry assay demonstrating that an extract from Hypericum perforatum (derived from a commercial source) causes dose-dependent inhibition of Alzheimer's disease A ⁇ 1-40 amyloid fibril formation.
  • FIGURE 2 is a black and white graph of a solid phase binding assay utilized to identify lead compounds which inhibit Alzheimer's A ⁇ -A ⁇ interactions (i.e. Alzheimer's amyloid fibril growth).
  • Hypericum perforatum derived either from an extract obtained from dried plant materials, or from an extract obtained from the powder present in commercially available gelatin-capsules which contain 0.3% hypericin are potent inhibitors of Alzheimer's amyloid fibril growth.
  • FIGURE 3 is a black and white graph of a solid phase binding immunoassay utilized to determine the potential dose-dependent effects of Hypericum perforatum (derived either from an extract obtained from dried plant materials, or from an extract obtained from the powder present in commercially available gelatin-capsules which contain 0.3% hypericin) on inhibition of A ⁇ -Perlecan GAG interactions. Significant dose-dependent inhibition of A ⁇ - perlecan GAG interactions is observed with treatment of Hypericum perforatum.
  • Hypericum perforatum derived either from an extract obtained from dried plant materials, or from an extract obtained from the powder present in commercially available gelatin-capsules which contain 0.3% hypericin
  • FIGURE 4 is a black and white graph of a Thioflavin T fluorometry assay utilized to determine the potential dose-dependent effects of Hypencum perforatum on dissolution/ disruption of pre-formed Alzheimer's A ⁇ 1-40 amyloid fibnls with a 2 hour incubation penod.
  • Hype ⁇ cum perforatum (de ⁇ ved either from an extract obtained from d ⁇ ed plant matenals, or from an extract obtained from the powder present in commercially available gelatin-capsules which contain 0.3% hypencin) causes dissolution of pre-formed
  • Alzheimer ' s A ⁇ 1-40 amyloid fibnls in a dose-dependent manner.
  • FIGURE 5 is a black and white graph of a Thioflavin T fluorometry assay utilized to determine the potential dose-dependent effects of Hype ⁇ cum perforatum on dissolution/ disruption of pre-formed Alzheimer's A ⁇ 1-42 amyloid fibnls within a 2 hour incubation pe ⁇ od.
  • Hype ⁇ cum perforatum (de ⁇ ved either from an extract obtained from d ⁇ ed plant mate ⁇ als, or from an extract obtained from the powder present in commercially available gelatin-capsules which contain 0.3% hypencm) causes dissolution of pre-formed Alzheimer's A ⁇ 1-42 amyloid fibnls in a dose-dependent manner.
  • FIGURE 6 is a black and white graph of a Thioflavin T fluorometry assay utilized to show that an Hypencum perforatum (de ⁇ ved either from an extract obtained from dned plant mate ⁇ als, or from an extract obtained from the powder present in commercially available gelatin-capsules which contain 0.3% hypencin) is also able to cause a significant dissolution of pre-formed islet amyloid fibnls (le. amylin).
  • Amyloid is a gene ⁇ c term refernng to a group of diverse, but specific extracellular protein deposits which all have common morphological properties, staining charactenstics, and x-ray diffraction spectra. Regardless of the nature of the amyloid protein deposited all amyloids have the following characte ⁇ stics: 1) an amorphous appearance at the light microscopic level and appear eosmophilic using hematoxy n and eosm stains; 2) all stain with Congo red and demonstrate a red green biref ⁇ ngence as viewed under polanzed light (Puchtler et al., J.
  • amyloid usually consist of non-branching fibnls of indefinite length and with a diameter of 7-10 nm.
  • Amyloid today is classified according to the specific amyloid protein deposited.
  • the amyloid diseases include, but are not limited to, the amyloid associated with Alzheimer's disease, Down's syndrome and Hereditary cerebral hemorrhage with amyloidosis of the Dutch type (wherein the specific amyloid is referred to as beta-amyloid protein or A ⁇ ), the amyloid associated with chronic inflammation, vanous forms of malignancy and Familial
  • Mediterranean Fever wherein the specific amyloid is refened to as AA amyloid or inflammation-associated amyloidosis
  • the amyloid associated with multiple myeloma and other B-cell dyscrasias wherein the specific amyloid is referred to as AL amyloid
  • the amyloid associated with type II diabetes wherein the specific amyloid is referred to as amylin or islet amyloid
  • the amyloid associated with the p ⁇ on diseases including
  • PrP amyloid the amyloid associated with long-term hemodialysis and carpal tunnel syndrome
  • beta 2 -m ⁇ croglobuhn amyloid the amyloid associated with senile cardiac amyloid and Familial Amyloidotic Polyneuropathy
  • prealbumin or transthyretin amyloid the amyloid associated with endoc ⁇ ne tumors such as medullary carcinoma of the thyroid
  • amyloid deposits m clinical conditions share common physical properties relating to the presence of a beta-pleated sheet conformation
  • many different chemical types exist and additional ones are likely to be descnbed in the future.
  • a circulating precursor protein may result from overproduction of either intact or aberrant molecules (ex. plasma cell dyscrasias), reduced degradation or excretion (serum amyloid A in some secondary amyloid syndromes and beta 2 -m ⁇ croglobuhn in long-term hemodialysis), or genetic abnormalities associated with vanant proteins (ex. familial amyloidotic polyneuropathy).
  • Proteolysis of a larger protein precursor molecule occurs in many types of amyloidosis, resulting in the production of lower molecular weight fragments that polymenze and assume a beta-pleated sheet conformation as tissue deposits, usually in an extracellular location. What are the precise mechanisms involved, and the aberrant causes leading to changes in proteolytic processing and/or translational modifications is not known most amyloids.
  • Systemic amyloids which include the amyloid associated with chronic inflammation, va ⁇ ous forms of malignancy and Familial Mediterranean Fever (le. AA amyloid or inflammation-associated amylo ⁇ dos ⁇ s)(Benson and Cohen, Arth Rheum. 22:36-42, 1979; Kamei et al, Acta Path. Jpn. 32.123-133, 1982; McAdam et al, Lancet 2:572-573, 1975; Metaxas, Kidney Int. 20:676-685, 1981), and the amyloid associated with multiple myeloma and other B-cell dyscrasias (le. AL amylo ⁇ d)(Harada et al, J. Histochem.
  • Cytochem 19:1- 15, 1971 are known to involve amyloid deposition in a vanety of different organs and tissues generally lying outside the central nervous system.
  • Amyloid deposition in these diseases may occur, for example, m liver, heart, spleen, gastrointestinal tract, kidney, skin, and/or lungs (Johnson et al, N. Engl J. Med. 321:513-518, 1989).
  • amyloid deposition in kidney may lead to renal failure
  • amyloid deposition in heart may lead to heart failure.
  • amyloid accumulation in systemic organs leads to eventual death generally within 3-5 years.
  • Other amyloidoses may affect a single organ or tissue such as observed with the A ⁇ amyloid deposits found in the brains of patients with Alzheimer's disease and Down's syndrome- the PrP amyloid deposits found in the brains of patients with
  • Alzheimer's disease is a leading cause of dementia in the elderly, affecting 5-10% of the population over the age of 65 years (A Guide to Understanding Alzheimer's Disease and
  • Alzheimer's disease the parts of the brain essential for cognitive processes such as memory, attention, language, and reasoning degenerate, robbing victims of much that makes us human, including independence.
  • onset is m middle age, but more commonly, symptoms appear from the m ⁇ d-60's onward.
  • Alzheimer's disease today affects 4-5 million Ame ⁇ cans, with slightly more than half of these people receiving care at home, while the others are in many different health care institutions.
  • Alzheimer's disease and other dementias doubles every 5 years beyond the age of 65, and recent studies indicate that nearly 50% of all people age 85 and older have symptoms of Alzheimer's disease (1997 Progress Report on Alzheimer's Disease. National Institute on Aging/National Institute of Health) 13% (33 million people) of the total population of the United States are age 65 and older, and this % will climb to 20% by the year 2025 (1997 Progress Report on Alzheimer ' s Disease. National Institute on Aging/National Institute of Health)
  • Alzheimer's disease also puts a heavy economic burden on society as well
  • the overall cost of Alzheimer's disease to families and to society is stagge ⁇ ng.
  • the annual economic toll of Alzheimer's disease in the United States in terms of health care expenses and lost wages of both patients and their caregivers is estimated at $80 to $100 billion (1997 Progress Report on Alzheimer's Disease. National Institute on Aging/National Institute of Health).
  • Tacnne hydrochlo ⁇ de (“Cognex"), the first FDA approved drug for Alzheimer's disease is a acetylchohnesterase inhibitor (Cutler and Sramek, N Engl. J. Med 328-808- 810, 1993) However, this drug has showed limited success in the cognitive improvement in Alzheimer's disease patients and initially had major side effects such as liver toxicity.
  • the second more recently FDA approved drug, donepezil (also known as "Ancept”) which is also an acetylchohnesterase inhibitor, is more effective than tacnne, by demonstrating slight cognitive improvement in Alzheimer's disease patients (Barner and Gray, Ann Pharmacotherapy 32:70-77, 1998; Rogers and Fnedhoff, Eur. Neuropsvch. 8.67-75, 1998), but is not believed to be a cure. Therefore, it is clear that there is a need for more effective treatments for Alzheimer's disease patients
  • Amyloid as a Therapeutic Target for Alzheimer's Disease
  • Alzheimer's disease is characte ⁇ zed by the deposition and accumulation of a 39-43 ammo acid peptide termed the beta-amyloid protein, A ⁇ or ⁇ /A4 (Glenner and Wong, Biochem Biophvs Res Comm 120:885-890, 1984, Masters et al, Proc. Natl. Acad. Sci USA 82 4245-4249, 1985; Husby et al, Bull WHO 71:105-108, 1993).
  • a ⁇ is denved from larger precursor proteins termed beta-amyloid precursor proteins (or BPPs) of which there are several alternatively spliced vanants
  • BPPs beta-amyloid precursor proteins
  • the most abundant forms of the BPPs include proteins consisting of 695, 751 and 770 amino acids (Tanzi et al, Nature 331:528-530,
  • the small A ⁇ peptide is a major component which makes up the amyloid deposits of "plaques" in the brains of patients with Alzheimer's disease.
  • Alzheimer's disease is characte ⁇ zed by the presence of numerous neurofib ⁇ llary "tangles", consisting of paired helical filaments which abnormally accumulate the neuronal cytoplasm (Grundke-Iqbal et al, Proc Natl Acad Sci USA 83.4913-4917, 1986; Kosik et al, Proc Natl Acad.
  • Alzheimer's disease The pathological hallmarks of Alzheimer's disease is therefore the presence of "plaques” and "tangles", with amyloid being deposited m the central core of plaques.
  • the other major type of lesion found in the Alzheimer's disease bram is the accumulation of amyloid the walls of blood vessels, both within the brain parenchyma and in the walls of meningeal vessels which lie outside the brain
  • the amyloid deposits localized to the walls of blood vessels are referred to as cerebrovascular amyloid or congophi c angiopathy (Mandybur, J. Neuropath. Exp. Neurol 45:79-90, 1986; Pard ⁇ dge et al, J Neurochem. 49.1394-1401, 1987).
  • Alzheimer's A ⁇ protein in cell culture has been shown to cause degeneration of nerve cells withm short penods of time
  • Alzheimer's A ⁇ Injection of the Alzheimer's A ⁇ into rat brain also causes memory impairment and neuronal dysfunction (Flood et al, Proc Natl. Acad Sci. 88:3363-3366, 1991; Br Res. 663.271-276, 1994) Probably, the most convincing evidence that A ⁇ amyloid is directly involved in the pathogenesis of Alzheimer's disease comes from genetic studies. It has been discovered that the production of A ⁇ can result from mutations in the gene encoding, its precursor, beta- amyloid precursor protein (Van Broeckhoven et al.
  • Hypencum perforatum St. John's Wort, whose latin name is Hypencum perforatum belongs to the family
  • Hype ⁇ caceae which consists of eight genuses and about 350 species.
  • the species of the Hype ⁇ cum genus are widely distnaded throughout the world, including more than 70 different species from India alone.
  • Hype ⁇ cum perforatum is native to the temperate zones of Europe and western Asia, but has become naturalized in North and South Ame ⁇ ca as well as in Australia. This is a perennial plant, which by the second year of growth from seed reaches a height of about 30 inches.
  • the many b ⁇ ght yellow flowers each contain about 50 stamens, which tend to group into three loosely-defined clumps.
  • the lance-shaped, opposite leaves with their numerous pellucid dots and dark spots of hypencin are also charactenstic. If you squeeze a fresh bud between your thumbnails, it will produce a freely exuding liquid, a b ⁇ ght red, resinous pigment which contains multiple bioactive compounds.
  • Hype ⁇ cum perforatum- the dianthrones and the flavanoids There are two main types of active compounds in Hype ⁇ cum perforatum- the dianthrones and the flavanoids.
  • the activity of the whole herb is best represented by a liquid extract of the fresh or recently d ⁇ ed flowe ⁇ ng tops.
  • the herb yields its properties to hot water, alcohol and oil, with alcohol providing the most complete extraction.
  • the red pigments are located in the buds and the flowers, as well as in the ldioblasts of the leaves. These pigments denote the presence of dianthrones (hypencins), a rather unstable but extremely active class of constituents. In chromatographic analysis of this herb, the fluorescent red pigments gravitate to one spot, resting above layer after layer of verticolored flavanoids.
  • the flavanoids (hyperoside, rutin, quercitin, lsoquercitnn, luteolin, etc.) are located in the flowers and in the leaves. These flavanoids provide a slightly sedative, diuretic and anti-inflammatory effect. They work closely with the hypencins, and are a highly significant part of this whole herb The tann content of the herb is about 10%, accounting for its histo ⁇ cal use as an ast ⁇ ngent and antidiarrheal.
  • the macerated oil of fresh Hypencum perforatum flowers contains hyperfonn, which provides a wound-healing and antibiotic influence (Brondz et al, Tetrahedron Lett 23, 1982). This is a compound which increases in concentration in the plant du ⁇ ng the development of fruit and seeds, therefore oil extractions are best performed using herb harvested as the plant begins to mature its fruit and seed
  • Hype ⁇ cum extracts have been shown to cause a 50% inhibition of serotonin uptake by rat brain cells Whenever a brain impulse occurs, serotonin is released from one brain cell, influences a receptor on an adjoining cell, and then much of it is returned to the o ⁇ ginal cell to be used again or degraded.
  • Prozac, and other similar anti depressants such as Zoloft and Paxil, work by inhibiting the re-uptake of serotonin. This is why they are referred to as serotonin re-uptake inhibitors. As a consequence of this re-uptake inhibition, more serotonin stays around to influence brain cells, and mood is elevated.
  • MAO monoamine oxidase
  • Hype ⁇ cum perforatum is used p ⁇ ma ⁇ ly for treating mild to moderate depression and related disorders (Lmde et al, British Med. J. 313:253-258, 1996).
  • Viruses studies include HIV (Hudson et al, Science 254:522, 1991), herpes simplex virus type I and II, Epstein-Barr virus and influenza types A and B. Hype ⁇ cum perforatum also appears to have broad spectrum anti-microbial activity.
  • the organisms studies include Staphylococcus aureus (Staph), Streptococcus mutans (Strep) and Eschenchia coli (E. coli).
  • Staph Staphylococcus aureus
  • Streptococcus mutans Strep
  • Eschenchia coli E. coli
  • Hypencin one of the active agents from Hypencum perforatum, has been also shown in vanous studies to work effectively against cancerous cells and tumors of varying kinds.
  • Werf et al Lanrvngvscope 106:479-483 reported that hypencin shows great potential in targeting human cancer growths through what is called "phototargetmg", a process that uses laser activation of hype ⁇ cm, along with chemotherapy, for improved results in inhibiting the growth of cancerous cells.
  • Hype ⁇ cum perforatum may be used to treat a vanety of ailments, such as those descnbed above, nowhere has there been any use, or suggestion of use, of this compound for the treatment of amyloid formation . deposition, accumulation and/or persistence, such as that which occurs in the amyloidoses, including Alzheimer's disease.
  • the present invention clearly demonstrates the effectiveness of Hype ⁇ cum perforatum and de ⁇ vatives thereof obtained from different commercial sources for the 1) inhibition of Alzheimer's A ⁇ amyloid fibnl formation (important for patients in early to mid-stage Alzheimer's disease), 2) inhibition of Alzheimer's amyloid fib ⁇ l growth
  • the present invention demonstrates that Hypencum perforatum is effective in causing the dissolution of islet amyloid fibnls (le. amylin) and therefore may serve as an effective treatment for -90% of type II diabetic patients who have islet amyloid accumulation in the pancreas
  • Hype ⁇ cum perforatum has the ability to inhibit the formation of brain amyloid deposits that occur du ⁇ ng normal aging and in a va ⁇ ety of bram disorders including Alzheimer's disease
  • Hypencum perforatum has the ability to reduce, eliminate, prevent, inhibit or disrupt dissolve amyloid fibnl or protein deposits, brain associated amyloid fibnl deposits or brain associated amyloid protein deposits, as well as amyloid fibnl formation and growth or age associated amyloid fibnl formation and growth, brain associated amyloid fibnl formation and growth, and interaction of amyloid protein with glycosaminoglycans, or with proteoglycans.
  • Hypencum perforatum has the ability to reduce, eliminate, prevent, inhibit or disrupt/dissolve amyloid fibnl or protein deposits, pancreas associated amyloid fibnl or protein deposits, as well as amyloid fibnl formation and growth, pancreas associated amyloid fibnl formation and growth, and pancreas interactions of amyloid protein with glycosaminoglycans or with proteoglycans
  • Example 1 Hypericum perforatum Causes a Dose-Dependent Inhibition of Alzheimer's A ⁇ (1-40) Amyloid Fibril Formation
  • Hypencum perforatum For the procedure to generate water extracts of Hypencum perforatum, 500mg of either a) standardized Hypencum perforatum obtained from the contents of gelatin-capsules of a commercial source of Hype ⁇ cum perforatum (containing 0.3% hypencin standardized extract) or b) freeze-powdered dned whole plant (i.e. leaves, buds and flowers) of Hypencum perforatum, were extracted with 3 ml of distilled water (Baxter) and placed in microcent ⁇ fuge tubes. The microcent ⁇ fuge tube contents were then vortexed by hand for 3-4 minutes, and then allowing to stand for 1-2 minutes.
  • Hype ⁇ cum perforatum was a potent inhibitor of Alzheimer's amyloid fibnl formation and exerted its effects in a dose-dependent manner.
  • amyloid fibnl growth is believed to involve amyloid protein self-interactions (le. A ⁇ -A ⁇ interactions)
  • Any potential effective therapeutic agent for amyloid deposition, accumulation and/or persistence should also be capable of causing an inhibition of amyloid protein self-interactions. This is important for preventing any new amyloid fibnl formation when treating Alzheimer's disease patients at early stages of the disease.
  • ELISA methodologies i.e. solid phase binding assays
  • a ⁇ (1-40) was first labelled with biotin according to the following protocol. 1 mg of AB (1-40) (Bachem Inc., Torrance, CA. USA, Lot #WL934) was dissolved in 200 ⁇ l of PBS (pH 8.0) and incubated for 1 week at 37°C. The fibnllar A ⁇ solution was then added to 0.2mg of a biotinylation agent [(sulfosucc ⁇ n ⁇ m ⁇ dyl-6-(b ⁇ ot ⁇ nam ⁇ do) hexanoate)](sulfo-NHS-
  • the pellet which contained fibnl zed A ⁇ which was biotmylated (at the non self- interacting region of A ⁇ ) was then resuspended in 1 ml of distilled deionized water.
  • the amount of biotin incorporated was then determined using the HABA (2-(4'-hydroxyazo- benzene)benzo ⁇ c acid) method (according to the manufacturer's protocol; Pierce).
  • Hype ⁇ cum perforatum obtained from a water extract (extracted as descnbed in example 1) of freeze-powdered d ⁇ ed plant mate ⁇ als (whole plant including leaves, buds and flowers; obtained fron The Herbalist, Seattle, WA, U.S.A.) or from the powdered contents of gelatin-coated capsules containing standardized Hypencum perforatum extract (i.e. 0.3% hypencin) obtained commercially (Sundown Herbals, Lot #04228 04-01)
  • Hype ⁇ cum perforatum extract obtained from freeze-powdered d ⁇ ed plant mate ⁇ als, and from a commercial source (containing 0.3% standardized extract) were effective in causing a significant reduction in A ⁇ -A ⁇ interactions.
  • Commercially available standardized Hype ⁇ cum perforatum extract (containing 0.3% hypencin) caused a significant (p ⁇ 0.001) 88% inhibition of A ⁇ -A ⁇ interactions
  • Hype ⁇ cum perforatum extract obtained from freeze-powdered d ⁇ ed plant mate ⁇ als caused a significant (p ⁇ 0.001) 66% inhibition of A ⁇ -A ⁇ interactions.
  • Hype ⁇ cum perforatum was an effective inhibitor of A ⁇ -proteoglycan/glycosaminoglycan (PG/GAG) interactions. Since PGs/GAGs have been found to accumulate in amyloid deposits and are believed to prevent the body's natural ability to remove unwanted "amyloid" (reviewed in Snow and Wight,
  • Hypencum perforatum obtained from a water extract (extracted as descnbed in example 1) of dned plant matenals (whole plant including leaves, buds and flowers; obtained fron The Herbalist, Seattle, WA, U.S.A.) oi from the powdered contents of gelatin-coated capsules containing standardized Hype ⁇ cum perforatum extract (i.e. 0.3% hypencin) obtained commercially (Sundown Herbals, Lot #04228 04-01).
  • Hypericum perforatum causes a Dissolution/Disruption of Pre-Formed Alzheimer's Disease Amyloid 1-40 Fibrils in a Dose-Dependent Manner and Within a 2-Hour Period "
  • Hype ⁇ cum perforatum extracts were capable of causing a "dissolution” or “disruption” of pre-formed Alzheimer's disease amyloid fibnls.
  • This type of activity would be important for any potential anti-amyloid drug which can be used in patients who already have substantial amyloid deposition in organs and/or tissues
  • Alzheimer's disease patients in mid-to late stage disease have abundant amyloid deposits in their brains as part of both neu ⁇ tic plaques and cerebrovascular amyloid deposits
  • a natural therapeutic agent capable of causing dissolution of pre-existing amyloid would be advantageous for use in these patients who are at latter stages of the disease process
  • a ⁇ (l-40)(Bachem Inc., Tonance, CA, USA, Lot #T20824) was dissolved in 1 0 ml of double distilled water (1 mg/ml solution) and then incubated at 37°C for 1 week to cause abundant Alzheimer's amyloid fibnl formation. 25 ⁇ M of fibnlhzed A ⁇ was then incubated in tnphcate for 2 hours at 37°C in a total final volume of 60 ⁇ l TBS, in the absence or presence of increasing concentrations (i.e.
  • Hype ⁇ cum perforatum water extracts de ⁇ ved from either freeze-powdered whole d ⁇ ed plant mate ⁇ als (whole plant including leaves, buds and flowers; obtained fron The Herbalist, Seattle, WA, U.S. A ) or from the powdered contents of gelatin-coated capsules containing standardized Hype ⁇ cum perforatum extract (i.e. 0.3% hypencin) obtained commercially (Sundown Herbals, Lot #04228 04-01) Following a 2 hour incubation, 50 ⁇ l aliquots were added to 1.2ml of lOO ⁇ M Thioflavin T (Sigma Chemical Co., St. Louis, MO) in 50mM NaPO 4 (pH 6.0) for fluorometry readings as descnbed in Example 1 above.
  • the compounds tested included increasing concentrations of a water extract of Hypencum perforatum obtained from freeze-powdered whole dned plant matenals or from the powdered contents of gelatin-coated capsules obtained commercially, as desc ⁇ bed in example 1
  • Hypericum perforatum causes a Dissolution/Disruption of A ⁇ (1-42) Alzheimer's Amyloid Fibrils
  • amyloid fibnls of Alzheimer's disease p ⁇ ma ⁇ ly consist of A ⁇ in a form containing residues 1-40 or 1-42
  • the longer vanant of A ⁇ contains two hydrophobic residues which cause substantial fibnl formation almost immediately (Castillo et al, J Neurochem 69 2452-2465, 1997)
  • a ⁇ 1-42 is also believed to be the predominant form of A ⁇ existing in Alzheimer's amyloid plaques
  • a ⁇ 1-40 is believed to be the predominant form of A ⁇ existing in Alzheimer's cerebrovascular amyloid deposits (Tamaoka et al, Br Res 679 151-156, 1995, Biochem Biophvs Res Comm 205 834-842, 1994)
  • Hypencum perforatum also causes dissolution/disruption of pre-formed A ⁇ (1-42) amyloid fibnls and whether this effect was long-lasting
  • the compounds tested included increasing concentrations of a water extract of Hypencum perforatum obtained from freeze-powdered whole d ⁇ ed plant mate ⁇ als (leaves, buds and flowers obtained from The Herbalist, Seattle, WA, U S A ) or from the powdered contents of gelatin-coated capsules containing standardized extract (I e 0 3% hypencin) and obtained commercially (Sundown Herbals, Lot #04228 04-01) Water extracts were prepared as desc ⁇ bed in example 1
  • amyloid fibnls in the islets of Langerhans in the pancreas (Cooper et al, Proc Natl Acad. Sci USA 84.8628-8632, 1987)
  • This amyloid protein involved consists of a 37 amino acid protein known as islet amyloid polypeptide or amylin. Islet amyloid is believed to cont ⁇ bute to the destruction of the beta-cells of the pancreas, thus eventually leading many patients to become insulin-dependent (le. type I diabetes).
  • Amylin has the ability to also form substantial amyloid fibnls immediately when placed m solution. The next study was therefore implemented to determine whether Hype ⁇ cum perforatum also causes dissolution/disruption of another type of amyloidosis, and whether this effect was also long-lasting.
  • Example 5 the method of Thioflavin T fluorometry as desc ⁇ bed in Example 5 was used. Bnefly, 60 ⁇ l (final volume) of 25 ⁇ M of human amylin (Bachem Inc,Torrance, CA, USA, Lot # WL934) in TBS (pH 7 0) was incubated in microcent ⁇ fuge tubes at 37°C for 2 days (in tnphcate), either alone, or in the presence of increasing amounts (i.e. O.Ol ⁇ l, O.l ⁇ l, 0.5 ⁇ l and l.O ⁇ l) of Hypencum perforatum, water extracts
  • the compounds tested included increasing concentrations of a water extract of Hypencum perforatum obtained from freeze-powdered whole dned plant mate ⁇ als (leaves, buds and flowers obtained from The Herbalist, Seattle, WA, U.S.A.) or from the powdered contents of gelatm-coated capsules containing standardized extract (i.e. 0.3% hypencin) and obtained commercially (Sundown Herbals, Lot #04228 04-01). Water extracts were prepared as descnbed in example 1.
  • Hype ⁇ cum perforatum on amylin fibnls was demonstrated by Congo red staining assays, whereby a marked reduction of congophiha (i.e. red/green birefnngence when viewed under polanzed light, and which represents a dissolution/disruption of the amyloid fibnllar structure) was observed when amylin amyloid fibnls were treated with Hypencum perforatum (from either source) for 2 hours (not shown) This study demonstrated that
  • Hype ⁇ cum perforatum is capable of causing significant dissolution/disruption of other forms of amyloid (such as islet amyloidosis)
  • One embodiment of the present invention is to formulate p ⁇ or to administration in a patient, a pharmaceutical formulation comp ⁇ sing Hype ⁇ cum perforatum (and or its active ingredients) in one or more pharmaceutical acceptable earners, diluents or excipients.
  • a patient who has Alzheimer's disease, type II diabetes or any other amyloidosis would orally consume commercially available Hypencum perforatum m pill, tablet, caplet, soft and hard gelatin capsule, lozenge, vegicap, liquid drop, solution, syrup, tea bag, and/or powder form.
  • Hypencum perforatum obtained commercially in any form could be further modulated using suitable earners, excipients and diluents including lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, algmates, tragacanth, gelatin, calcium silicate, microcrystallme cellulose, polyvinylpyrrohdone, cellulose, water syrup, methyl cellulose, methyl and propylhydroxybenzoates, talc, magnesium stearate and mineral oil.
  • suitable earners, excipients and diluents including lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, algmates, tragacanth, gelatin, calcium silicate, microcrystallme cellulose, polyvinylpyrrohdone, cellulose, water syrup, methyl cellulose, methyl and propy
  • compositions of the invention may be formulated so as to provide quick, sustained or delayed response of the active ingredient after administration to the patient.
  • the compositions are preferably formulated m a unit dosage form, each dosage containing from about 1 to about 1000 mg of Hypencum perforatum (or its active ingredients), more usually about 400 to about 750 mg of Hype ⁇ cum perforatum (or its active ingredients).
  • the therapeutic dosage administered will be determined by the physician m the light of the relevant circumstances including the clinical condition to be treated, the organ or tissues affected or suspected to be affected with amyloid accumulation, and the chosen route of administration
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active matenal calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical earner
  • Hard gelatin capsules may be prepared by using 500mg of Hype ⁇ cum perforatum (or its active ingredients), 400mg of starch, and 20 mg of magnesium stearate. The above ingredients are mixed and filled into hard gelatin capsules in 920mg quantities.
  • a tablet is prepared by using 500 mg of Hype ⁇ cum perforatum (or its active ingredients), 800 mg of microcrystallme cellulose, 20 mg of fumed silicon dioxide and 10 mg of stea ⁇ c acid. The components are blended and compressed to form tablets each weighing 1230mg
  • An aerosol solution is prepared by using 0.25 active ingredient, 29.75 ethanol, and 70 of propellent 22 (chlorodifluoromethane).
  • the Hype ⁇ cum perforatum (or its active ingredients) is mixed with ethanol The mixture is added to a portion of the Propellent 22, cooled to -30°C, and transferred to a filling device. The required amount is then fed to a stainless steel container and diluted with the remainder of the propellent. The value units (listed above) are then fitted to the container.
  • Such an aerosol form of Hype ⁇ cum perforatum (or its active ingredients) may be useful for the treatment of amyloids involving the brain (such as Alzheimer's disease. Down's syndrome, p ⁇ on diseases etc) by using an aerosol or nasal spray. Previous studies have suggested that in these central nervous system amyloidoses the initial form of entry of a possible environmental agent which may be playing a role in pathogenesis may be denved from the outside world through the nasal passages.
  • Hypencum perforatum or its active ingredients
  • starch 70mg of microcrystallme cellulose
  • polyvmylpynolidone 9 mg of sodium carboxymethyl starch
  • Hype ⁇ cum perforatum (or its active ingredients) starch and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly
  • the solution of polyvmylpynolidone is mixed with the resultant powders which are then passed through a No 14 mesh U.S. sieve.
  • the granules so produced are d ⁇ ed at
  • the Hypencum perforatum (or its active ingredients), cellulose, starch and magnesium stearate are blended, passed through a No. 45 mesh U.S sieve, and filled into hard gelatin capsules in 400 mg quantities.
  • the Hypencum perforatum (or its active ingredients) are passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glyce ⁇ des previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2 g capacity and allowed to cool.
  • Suspensions each containing 50 mg of medicant per 5 ml dose are made by using 50mg of Hype ⁇ cum perforatum (or its active ingredients), 50 mg of sodium carboxymethyl cellulose, 1.25ml of syrup, 0 10ml of benzoic acid solution, flavor, color, and pu ⁇ fied water to total 5 ml.
  • the medicant is passed though a No. 45 mesh U.S. sieve and mixed with the sodium carboxymethyl cellulose and syrup to form a smooth paste.
  • the benzoic acid solution, flavor and color are diluted with some of the water and added, with stirnng. Sufficient water is then added to produce the required volume.
  • An intravenous formulation is prepared by using 250mg of Hypencum perforatum
  • the therapeutic compound of the invention can be administered in any pharmaceutically acceptable vehicle.
  • pharmaceutically acceptable vehicle includes, but is not limited to, any and all solvents, stenle liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic o ⁇ gin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, dispersion media, coatings, antibacte ⁇ al and antifungal agents, isotonic and adsorption delaying agents, and the like which are compatible with the activity of the compound and are physiologically acceptable to the subject.
  • An example of a pharmaceutically acceptable vehicle is buffered normal saline (0.15 molar NaCl).
  • Supplementary active compounds can also be incorporated into the compositions.
  • suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, ⁇ ce, fluor, chalk, silica gel. magnesium carbonate, magnesium stearate, sodium stearate. glycerol monostearate. talc, sodium chlonde, d ⁇ ed sk m milk, glycerol, propylene, glycol, water, ethanol and the hke.
  • These compositions can take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • amyloid formation, deposition, accumulation and/or persistence in a subject is inhibited by administrating Hypencum perforatum (or its active ingredients) in a therapeutic dosage to the subject.
  • the term subject is intended to include living organisms in which amyloidosis can occur. Examples of subjects include humans, monkeys, cows, dogs, sheep, cats, mice.
  • compositions of the present invention to a subject to be treated can be earned out using known procedures, at dosages and for penods of time effective to inhibit amyloidosis in the subject
  • An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the amount of amyloid already deposited at the organ or tissue site in the subject, the age, sex and weight of the subject, and the ability of the therapeutic compound to inhibit amyloid formation, deposition, accumulation, persistence, and/or to cause dissolution of pre-formed amyloid in the subject Dosage regimens can therefore be adjusted to provide the optimum therapeutic response.
  • Hypencum perforatum or its active ingredients
  • a non- mitmg example of an effective dose range for Hypencum perforatum (or its active ingredients) is between 400 and lOOOmg/kg of body weight/per day
  • Hypencum perforatum or its active ingredients
  • a prefened route of administration is oral administration
  • Hypencum perforatum (or its active ingredients) may be administered by other suitable routes such as subcutaneous, intravenous, mtrapentoneal, all routes administered by injection.
  • the active compound may be coated m a matenal to protect the compound from the action of acids and other natural conditions which may inactivate the compound
  • the therapeutic compound may be administered to a subject in an approp ⁇ ate earner, for example, liposomes or a diluent
  • Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes
  • Hype ⁇ cum perforatum (or its active ingredients) may also be administered parenterally or intrapentoneally.
  • Dispersions can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms
  • Pharmaceutical compositions suitable for injectable use include ste ⁇ le aqueous solutions or dispersions and ste ⁇ le powders for the preparation of ste ⁇ le injectable solutions or dispersion. In all cases, the composition must be stenle and must be fluid to the extent that easy use in the synnge exists It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, prabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
  • Sterile injectable solutions can be prepared by incorporating the therapeutic compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the therapeutic compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the therapeutic agent plus any desired ingredients from a previously sterile-filtered solution thereof.
  • the Hypericum perforatum (or its active ingredients) for Alzheimer's disease and other central nervous system amyloidoses may be optimized to cross the blood-brain barrier.
  • Methods of introductions include but are not limited to systemic administration, parenteral administration i.e., via an intraperitoneal, intravenous, perioral, subcutaneous, intramuscular, intraarterial, intradermal, intramuscular, intranasal, epidural and oral routes.
  • Hypericum perforatum (or its active ingredients) may be directly administered to the cerebrospinal fluid by intraventricular injection.
  • Hypericum perforatum or its active ingredients
  • Hypericum perforatum (or its active ingredients) may be delivered in a controlled release system, such as an osmotic pump.
  • a controlled release system can be placed in proximity to the therapeutic target, ie. the brain, thus requiring only a fraction of the systemic dose.

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Abstract

This invention relates to compositions and methods for treating Alzheimer's Disease and other amyloidoses using an extract from Hypericum perforatum.

Description

Title Methods of Treating Alzheimer's Disease and Other
Amyloidoses using Hypericum perforatum and Derivatives Thereof
TECHNICAL FIELD
The invention relates to compositions and methods for treating Alzheimer's Disease and other amyloidoses, and to methods for isolating pharmaceutical agents from plant matter; more particularly, it relates to compositions and methods for therapeutic intervention in Alzheimer's disease and other amyloidoses using plant matter and deπvatives thereof
BACKGROUND OF THE INVENTION Alzheimer's disease is characteπzed by the accumulation of a 39-43 ammo acid peptide termed the beta-amyloid protein or Aβ, in a fibπllar form, existing as extracellular amyloid plaques and as amyloid within the walls of cerebral blood vessels. Fibπllar Aβ amyloid deposition in Alzheimer's disease is believed to be detπmental to the patient and eventually leads to toxicity and neuronal cell death, characteπstic hallmarks of Alzheimer's disease. Accumulating evidence implicates amyloid as a major causative factor of Alzheimer's disease pathogenesis
A vaπety of other human diseases also demonstrate amyloid deposition and usually involve systemic organs (I e oigans or tissues lying outside the central nervous system), with the amyloid accumulation leading to organ dysfunction or failure. n Alzheimer's disease and "systemic" amyloid diseases, there is currently no cure or effective treatment, and the patient usually dies within 3 to 10 years from disease onset.
New compounds or agents for therapeutic regimes to arrest or reverse amyloid formation, deposition, accumulation and/or persistence that occurs in Alzheimer's disease and other amyloidoses are therefore desperately sought.
DISCLOSURE OF THE INVENTION
A pπmary object of the present invention is to establish new methods for the treatment of the amyloid diseases The amyloid diseases include, but are not limited to, the amyloid associated with Alzheimer's disease, Down's syndrome and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (wherein the specific amyloid is referred to as beta-amyloid protein or Aβ), the amyloid associated with chronic inflammation, vaπous forms of malignancy and Familial Mediterranean Fever (wherein the specific amyloid is referred to as AA amyloid or inflammation-associated amyloidosis), the amyloid associated with multiple myeloma and other B-cell dyscrasias (wherein the specific amyloid is referred to as AL amyloid), the amyloid associated with type II diabetes (wherein the specific amyloid is referred to as amylin or islet amyloid), the amyloid associated with the pπon diseases including Creutzfeldt- Jakob disease, Gerstmann-Straussler syndrome, kuru and animal scrapie (wherein the specific amyloid is referred to as PrP amyloid), the amyloid associated with long-term hemodialysis and carpal tunnel syndrome (wherein the specific amyloid is referred to as beta2-mιcroglobuhn amyloid), the amyloid associated with senile cardiac amyloid and Familial Amyloidotic Polyneuropathy (wherein the specific amyloid is referred to as transthyretin or prealbumin), and the amyloid associated with endocπne tumors such as medullary carcinoma of the thyroid (wherein the specific amyloid is referred to as vanants of procalcitomn)
Another object of the present invention is to use the plant Hypeπcum perforatum
(also referred to as St John's Wort) and/or its constituents thereof (including but not limited to its leaves, buds and flowers) for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes and other amyloidoses Hypeπcum perforatum is also referred to as, but not limited to, St John's
Wort, Hypeπci herba, Amber, Goatweed, Johnswort, Klamath Weed, and Tipton Weed Another object of the present invention is to use extracts and/or deπvatives thereof from plant matter related to the family Hypeπcaceae, which includes, but is not limited to the genus Hypeπcum, for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes and other amyloidoses
Another object of the present invention is to use extracts and/or deπvatives thereof from plant matter related to the vanous Hypeπcum species, which may include but not limited to, Hypeπcum perforatum, Hypeπcum calycmum, Hypeπcum formosum,
Hypeπcum hirsutum, Hypeπcum patulum, and Hypeπcum olympicum
Another object of the present invention is to use commercially available pills, tablets, caplets. soft and hard gelatin capsules lozenges, sachets, cachets, vegicaps, liquid drops, elixers, suspensions, emulsions, solutions, syrups, tea bags, aerosols (as a solid or in a liquid medium), suppositoπes, steπle injectable solutions, steπle packaged powders, dπed leaves, dπed buds, dπed flowers which contain Hypeπcum perforatum to treat patients with Alzheimer's disease, type II diabetes and other amyloidoses
Another object of the present invention is to use Hypeπcum perforatum, and/or the hypeπcins contained within Hypeπcum perforatum for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes and other amyloidoses
Yet another object of the present invention is to use the flavanoids contained within Hypeπcum perforatum for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes and other amyloidoses. Such flavanoids include, but are not limited to, hyperoside, biapigenm, rutm, quercetin, quercitin, isoquercitπn, pseudohypeπcin, hyperfoπn, procyanidines, amentoflavme, and luteohn
Yet another object of the present invention is to use the xanthones contained within Hypencum perforatum for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes and other amyloidoses.
Yet another object of the present invention is to use the proanthocyamdins contained within Hypeπcum perforatum for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes and other amyloidoses. Yet another object of the present invention is to use the dianthrones contained within
Hypeπcum perforatum for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes and other amyloidoses.
Yet another object of the present invention is to use the tannins contained within Hypeπcum perforatum for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes and other amyloidoses.
Yet another object of the present invention is to use the carbohydrates contained within Hypeπcum perforatum for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes and other amyloidoses. Such carbohydrates include, but are not limited to, pectin. Yet another object of the present invention is to use the hpids contained within
Hypeπcum perforatum for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes and other amyloidoses. Such hpids include, but are not limited to, monoterpenes.
Yet another object of the present invention is to use the vitamins contained within Hypeπcum perforatum for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes and other amyloidoses. Such vitamins include, but are not limited to, vitamin A and vitamin C
Yet another object of the present invention is to provide methods to isolate the active ingredients present within Hypeπcum perforatum for use as potent agents which inhibit amyloid formation, amyloid deposition, amyloid accumulation, amyloid persistence, amyloid protein-amyloid protein interactions, and/or cause a dissolution/disruption of preformed or pre-deposited amyloid fibnls in Alzheimer's disease, type II diabetes and other amyloidoses. Methods for isolation of the active ingredients within Hypeπcum perforatum include application of some standard techniques known to those skilled in the art, including, but not limited to, thin layer chromatography using silica-coated plates, and separation and isolation using high pressure liquid chromatography (HPLC). Unknown active ingredients within Hypeπcum perforatum found to be potent inhibitors of amyloid formation, amyloid deposition, amyloid accumulation, amyloid persistence, amyloid protein-amyloid protein interactions, and/or cause a dissolution/disruption of pre-formed or pre-deposited amyloid fibnls in Alzheimer's disease, type II diabetes and other amyloidoses, are identified by re- testing of individual bands or fractions (separated by thin layer chromatography, column chromatography and/or HPLC) using specific assay tests as descπbed in the examples of the present invention. Sufficient isolation of these active ingredients contained within individual bands and/or fractions are then sent out for specific analyses which may include, but are not limited to, scanning electron microscope equipped with energy dispersive x-ray analyzer to detect and spatially map some elements present in each sample, elemental analysis by combustion to determine the relative % of carbon, hydrogen and nitrogen, high resolution mass spectroscopy to determine molecular weight and elemental composition, fouπer transform infrared spectroscopy to determine functional groups and make compaπsons to the spectra of known compounds, differential scanning caloπmetry to determine melting point, atomic absorption, gel chromatography. high performance liquid chromatography, proton and C13 nuclear magnetic resonance spectroscopy for matenal characteπzation and to provide information regarding the position of atoms relative to each other, and UN/NIS spectroscopy It is expected that additional techniques will be developed as part of the further isolation of potent active ingredients within Hypeπcum perforatum
Yet another object of the present invention is to provide the use of Hypeπcum perforatum and/or its ingredients [(regardless of commercial source and regardless of final form for consumption by humans, i.e. pills, tablets, caplets, soft and hard gelatin capsules, lozenges, sachets, cachets, vegicaps, liquid drops, elixers, suspensions, emulsions, solutions, syrups, tea bags, aerosols (as a solid or m a liquid medium), suppositones, steπle injectable solutions, steπle packaged powders, dπed leaves, dπed buds, and dπed flowers] for inhibition of amyloid formation, deposition, accumulation, and/or persistence, regardless of its clinical setting.
Yet another object of the present invention is to provide compositions and methods involving admmisteπng to a subject a therapeutic dose of Hypeπcum perforatum (or its active ingredients) which inhibits amyloid deposition. Accordingly, the compositions and methods of the invention are useful for inhibiting amyloidosis in disorders in which amyloid deposition occurs. The compounds of the invention can be used therapeutically to treat amyloidosis or can be used prophylactically in a subject susceptible to amyloidosis. The methods of the invention are based, at least in part, in directly inhibiting amyloid fibπl formation, inhibiting amyloid fibπl growth, and/or causing dissolution/disruption of preformed amyloid fibnls. Yet another object of the present invention is to provide pharmaceutical compositions for treating amyloidosis. The pharmaceutical compositions include a therapeutic compound of the invention in an amount effective to inhibit amyloid deposition and a pharmaceutically acceptable vehicle.
Yet another object of the present invention is the use of any and all synthetic compounds made similar to Hypencum perforatum and/or its active ingredients for use as potent agents which inhibit amyloid formation, amyloid deposition, amyloid accumulation, amyloid persistence, amyloid protein-amyloid protein interactions, and/or cause a dissolution/ disruption of pre-formed or pre-deposited amyloid fibnls in Alzheimer's disease, type II diabetes and other amyloidoses. It is yet another object of the invention to meet any and all of the needs summanzed above.
These and such other objects of the invention will become evident from the disclosure below are met by the invention disclosed herein.
Application of the invention to these needs is especially beneficial in that the invention is the only system that effectively provides for use of extracts from the leaves, flowers and/or buds of Hypeπcum perforatum. and use of the ingredients contained within the vanous commercial preparations of Hypeπcum perforatum, to benefit human patients with Alzheimer's disease and other amyloidoses due to Hypencum perforatum' s newly discovered ability to inhibit amyloid fibnl formation, inhibit amyloid fibπl growth, inhibit amyloid-proteoglycan interactions, amyloid-glycosaminoglycan interactions, and cause dissolution and/or disruption of preformed amyloid fibnls.
The present invention pertains to the identification and surpnsing discovery that an extract from the dπed upper plant parts (i.e. leaves, flowers and/or buds) of Hypeπcum perforatum, otherwise known as St. John's Wort, act as an impressive inhibitor of
Alzheimer's disease amyloid formation and growth. In addition, Hypeπcum perforatum also has the ability to inhibit amyloid protein-amyloid protein interactions, which are believed to be important for the growth of amyloid deposits in tissues. Furthermore, Hypencum perforatum also has the ability to inhibit amyloid protem-proteoglycan (PG)/ glycosaminoglycan (GAG) interactions, which are believed to be important for the formation and persistence of all amyloid deposits in tissues. In addition, Hypencum perforatum has the ability to dissolve/disrupt pre-formed amyloid fibnls of the Alzheimer's and type II diabetes types, suggesting that this agent may be useful for patients at latter stages of both Alzheimer's disease, type II diabetes and other amyloidoses. Hypeπcum perforatum extracted from different commercial sources (extracts isolated from dned whole plant mateπals or from powder obtained from gelatin capsules containing a concentrated extract of 0.3% hypencin) were all found to serve as potent inhibitors of Alzheimer's disease amyloid fibπllogenesis.
While results are exemplified with Hypencum perforatum, other species of Hypeπcum are believed to have similar effect
Commercially available Hypeπcum perforatum (extracts obtained from dπed whole plant mateπals, or from gelatin-capsules containing 0.3% hypencin) caused a marked significant inhibition of Aβ amyloid fibnl formation as determined using a Thioflavin T fluorometry assay. Extracts of Hypeπcum perforatum obtained from different commercial sources inhibited Aβ amyloid fibπllogenesis in a dose-dependent manner. Hypeπcum perforatum extract also inhibited Alzheimer's Aβ-Aβ interactions as determined using a solid phase binding assay demonstrating that Hypencum perforatum is additionally an effective inhibitor of Alzheimer's amyloid fibnl growth. Furthermore, Hypeπcum perforatum was effective in the inhibition of Aβ-proteoglycan/ glycosaminoglycan (PG/GAG) interactions (an important therapeutic target for all amyloidoses) as determined using a solid phase binding immunoassay. Hypeπcum perforatum extracts deπved from different commercial sources was also a potent dissolving/inhibiting agent of pre-formed Aβ (1-40) or Aβ (1-42) containing amyloid fibnls, and pre-formed amylin fibnls, as determined using a Thioflavin T fluorometry and Congo red staining assays. This latter effect occurred in a dose-dependent
SUBSTΠTJTE SHEET (RULE 26) manner, causing a significant dissolution of both Aβ 1-40 and 1-42 pre-formed Alzheimer's fibrils within a 2 hour incubation duration. Hypencum perforatum which was effective in all of the studies described above were all derived from Hypericum perforatum extract obtained from whole plant (i.e. containing dried flowers, leaves and buds), and gelatin-capsule form, and were both currently available commercially for oral use in humans. Therefore, the present invention claims the use of Hypericum perforatum (in a pill, tablet or liquid form, and from dried plant materials) and derivatives thereof from different commercial sources for the treatment of amyloidosis in Alzheimer's disease, type II diabetes and other amyloidoses. Also disclosed are methods of isolation to identify and purify the key amyloid inhibitory ingredients within the plant material. Identification of the "active" amyloid inhibitory ingredients within the extracted plant materials are anticipated to lead to new drug design for anti-amyloid therapeutics of the future. Current use of Hypericum perforatum and its ingredients contained within different commercial preparations are anticipated to benefit human patients at all stages of Alzheimer's disease due to Hypericum perforatu 's inherent ability to inhibit Aβ amyloid fibril formation (early to mid-stage Alzheimer's disease), inhibit amyloid fibril growth (early to mid-stage Alzheimer's disease), inhibit amyloid-PG/GAG interactions (all stages of Alzheimer's disease) and cause dissolution/disruption of preformed amyloid fibrils (mid to late stages of Alzheimer's disease). Similarly, Hypericum perforatum is anticipated to benefit patients with different systemic amyloid diseases such as type II diabetes, regardless of the stage of amyloid accumulation and the organ (or tissue) involved.
These and other features and advantages of the present invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying figures.
In other aspects of the invention, a pharmaceutical agent is disclosed for treating an amyloid disease in a patient, wherein the pharmacological agent comprises a therapeutically effect amount of plant matter from the genus Hypericum. The pharmaceutical agent is preferably from a plant of the genus Hypericum, species perforatum. The pharmacological agent is preferably an extract obtained from Hypericum perforatum, the extract being derived from the dried leaves, flowers and buds of Hypericum perforatum, and advantageously taken from some commercially available source such as pills, tablets, caplets, soft and hard gelatin capsules, lozenges, sachets, cachets, vegicaps, liquid drops, elixers, suspensions, emulsions, solutions, syrups, tea bags, aerosols (as a solid or in a liquid medium), suppositories, sterile injectable solutions, sterile packaged powders, or plant powder obtained from dried leaves, buds, and/or flowers. In a preferred embodiment, the pharmaceutical agent is an amyloid inhibitory ingredient selected from the group consisting of, but not limited to, flavanoids, xanthones, proanthocyanidins, dianthrones, tannins, monoterpenes, hyperoside, biapigenin, rutin, quercetin, quercitin, isoquercitrin, pseudohypericin, hyperforin, procyanidines, amentoflavine, luteolin, pectin, vitamin A, and vitamin C.
The pharmacological agent preferably has a therapeutically effective amount of Hypericum perforatum in a dosage in the range of from about 10 to 1,000 mg/kg of body weight of the patient, and more preferably in the range of about 10 to 100 mg/kg of body weight of the patient. The pharmacological agent preferably has a therapeutically effective amount of hypericin, standardized to contain hypericin at a range of 0.05% to 2%, but more preferably in the range of about 0.1% to 0.5%, per 250mg to 500mg capsule containing Hypericum perforatum.
The amyloid disease for treatment with the pharmacological agent is selected from the group consisting of the amyloid associated with Alzheimer's disease, Down's syndrome and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (wherein the specific amyloid is referred to as beta-amyloid protein or Aβ), the amyloid associated with chronic inflammation, various forms of malignancy and Familial Mediterranean Fever (wherein the specific amyloid is referred to as AA amyloid or inflammation-associated amyloidosis), the amyloid associated with multiple myeloma and other B-cell dyscrasias (wherein the specific amyloid is referred to as AL amyloid), the amyloid associated with type II diabetes (wherein the specific amyloid is refened to as amylin or islet amyloid), the amyloid associated with the prion diseases including Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, kuru and animal scrapie (wherein the specific amyloid is refened to as PrP amyloid), the amyloid associated with long-term hemodialysis and carpal tunnel syndrome (wherein the specific amyloid is referred to as beta2-microglobulin amyloid), the amyloid associated with senile cardiac amyloid and Familial Amyloidotic Polyneuropathy (wherein the specific amyloid is referred to as transthyretin or prealbumin), and the amyloid associated with endocrine tumors such as medullary carcinoma of the thyroid (wherein the specific amyloid is referred to as variants of procalcitonin).
Preferred pharmaceutical agents have a weight percentage of plant extract in the agent is in the range of from about 70% to about 95%, and may also have a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical agent preferably has an amyloid inhibitory activity or efficacy greater than 50%. In addition, Hypencum perforatum has the ability to inhibit the formation of brain amyloid deposits in patients who accumulate brain amyloid deposits that occur dunng normal aging and in a vanety of brain disorders including Alzheimer's disease; it will therefore promote mental alertness in such patients. Hypeπcum perforatum has the ability to reduce, eliminate, prevent or inhibit or disrupt/dissolve amyloid fibnl or protein deposits, brain associated amyloid fibnl deposits or brain associated amyloid protein deposits, as well as amyloid fibnl formation and growth or age associated amyloid fibnl formation and growth, bram associated amyloid fibπl formation and growth, and interaction of amyloid protein with glycosaminoglycans or proteoglycans; it will therefore promote mental acuity, promote mental alertness, provide nutπtional support for age or related cognitive or memory decline, promote cognitive well being, support brain function, improve cognitive ability, mental performance or memory, promote concentration and mental sharpness, improve mental vitality, promote greater mental clanty and alertness, improve short term memory, reduce or reverse age associated cognitive or memory decline, support normal bra function, enhance learning or memory; improve concentration, enhance mental performance, reduce mental decline, reduce likelihood of age related bram disorders, and maintain good brain health.
Hypeπcum perforatum further has the ability to reduce, eliminate, prevent, inhibit or disrupt/dissolve amyloid fibnl or protein deposits, as well as amyloid fibnl formation and growth, pancreas associated amyloid fibπl formation and growth, and interaction of amyloid protein with glycosaminoglycans or with proteoglycans; it will therefore support healthy pancreatic function and promote pancreatic function by helping to promote normal insulin function
Another aspect of the invention is a method for isolating amyloid inhibitory constituents within Hypencum perforatum plant matter, the method compnsing the following steps: a) extracting the plant matter with an organic solvent or water, b) concentrating the extract, c) removing insoluble mateπals, d) precipitating amyloid inhibitory constituents with organic solvent or water, e) recoveπng and redissolvmg the amyloid inhibitory constituents obtained in organic solvent or water, and f) injecting and separation by HPLC.
The plant matter is preferably compnsed of commercially obtained pills, tablets, caplets, soft and hard gelatin capsules, lozenges, sachets, cachets, vegicaps, liquid drops, elixers, suspensions, emulsions, solutions, syrups, tea bags, aerosols (as a solid or in a liquid medium), suppositoπes, steπle injectable solutions, stenle packaged powders, or plant matter which contain Hypericum perforatum, extracts or derivatives thereof, and may be taken from commercially available gelatin-coated capsules which contain dried-plant material (preferably leaves, buds and flowers) of Hypericum perforatum, extracts or derivatives thereof. A method is also disclosed for treating an amyloid disease in a patient, comprising the step of administering to the patient a therapeutically effective amount of plant matter from the plant of the genus Hypericum, species perforatum. The plant matter is preferably administered orally or by aerosal spray or in a parenterally injectable or infusible form. The therapeutically effective amount of plant matter is preferably an amyloid inhibitory ingredient selected from the group consisting of but not limited to, flavanoids, xanthones, proanthocyanidins, dianthrones, tannins, monoterpenes, hyperoside, biapigenin, rutin, quercetin, quercitin, isoquercitrin, pseudohypericin, hyperforin, procyanidines, amentoflavine, luteolin, pectin, vitamin A, and vitamin C.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings are illustrative of the invention and are not meant to limit the scope of the invention.
FIGURE 1 is a black and white graph of a 1 week Thioflavin T fluorometry assay demonstrating that an extract from Hypericum perforatum (derived from a commercial source) causes dose-dependent inhibition of Alzheimer's disease Aβ 1-40 amyloid fibril formation.
FIGURE 2 is a black and white graph of a solid phase binding assay utilized to identify lead compounds which inhibit Alzheimer's Aβ-Aβ interactions (i.e. Alzheimer's amyloid fibril growth). Hypericum perforatum (derived either from an extract obtained from dried plant materials, or from an extract obtained from the powder present in commercially available gelatin-capsules which contain 0.3% hypericin) are potent inhibitors of Alzheimer's amyloid fibril growth.
FIGURE 3 is a black and white graph of a solid phase binding immunoassay utilized to determine the potential dose-dependent effects of Hypericum perforatum (derived either from an extract obtained from dried plant materials, or from an extract obtained from the powder present in commercially available gelatin-capsules which contain 0.3% hypericin) on inhibition of Aβ-Perlecan GAG interactions. Significant dose-dependent inhibition of Aβ- perlecan GAG interactions is observed with treatment of Hypericum perforatum.
FIGURE 4 is a black and white graph of a Thioflavin T fluorometry assay utilized to determine the potential dose-dependent effects of Hypencum perforatum on dissolution/ disruption of pre-formed Alzheimer's Aβ 1-40 amyloid fibnls with a 2 hour incubation penod. Hypeπcum perforatum (deπved either from an extract obtained from dπed plant matenals, or from an extract obtained from the powder present in commercially available gelatin-capsules which contain 0.3% hypencin) causes dissolution of pre-formed
Alzheimer's Aβ 1-40 amyloid fibnls in a dose-dependent manner.
FIGURE 5 is a black and white graph of a Thioflavin T fluorometry assay utilized to determine the potential dose-dependent effects of Hypeπcum perforatum on dissolution/ disruption of pre-formed Alzheimer's Aβ 1-42 amyloid fibnls within a 2 hour incubation peπod. Hypeπcum perforatum (deπved either from an extract obtained from dπed plant mateπals, or from an extract obtained from the powder present in commercially available gelatin-capsules which contain 0.3% hypencm) causes dissolution of pre-formed Alzheimer's Aβ 1-42 amyloid fibnls in a dose-dependent manner.
FIGURE 6 is a black and white graph of a Thioflavin T fluorometry assay utilized to show that an Hypencum perforatum (deπved either from an extract obtained from dned plant mateπals, or from an extract obtained from the powder present in commercially available gelatin-capsules which contain 0.3% hypencin) is also able to cause a significant dissolution of pre-formed islet amyloid fibnls (le. amylin).
BEST MODE OF CARRYING OUT THE INVENTION
Turning now to the drawings, the invention will be descπbed in a preferred embodiment by reference to the numerals of the drawing figures wherein like numbers indicate like parts.
Amyloid and Amyloidosis
Amyloid is a geneπc term refernng to a group of diverse, but specific extracellular protein deposits which all have common morphological properties, staining charactenstics, and x-ray diffraction spectra. Regardless of the nature of the amyloid protein deposited all amyloids have the following characteπstics: 1) an amorphous appearance at the light microscopic level and appear eosmophilic using hematoxy n and eosm stains; 2) all stain with Congo red and demonstrate a red green birefπngence as viewed under polanzed light (Puchtler et al., J. Histochem Cytochem 10:355-364, 1962), 3) all contain a predominant beta-pleated sheet secondary structure, and 4) ultrastructurally amyloid usually consist of non-branching fibnls of indefinite length and with a diameter of 7-10 nm. Amyloid today is classified according to the specific amyloid protein deposited. The amyloid diseases include, but are not limited to, the amyloid associated with Alzheimer's disease, Down's syndrome and Hereditary cerebral hemorrhage with amyloidosis of the Dutch type (wherein the specific amyloid is referred to as beta-amyloid protein or Aβ), the amyloid associated with chronic inflammation, vanous forms of malignancy and Familial
Mediterranean Fever (wherein the specific amyloid is refened to as AA amyloid or inflammation-associated amyloidosis), the amyloid associated with multiple myeloma and other B-cell dyscrasias (wherein the specific amyloid is referred to as AL amyloid), the amyloid associated with type II diabetes (wherein the specific amyloid is referred to as amylin or islet amyloid), the amyloid associated with the pπon diseases including
Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, kuru and animal scrapie (wherein the specific amyloid is referred to as PrP amyloid), the amyloid associated with long-term hemodialysis and carpal tunnel syndrome (wherein the specific amyloid is referred to as beta2-mιcroglobuhn amyloid), the amyloid associated with senile cardiac amyloid and Familial Amyloidotic Polyneuropathy (wherein the specific amyloid is referred to as prealbumin or transthyretin amyloid), and the amyloid associated with endocπne tumors such as medullary carcinoma of the thyroid (wherein the specific amyloid is referred to as vanants of procalcitonin).
Although amyloid deposits m clinical conditions share common physical properties relating to the presence of a beta-pleated sheet conformation, it is now clear that many different chemical types exist and additional ones are likely to be descnbed in the future. It is currently thought that there are several common pathogenetic mechanisms that may be operating in amyloidosis in general. In many cases, a circulating precursor protein may result from overproduction of either intact or aberrant molecules (ex. plasma cell dyscrasias), reduced degradation or excretion (serum amyloid A in some secondary amyloid syndromes and beta2-mιcroglobuhn in long-term hemodialysis), or genetic abnormalities associated with vanant proteins (ex. familial amyloidotic polyneuropathy). Proteolysis of a larger protein precursor molecule occurs in many types of amyloidosis, resulting in the production of lower molecular weight fragments that polymenze and assume a beta-pleated sheet conformation as tissue deposits, usually in an extracellular location. What are the precise mechanisms involved, and the aberrant causes leading to changes in proteolytic processing and/or translational modifications is not known most amyloids.
Systemic amyloids which include the amyloid associated with chronic inflammation, vaπous forms of malignancy and Familial Mediterranean Fever (le. AA amyloid or inflammation-associated amyloιdosιs)(Benson and Cohen, Arth Rheum. 22:36-42, 1979; Kamei et al, Acta Path. Jpn. 32.123-133, 1982; McAdam et al, Lancet 2:572-573, 1975; Metaxas, Kidney Int. 20:676-685, 1981), and the amyloid associated with multiple myeloma and other B-cell dyscrasias (le. AL amyloιd)(Harada et al, J. Histochem. Cytochem 19:1- 15, 1971), as examples, are known to involve amyloid deposition in a vanety of different organs and tissues generally lying outside the central nervous system. Amyloid deposition in these diseases may occur, for example, m liver, heart, spleen, gastrointestinal tract, kidney, skin, and/or lungs (Johnson et al, N. Engl J. Med. 321:513-518, 1989). For most of these amyloidoses, there is no apparent cure or effective treatment and the consequences of amyloid deposition can be detπmental to the patient. For example, amyloid deposition in kidney may lead to renal failure, whereas amyloid deposition in heart may lead to heart failure. For these patients, amyloid accumulation in systemic organs leads to eventual death generally within 3-5 years. Other amyloidoses may affect a single organ or tissue such as observed with the Aβ amyloid deposits found in the brains of patients with Alzheimer's disease and Down's syndrome- the PrP amyloid deposits found in the brains of patients with
Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, and kuru; the islet amyloid (amylin) deposits found in the islets of Langerhans in the pancreas of 90% of patients with type II diabetes (Johnson et al, N Engl. J Med. 321:513-518, 1989; Lab. Invest. 66:522- 535, 1992); the beta2-mιcroglobuhn amyloid deposits in the medial nerve leading to carpal tunnel syndrome as observed in patients undergoing long-term hemodialysis (Geyjo et al,
Biochem. Biophvs Res Comm 129:701-706, 1985; Kidnev Int. 30:385-390, 1986); the prealbumin/ transthyretin amyloid observed in the hearts of patients with senile cardiac amyloid, and the prealbumin/ transthyretin amyloid observed in peπpheral nerves of patients who have Familial Amyloidotic Polyneuropathy (Skinner and Cohen, Biochem. Biophys Res Comm. 99: 1326-1332. 1981 ; Saraiva et al, J. Lab Clin. Med. 102:590-603. 1983, 1
Chn. Invest. 74: 104-119. 1984; Tawara et al. J. Lab. Clin. Med. 98:811-822. 1989)
Alzheimer's Disease and the Aging Population
Alzheimer's disease is a leading cause of dementia in the elderly, affecting 5-10% of the population over the age of 65 years (A Guide to Understanding Alzheimer's Disease and
Related Disorders, edited by Jorm, New York University Press, New York, 1987). In Alzheimer's disease, the parts of the brain essential for cognitive processes such as memory, attention, language, and reasoning degenerate, robbing victims of much that makes us human, including independence. In some mhented forms of Alzheimer's disease, onset is m middle age, but more commonly, symptoms appear from the mιd-60's onward. Alzheimer's disease today affects 4-5 million Ameπcans, with slightly more than half of these people receiving care at home, while the others are in many different health care institutions. The prevalence of Alzheimer's disease and other dementias doubles every 5 years beyond the age of 65, and recent studies indicate that nearly 50% of all people age 85 and older have symptoms of Alzheimer's disease (1997 Progress Report on Alzheimer's Disease. National Institute on Aging/National Institute of Health) 13% (33 million people) of the total population of the United States are age 65 and older, and this % will climb to 20% by the year 2025 (1997 Progress Report on Alzheimer's Disease. National Institute on Aging/National Institute of Health)
Alzheimer's disease also puts a heavy economic burden on society as well A recent study estimated that the cost of canng for one Alzheimer's disease patient with severe cognitive impairments at home or in a nursing home, is more than $47,000 per year (A Guide to Understanding Alzheimer's Disease and Related Disorders, edited by Jorm, New York University Press, New York, 1987). For a disease that can span from 2 to 20 years, the overall cost of Alzheimer's disease to families and to society is staggeπng. The annual economic toll of Alzheimer's disease in the United States in terms of health care expenses and lost wages of both patients and their caregivers is estimated at $80 to $100 billion (1997 Progress Report on Alzheimer's Disease. National Institute on Aging/National Institute of Health).
Tacnne hydrochloπde ("Cognex"), the first FDA approved drug for Alzheimer's disease is a acetylchohnesterase inhibitor (Cutler and Sramek, N Engl. J. Med 328-808- 810, 1993) However, this drug has showed limited success in the cognitive improvement in Alzheimer's disease patients and initially had major side effects such as liver toxicity. The second more recently FDA approved drug, donepezil (also known as "Ancept"), which is also an acetylchohnesterase inhibitor, is more effective than tacnne, by demonstrating slight cognitive improvement in Alzheimer's disease patients (Barner and Gray, Ann Pharmacotherapy 32:70-77, 1998; Rogers and Fnedhoff, Eur. Neuropsvch. 8.67-75, 1998), but is not believed to be a cure. Therefore, it is clear that there is a need for more effective treatments for Alzheimer's disease patients
Amyloid as a Therapeutic Target for Alzheimer's Disease
Alzheimer's disease is characteπzed by the deposition and accumulation of a 39-43 ammo acid peptide termed the beta-amyloid protein, Aβ or β/A4 (Glenner and Wong, Biochem Biophvs Res Comm 120:885-890, 1984, Masters et al, Proc. Natl. Acad. Sci USA 82 4245-4249, 1985; Husby et al, Bull WHO 71:105-108, 1993). Aβ is denved from larger precursor proteins termed beta-amyloid precursor proteins (or BPPs) of which there are several alternatively spliced vanants The most abundant forms of the BPPs include proteins consisting of 695, 751 and 770 amino acids (Tanzi et al, Nature 331:528-530,
1988; Kitaguchi et al, Nature 331:530-532, 1988; Ponte et al, Nature 331:525-527, 1988). The small Aβ peptide is a major component which makes up the amyloid deposits of "plaques" in the brains of patients with Alzheimer's disease. In addition, Alzheimer's disease is characteπzed by the presence of numerous neurofibπllary "tangles", consisting of paired helical filaments which abnormally accumulate the neuronal cytoplasm (Grundke-Iqbal et al, Proc Natl Acad Sci USA 83.4913-4917, 1986; Kosik et al, Proc Natl Acad. Sci USA 83:4044-4048, 1986, Lee et al, Science 251:675-678, 1991). The pathological hallmarks of Alzheimer's disease is therefore the presence of "plaques" and "tangles", with amyloid being deposited m the central core of plaques. The other major type of lesion found in the Alzheimer's disease bram is the accumulation of amyloid the walls of blood vessels, both within the brain parenchyma and in the walls of meningeal vessels which lie outside the brain The amyloid deposits localized to the walls of blood vessels are referred to as cerebrovascular amyloid or congophi c angiopathy (Mandybur, J. Neuropath. Exp. Neurol 45:79-90, 1986; Pardπdge et al, J Neurochem. 49.1394-1401, 1987). For many years there has been an ongoing scientific debate as to the importance of
"amyloid" in Alzheimer's disease and whether the "plaques" and "tangles" characteπstic of this disease, were a cause or merely the consequences of the disease. With the last few years, studies now indicate that amyloid is indeed a causative factor for Alzheimer's disease and should not be regarded as merely an innocent bystander. The Alzheimer's Aβ protein in cell culture has been shown to cause degeneration of nerve cells withm short penods of time
(Pike et al, BxJ es. 563:311-314, 1991. J Neurochem. 64.253-265. 1995). Studies suggest that it is the fibnllar structure (consisting of a predominant β-pleated sheet secondary structure), charactenstic of all amyloids, that is responsible for the neurotoxic effects Aβ has also been found to be neurotoxic in slice cultures of hippocampus (Harπgan et al, Neurobiol Aging 16:779-789, 1995) and induces nerve cell death m transgemc mice (Games et al,
Nature 373 523-527, 1995; Hsiao et al, Science 274-99-102, 1996). Injection of the Alzheimer's Aβ into rat brain also causes memory impairment and neuronal dysfunction (Flood et al, Proc Natl. Acad Sci. 88:3363-3366, 1991; Br Res. 663.271-276, 1994) Probably, the most convincing evidence that Aβ amyloid is directly involved in the pathogenesis of Alzheimer's disease comes from genetic studies. It has been discovered that the production of Aβ can result from mutations in the gene encoding, its precursor, beta- amyloid precursor protein (Van Broeckhoven et al. Science 248: 1120-1122, 1990: Murrell et al, Science 254:97-99, 1991; Haass et al, Nature Med. 1:1291-1296, 1995). The identification of mutations in the beta-amyloid precursor protein gene which causes early onset familial Alzheimer's disease is the strongest argument that amyloid is central to the pathogenetic process underlying this disease. Four reported disease-causing mutations have now been discovered which demonstrate the importance of Aβ in causing familial Alzheimer's disease (reviewed in Hardy, Nature Genet. 1:233-234, 1992). All of these studies suggest that providing a drug to reduce, eliminate or prevent fibπllar Aβ formation, deposition, accumulation and/or persistence in the brains of human patients is believed to serve as an effective therapeutic.
Hypencum perforatum (St John's Wort) St. John's Wort, whose latin name is Hypencum perforatum belongs to the family
Hypeπcaceae, which consists of eight genuses and about 350 species. The species of the Hypeπcum genus are widely distnbuted throughout the world, including more than 70 different species from India alone. Hypeπcum perforatum is native to the temperate zones of Europe and western Asia, but has become naturalized in North and South Ameπca as well as in Australia. This is a perennial plant, which by the second year of growth from seed reaches a height of about 30 inches. The many bπght yellow flowers each contain about 50 stamens, which tend to group into three loosely-defined clumps.The lance-shaped, opposite leaves with their numerous pellucid dots and dark spots of hypencin are also charactenstic. If you squeeze a fresh bud between your thumbnails, it will produce a freely exuding liquid, a bπght red, resinous pigment which contains multiple bioactive compounds.
There are two main types of active compounds in Hypeπcum perforatum- the dianthrones and the flavanoids. The activity of the whole herb is best represented by a liquid extract of the fresh or recently dπed floweπng tops. The herb yields its properties to hot water, alcohol and oil, with alcohol providing the most complete extraction. The red pigments are located in the buds and the flowers, as well as in the ldioblasts of the leaves. These pigments denote the presence of dianthrones (hypencins), a rather unstable but extremely active class of constituents. In chromatographic analysis of this herb, the fluorescent red pigments gravitate to one spot, resting above layer after layer of verticolored flavanoids. The flavanoids (hyperoside, rutin, quercitin, lsoquercitnn, luteolin, etc.) are located in the flowers and in the leaves. These flavanoids provide a slightly sedative, diuretic and anti-inflammatory effect. They work closely with the hypencins, and are a highly significant part of this whole herb The tann content of the herb is about 10%, accounting for its histoπcal use as an astπngent and antidiarrheal. The macerated oil of fresh Hypencum perforatum flowers contains hyperfonn, which provides a wound-healing and antibiotic influence (Brondz et al, Tetrahedron Lett 23, 1982). This is a compound which increases in concentration in the plant duπng the development of fruit and seeds, therefore oil extractions are best performed using herb harvested as the plant begins to mature its fruit and seed
Most extracts of Hypeπcum perforatum that are sold commercially provide a 300-mg dose that contains 0.3% hypencin, but this can vary. Many of the available prescπptions of St. John's Wort are standardized for their hypencin content, and not necessanly for the other compounds such as flavanoids. The reason most of the extracts purchased over-the-counter have 0.3% hypencin in a 300-mg dose is because they are based on a European formulation that has been used as a standard in the vanous studies conducted over the years. This formulation is called Jarsin, or LI 160
Hypeπcum extracts have been shown to cause a 50% inhibition of serotonin uptake by rat brain cells Whenever a brain impulse occurs, serotonin is released from one brain cell, influences a receptor on an adjoining cell, and then much of it is returned to the oπginal cell to be used again or degraded. Prozac, and other similar anti depressants, such as Zoloft and Paxil, work by inhibiting the re-uptake of serotonin. This is why they are referred to as serotonin re-uptake inhibitors. As a consequence of this re-uptake inhibition, more serotonin stays around to influence brain cells, and mood is elevated. Another way that pharmaceutical antidepressants work is by inhibiting an enzyme called monoamine oxidase (MAO). This enzyme degrades many bram chemicals including serotonin, norepinephπne, epinephnne and especially dopamine. By inhibiting this enzyme, most of the brain chemicals stay in our brain, leading to enhanced alertness and mood elevation. It seems that some compounds within Hypencum perforatum (probably hypencins and flavanols) also have the ability to inhibit this enzyme (Bladt and Wahner, J. Geπatr
Psychiatry Neurol 7:S57-59, 1994, Thiede and Walper, J. Geπatr. Psychiatry Neurol 7:S54-56, 1994).
After you swallow a pill of Hypeπcum perforatum, it will start becoming apparent in your bloodstream roughly within an hour or two (Kerb, Antimicrob Agents Chemother 40:2087-2093, 1996). The higher the dose, the quicker it is found in the bloodstream. It is estimated that about 20% of the hypencin is absorbed. A dose of 0.75 mg of hypencin lasts m the bloodstream for more than a day
Today, Hypeπcum perforatum is used pπmaπly for treating mild to moderate depression and related disorders (Lmde et al, British Med. J. 313:253-258, 1996).
Germany's Kom msion E (the agency that regulates herbs and other natural remedies, equivalent to the FDA's regulation of pharmaceuticals in the U.S.A.) lists the indications for the use of Hypencum perforatum in the treatment of psychovegitative disturbances, depressive states, fear and nervous disturbances. In addition, to Hypencum perforatum's use as a natural remedy in the treatment of depression, the herb has demonstrated antiviral and antibactenal capabilities. Studies indicate that hypencin, the active component of Hypeπcum perforatum, inhibits the capability of the offspnng of certain viruses to replicate (Lavie et al, Transfusion 35:392-400, 1995). Additionally, it was found that hypencin also appeared to directly inactivate the replication process of certain viruses that previously had the capability. Viruses studies include HIV (Hudson et al, Science 254:522, 1991), herpes simplex virus type I and II, Epstein-Barr virus and influenza types A and B. Hypeπcum perforatum also appears to have broad spectrum anti-microbial activity. The organisms studies include Staphylococcus aureus (Staph), Streptococcus mutans (Strep) and Eschenchia coli (E. coli). A 1994 double-blind, placebo-controlled study showed that Hypeπcum extracts gave the benefit of increased deep sleep duπng the total sleeping penod of the patients (Schulz, J. Geπatrv Psvch. Neurol. 7.S39-43, 1994)
Hypencin, one of the active agents from Hypencum perforatum, has been also shown in vanous studies to work effectively against cancerous cells and tumors of varying kinds. In 1996, Werf et al ( Lanrvngvscope 106:479-483) reported that hypencin shows great potential in targeting human cancer growths through what is called "phototargetmg", a process that uses laser activation of hypeπcm, along with chemotherapy, for improved results in inhibiting the growth of cancerous cells.
Oils denved from Hypeπcum perforatum has long been held in high esteem for treatment of all types of abrasions and wounds. The oil, which does not contain hypencin, but another valuable compound known as hyperfonn, which is mainly responsible for the oil's therapeutic properties. Though somewhat difficult to isolate and preserve for extended penods of time, hyperfonn has shown considerable promise as a pπmary component m salves or dressings for topical and other wounds. It only makes sense that in being able to withstand and inhibit bacteπal and viral growth, Hypencum perforatum can effectively aid topical wounds in their healing and recovery
Although some health care providers have suggested that Hypeπcum perforatum may be used to treat a vanety of ailments, such as those descnbed above, nowhere has there been any use, or suggestion of use, of this compound for the treatment of amyloid formation . deposition, accumulation and/or persistence, such as that which occurs in the amyloidoses, including Alzheimer's disease. The present invention clearly demonstrates the effectiveness of Hypeπcum perforatum and deπvatives thereof obtained from different commercial sources for the 1) inhibition of Alzheimer's Aβ amyloid fibnl formation (important for patients in early to mid-stage Alzheimer's disease), 2) inhibition of Alzheimer's amyloid fibπl growth
(important for patients in early to mid-stage Alzheimer's disease), 3) inhibition of Alzheimer's amyloid-PG/GAG interactions (important for patients in all stages of Alzheimer's disease) and 4) causing the dissolution/disruption of preformed Alzheimer's disease amyloid fibnls. In addition, the present invention demonstrates that Hypencum perforatum is effective in causing the dissolution of islet amyloid fibnls (le. amylin) and therefore may serve as an effective treatment for -90% of type II diabetic patients who have islet amyloid accumulation in the pancreas
The Examples illustrated below all serve well to establish that, at least in vitro, Hypeπcum perforatum has the ability to inhibit the formation of brain amyloid deposits that occur duπng normal aging and in a vaπety of bram disorders including Alzheimer's disease
In addition, it is known that patients who accumulate brain amyloid deposits eventually lose cognitive ability and memory function and sustain a marked reduction in mental clanty in general. Therefore it follows that inhibition of such bram amyloid deposits will at least promote mental alertness in such patients The Examples also establish that again, at least in vitro, Hypencum perforatum has the ability to reduce, eliminate, prevent, inhibit or disrupt dissolve amyloid fibnl or protein deposits, brain associated amyloid fibnl deposits or brain associated amyloid protein deposits, as well as amyloid fibnl formation and growth or age associated amyloid fibnl formation and growth, brain associated amyloid fibnl formation and growth, and interaction of amyloid protein with glycosaminoglycans, or with proteoglycans. In addition, it is known that patients who accumulate amyloid fibnl or protein deposits, brain associated amyloid fibnl deposits or brain associated amyloid protein deposits, or who display symptoms of amyloid fibnl formation and growth or age associated amyloid fibnl formation and growth, bram associated amyloid fibnl formation and growth, or interaction of amyloid protein with glycosaminoglycans, or with proteoglycans. in general will eventually lose mental acuity, mental alertness, concentration, cognitive well being, or some measure of brain function, or cognitive ability, mental performance or memory, or concentration and mental sharpness, or mental vitality, or mental clanty and alertness, short term memory, or some of the ability to learn or remember It is also known that such patients are subject to age associated or related cognitive or memory decline, or will sustain a marked reduction in mental clanty It follows that inhibition, reduction, elimination, prevention, disruption, or dissolution of such amyloid fibnl or protein deposits, brain associated amyloid fibnl deposits or brain associated amyloid protein deposits, or amyloid fibnl formation and growth or age associated amyloid fibnl formation and growth, brain associated amyloid fibnl formation and growth, or interaction of amyloid protein with glycosaminoglycans or with proteoglycans, will improve mental acuity, promote mental alertness, provide nutntional support for age related cognitive or memory decline, promote cognitive well being, support brain function, improve cognitive ability, mental performance or memory, promote concentration and mental sharpness. improve mental vitality, promote greater mental clanty and alertness, improve short term memory, reduce or reverse age associated cognitive or memory decline, support normal brain function, enhance learning or memory, improve concentration, enhance mental performance, reduce mental decline, reduce likelihood of age related brain disorders, and maintain good brain health, in such patients The Examples further establish that, at least in vitro, Hypencum perforatum has the ability to reduce, eliminate, prevent, inhibit or disrupt/dissolve amyloid fibnl or protein deposits, pancreas associated amyloid fibnl or protein deposits, as well as amyloid fibnl formation and growth, pancreas associated amyloid fibnl formation and growth, and pancreas interactions of amyloid protein with glycosaminoglycans or with proteoglycans In addition, it is known that patients who accumulate amyloid fibπl or protein deposits, pancreas associated amyloid fibnl or protein deposits, or who display symptoms of amyloid fibnl formation and growth, pancreas associated amyloid fibnl formation and growth, or pancreas associated interaction of amyloid protein with glycosaminoglycans or with proteoglycans, in general lose healthy pancreatic function, or sustain a reduction in normal insulin function, leading to loss or reduction of pancreatic function It therefore follows that inhibition, reduction, elimination, prevention, disruption, or dissolution of such amyloid fibnl or protein deposits, pancreas associated amyloid fibnl or protein deposits, or amyloid fibnl formation and growth, pancreas associated amyloid fibnl formation and growth, or pancreas associated interaction of amyloid protein with glycosaminoglycans or with proteoglycans. will support healthy pancreatic function and promote pancreatic function by helping to promote normal insulin function in such patients.
Examples The following examples are put forth so as to provide those with ordinary skill in the art with the disclosure and descπption of the identification and use of commercially available Hypeπcum perforatum to inhibit amyloid fibnl formation, inhibit amyloid fibnl growth, inhibit amyloid-PG/GAG interactions, and cause dissolution/disruption of preformed amyloid fibnls. However, it should not be construed that the invention is limited to these specific examples.
Example 1 Hypericum perforatum Causes a Dose-Dependent Inhibition of Alzheimer's Aβ (1-40) Amyloid Fibril Formation
A previously descnbed method of measuπng amyloid fibnl formation utilizing Thioflavin T fluorometry (H Naiki et al, Lab. Invest. 65:104-110, 1991; H Levine III, Protein Sci 2.404-410. 1993; H Levine HI. Amyloid: Int. J. Exp Clin. Invest. 2:1-6. 1995; H Naiki and K. Nakakuki. Lab Invest. 74:374-383, 1996) was employed initially to identify whether Hypeπcum perforatum was capable of inhibiting Alzheimer's Aβ 1-40 amyloid fibnl formation Using this sensitive assay, any decreases or increases in fluorescence was previously shown to correlate with a decrease or increase in the amount of amyloid fibnls (H Naiki et al. Lab Invest 65: 104-110, 1991; H Levine III. Protein Sci. 2:404-410, 1993; H Levine III. Amyloid- Int J Exp Clin Invest. 2: 1-6. 1995; H Naιkι and K Nakakuki, Lab Invest 74.374-383, 1996), allowing one to determine the identity and extent of potential inhibitors and or enhancers of amyloid fibnl formation.
In one study, the dose-dependent effects of Hypeπcum perforatum on Alzheimer's Aβ (1-40) fibπl formation was assessed by Thioflavin T fluorometry. Thioflavin T is known to bind to fibπllar amyloid proteins, and an increase in fluorescence correlates with an increase in amyloid fibnl formation, whereas a decrease in fluorescence correlates with a decrease in amyloid fibnl formation. The Alzheimer's Aβ protein (1-40) when incubated at
37°C tends to spontaneously form amyloid fibnls which increase in quantity over time. In this study, we tested for Hypeπcum perforatum's ability to inhibit the Alzheimer's amyloid Aβ protein from forming fibnls over a 1 week penod. For these studies, 300μl of 25 μM Aβ (l-40)(Bachem Inc., Torrance, CA, USA. Lot #T20824) in 150 mM TRIS, lOmM NaCl, pH 7.0 (TBS) was incubated in microcentπfuge tubes at 37°C for 1 week (in triplicate), either alone, or in the presence of increasing concentrations (i.e. O.Olμl, O.lμl, 0.5μl and 1 0 μl) of a water extract (descnbed below) obtained from the gelatin-capsule contents of a commercial source of Hypencum perforatum (Future Biotics, Lot #97V736). This commercial source of Hypencum perforatum contained 0 3% hypencin standardized extract in each capsule.
For the procedure to generate water extracts of Hypencum perforatum, 500mg of either a) standardized Hypencum perforatum obtained from the contents of gelatin-capsules of a commercial source of Hypeπcum perforatum (containing 0.3% hypencin standardized extract) or b) freeze-powdered dned whole plant (i.e. leaves, buds and flowers) of Hypencum perforatum, were extracted with 3 ml of distilled water (Baxter) and placed in microcentπfuge tubes. The microcentπfuge tube contents were then vortexed by hand for 3-4 minutes, and then allowing to stand for 1-2 minutes. The samples were then centπfuged on a microcentπfuge (Eppendorf, model 5415C) for 30 minutes at 14,000Xg (at room temperature). Following centπfugation, the supematants were collected and designated as the "water extracts". To determine dose-dependent effects of Hypeπcum perforatum, O.Olμl, O.lμl, 0.5μl and l.Oμl of the water extract was used for testing as descnbed above.
To assess the dose-dependent effects of each Hypencum perforatum on Aβ (1-40) fibnl formation, 50 μl aliquots were taken from each tube (as descnbed above) for analysis at 1 hr, 1 day, 3 days, and 1 week. For each determination descnbed above, following each incubation penod, 50μl of Aβ +/- increasing concentrations of a water extract of Hypencum perforatum were added to 1.2ml of lOOμM Thioflavin T (Sigma Chemical Co., St. Louis, MO) in 50mM NaPO4 (pH 6.0). Studies indicated that increasing concentrations of fibnlhzed Aβ gave a proportional increase m fluorescence in the presence of lOOμM
Thioflavin T, ruling out the presence of any disproportionate inner filter effects in these studies Fluorescence emission at 482 nm was measured on a Turner instrument-model 450 fluorometer at an excitation wavelength of 450 nm. For each determination, the fluorometer was calibrated by zeroing in the presence of the Thioflavin T reagent alone, and by seting the 50 ng/ml nboflavin (Sigma Chemical Co., St. Louis, Mo) in the Thioflavin T reagent to
1800 fluorescence units. All fluorescence determinations were based on these references and any fluorescence given off by any of the compounds tested in the presence of the Thioflavin T reagent was always subtracted from all pertinent readings.
For all fibπllogenesis studies utilizing Thioflavin T fluorometry, as disclosed herein. compansons of amyloid protein in the presence or absence of test compounds were based on paired Student's t tests with data shown as mean +/- standard deviation. Significance was reported at the 95% (p< 0.05), 99% (p<0.01) and 99.999% (p<0.001) confidence levels. As shown in Figure 1, the effects of vanous amounts (i.e. O.Olμl, O.lμl, 0.5μl and 1 Oμl) of Hypencum perforatum on Alzheimer's Aβ (1-40) amyloid fibnl formation was evaluated over a 1-week incubation peπod. Freshly suspended Aβ (1-40) alone, following a 1-hour incubation at 37°C, demonstrated an initial fluorescence of 126 +/- 10 fluorescence units. Dunng the 1-week incubation penod, there was a gradual increase m the fluorescence of Aβ (1-40) alone, increasing 5 4-fold from 1 hour to 3 days, with a peak fluorescence of 682 +/- 157 fluorescence units observed at 3 days (Figure 1). A significant inhibition
(p<0 001) of Aβ 1-40 amyloid fibnl formation by 0.5μl and 1 Oμl of Hypeπcum perforatum was detected as early as 1 hour of incubation. Significant dose-dependent inhibition by increasing concentrations of Hypeπcum perforatum on Aβ 1-40 amyloid fibnl formation was observed at all time points including 1 hour, 1 day, 3 days and 1 week. At 1 day, 0.5μl and l.Oμl of a water extract of Hypencum perforatum inhibited Aβ 1-40 amyloid fibnl formation by 88% and 93%, respectively At 3 days, 0.5μl and l.Oμl of a water extract of Hypencum perforatum inhibited Aβ 1-40 amyloid fibnl formation by 81% and 94%, respectively At 1 week, increasing concentrations of Hypeπcum perforatum inhibited Aβ 1-40 fibπl formation in a dose-dependent manner, such that 0 lμl, 0.5μl and 1 Oμl of a water extract of Hypencum perforatum inhibited Aβ 1-40 amyloid fibnl formation by 54%, 87% and 96%, respectively This initial data indicated that Hypeπcum perforatum was a potent inhibitor of Alzheimer's amyloid fibnl formation and exerted its effects in a dose-dependent manner.
Example 2
Hypericum perforatum Inhibits Alzheimer's Amyloid Fibril Growth
In Alzheimer's disease and other amyloidoses, amyloid fibnl growth is believed to involve amyloid protein self-interactions (le. Aβ-Aβ interactions) Any potential effective therapeutic agent for amyloid deposition, accumulation and/or persistence should also be capable of causing an inhibition of amyloid protein self-interactions. This is important for preventing any new amyloid fibnl formation when treating Alzheimer's disease patients at early stages of the disease. ELISA methodologies (i.e. solid phase binding assays) were therefore used to identify compounds which were capable of inhibiting Aβ-Aβ interactions (i.e. Alzheimer's amyloid fibnl growth)
Aβ (1-40) was first labelled with biotin according to the following protocol. 1 mg of AB (1-40) (Bachem Inc., Torrance, CA. USA, Lot #WL934) was dissolved in 200μl of PBS (pH 8.0) and incubated for 1 week at 37°C. The fibnllar Aβ solution was then added to 0.2mg of a biotinylation agent [(sulfosuccιnιmιdyl-6-(bιotιnamιdo) hexanoate)](sulfo-NHS-
LC-Biotin) and incubated for 45 minutes at room temperature (according to the manufacturer's protocol; Pierce). To remove excess sulfo-NHS-LC-Biotin not incorporated into Aβ, 25μl of 3M sodium acetate and 1 ml of ethanol were added to the solution, vortexed and then centπfuged at 14.000 Xg for 20 minutes. The supernatant was then discarded and the pellet was resuspended in 200μl of distilled water, and reprecipitated with ethanol containing 2.5% of 3M sodium acetate. The centπfugation steps (descnbed above) were then repeated. The pellet which contained fibnl zed Aβ which was biotmylated (at the non self- interacting region of Aβ) was then resuspended in 1 ml of distilled deionized water. The amount of biotin incorporated was then determined using the HABA (2-(4'-hydroxyazo- benzene)benzoιc acid) method (according to the manufacturer's protocol; Pierce).
2μg of unlabelled Aβ in 40μl of Tπs-buffered saline containing lOOmM Tπs-HCl, 50 mM NaCl, 3 mM NaN3, pH 7 0 (TBS) was allowed to bind overnight at 4°C to microtiter wells (Nunc plates, Maxιsorb).The next day all of the microtiter wells were blocked for 2 hours by incubating with 300 μl of TBS with 0.05% Tween-20 (TTBS) plus 2% bovine serum albumin (BSA)(obtaιned from the Sigma Chemical Company, St. Louis, MO, USA).
Then, 100 μl of 12.5 μM biotmylated Aβ 1-40 in TTBS, in the presence or absence of lμl of water extracts (descnbed above) were placed in wells (in tnphcate) containing substrate bound unlabelled Aβ or blank, and allowed to bind overnight at 4°C. The next day, the wells were nnsed 3 times with TTBS, and then probed for 2 hours with lOOμl of streptavidm- peroxidase or anti-biotinperoxidase (1:500 dilution of a 2μg/ml solutιon)(Sιgma Chemical
Co., St. Louis, MO) in TTBS containing 0 1% BSA. The wells were then nnsed 3 times with TTBS and lOOμl of a substrate solution (OPD-Sigma Fast from Sigma Chemical Co., St. Louis, MO) was added to each well and allowed to develop for 5 minutes or until a significant color change was observed The reaction was stopped with 50μl of 4N H2SO4 and read on a Model 450 microplate reader (Biorad, Hercules, CA, USA) at 490nm.
The compounds tested included Hypeπcum perforatum obtained from a water extract (extracted as descnbed in example 1) of freeze-powdered dπed plant mateπals (whole plant including leaves, buds and flowers; obtained fron The Herbalist, Seattle, WA, U.S.A.) or from the powdered contents of gelatin-coated capsules containing standardized Hypencum perforatum extract (i.e. 0.3% hypencin) obtained commercially (Sundown Herbals, Lot #04228 04-01)
As shown in Figure 2, Hypeπcum perforatum extract obtained from freeze-powdered dπed plant mateπals, and from a commercial source (containing 0.3% standardized extract) were effective in causing a significant reduction in Aβ-Aβ interactions. Commercially available standardized Hypeπcum perforatum extract (containing 0.3% hypencin) caused a significant (p<0.001) 88% inhibition of Aβ-Aβ interactions, whereas Hypeπcum perforatum extract obtained from freeze-powdered dπed plant mateπals caused a significant (p<0.001) 66% inhibition of Aβ-Aβ interactions. These data demonstrated that Hypeπcum perforatum was a potent inhibitor of Aβ-Aβ interactions, indicative of amyloid fibnl growth.
Example 3
Hypericum perforatum Inhibits AB-GIycosaminoglycan Interactions
One study was implemented to determine whether Hypeπcum perforatum was an effective inhibitor of Aβ-proteoglycan/glycosaminoglycan (PG/GAG) interactions. Since PGs/GAGs have been found to accumulate in amyloid deposits and are believed to prevent the body's natural ability to remove unwanted "amyloid" (reviewed in Snow and Wight,
Neurobiology Aging 10:481-497. 1989), an inhibitor of Aβ-PG/GAG interactions is a desirable additional target for an amyloid therapeutic. In this study a solid phase binding immunoassay was utilized to determine w hether Hypeπcum perforatum obtained from dπed plant mateπals or from the powdered contents of gelatin-coated capsules containing standardized Hypeπcum perforatum extract (i.e. 0.3% hypencin) obtained commercially was an effective inhibitor of Aβ-PG/GAG interactions.
12 μg of perlecan glycosaminoglycans (isolated from the Engelbreth-Holm-Swarm sarcoma as previously descπbed (Castillo et al, J. Neurochemistry 69:2452-2465, 1997) in 40μl of Tns-buffered saline containing 100 mM Tπs-HCl, 50 mM NaCl, 3 mM NaN3, pH 7.0 (TBS) was allowed to bind overnight at 4°C to microtiter wells (Nunc plates,
Maxisorb). The next day all of the microtiter wells were blocked for 2 hours by incubating with 300 μl of TBS with 0.05% Tween-20 (TTBS) plus 1% bovine serum albumin (BSA). 100 μl of Aβ 1-40 (12.5μM) (Bachem Inc., Torrance, CA, USA; Lot ##T20824)) in TTBS containing 1% albumin in the presence or absence of lμl of a water extract of Hypencum perforatum were placed in wells (in tnp cate) containing substrate bound perlecan GAGs or blank, and allowed to bind overnight at 4°C. The next day, the wells were nnsed 3 times with TTBS, and then probed for 2 hours with 100 μl of biotmylated antι-4G8 and antι-6E10 (Senetek, Maryland Heights. Missoun) diluted 1.2000 with TTBS. Bound antibodies were then probed with lOOμl of streptavidin-peroxidase or anti-biotinperoxidase (1:500 dilution of a 2μg/ml solution; Sigma Chemical Co., St. Louis, MO) in TTBS for 1 hour. The wells were then nnsed 3 times with TTBS and lOOμl of a substrate solution (OPD-Sigma Fast from Sigma Chemical Co., St. Louis, MO) was added to each well and allowed to develop for 5 minutes or until a significant color change was observed. The reaction was stopped with 50μl of 4N H2SO4 and read on a Model 450 microplate reader (Biorad, Hercules, CA,
USA) at 490nm
For the study, the compounds tested included Hypencum perforatum obtained from a water extract (extracted as descnbed in example 1) of dned plant matenals (whole plant including leaves, buds and flowers; obtained fron The Herbalist, Seattle, WA, U.S.A.) oi from the powdered contents of gelatin-coated capsules containing standardized Hypeπcum perforatum extract (i.e. 0.3% hypencin) obtained commercially (Sundown Herbals, Lot #04228 04-01).
As shown in Figure 3, Hypencum perforatum obtained from whole plant matenals significantly (p<0.001) inhibited Aβ-perlecan GAG interactions by 92%. In addition, Hypencum perforatum obtained from a standardized extract (containing 0.3% hypericin) was also a most potent inhibitor (by 97%; p<0.001) of Aβ-perlecan GAG interactions. These data demonstrated that Hypeπcum perforatum was also capable of inhibiting Aβ-PG/GAG interactions.
Example 4
Hypericum perforatum Causes a Dissolution/Disruption of Pre-Formed Alzheimer's Disease Amyloid 1-40 Fibrils in a Dose-Dependent Manner and Within a 2-Hour Period "
One study was implemented to determine whether Hypeπcum perforatum extracts were capable of causing a "dissolution" or "disruption" of pre-formed Alzheimer's disease amyloid fibnls. This type of activity would be important for any potential anti-amyloid drug which can be used in patients who already have substantial amyloid deposition in organs and/or tissues For example, Alzheimer's disease patients in mid-to late stage disease have abundant amyloid deposits in their brains as part of both neuπtic plaques and cerebrovascular amyloid deposits A natural therapeutic agent capable of causing dissolution of pre-existing amyloid would be advantageous for use in these patients who are at latter stages of the disease process
For this study, 1 mg of Aβ (l-40)(Bachem Inc., Tonance, CA, USA, Lot #T20824) was dissolved in 1 0 ml of double distilled water (1 mg/ml solution) and then incubated at 37°C for 1 week to cause abundant Alzheimer's amyloid fibnl formation. 25μM of fibnlhzed Aβ was then incubated in tnphcate for 2 hours at 37°C in a total final volume of 60μl TBS, in the absence or presence of increasing concentrations (i.e. O.Olμl, O.lμl, 0.5μl, and 1 Oμl) of Hypeπcum perforatum water extracts deπved from either freeze-powdered whole dπed plant mateπals (whole plant including leaves, buds and flowers; obtained fron The Herbalist, Seattle, WA, U.S. A ) or from the powdered contents of gelatin-coated capsules containing standardized Hypeπcum perforatum extract (i.e. 0.3% hypencin) obtained commercially (Sundown Herbals, Lot #04228 04-01) Following a 2 hour incubation, 50 μl aliquots were added to 1.2ml of lOOμM Thioflavin T (Sigma Chemical Co., St. Louis, MO) in 50mM NaPO4 (pH 6.0) for fluorometry readings as descnbed in Example 1 above.
For this study, the compounds tested included increasing concentrations of a water extract of Hypencum perforatum obtained from freeze-powdered whole dned plant matenals or from the powdered contents of gelatin-coated capsules obtained commercially, as descπbed in example 1
As shown in Figure 4, both extracts deπved from whole dned plant matenals and from a commercial source (containing 0 3% hypencin) caused a dose-dependent dissolution/disruption of pre-formed Aβ 1-40 fibnls within a 2-hour incubation peπod For example, 0 5 μl and 1 Oμl of water extracts deπved from a commercial standardized extract of Hypeπcum perforatum caused a significant (p<0.001) 77% and 83% dissolution/disruption of Aβ 1-40 amyloid fibnls, respectively. Similarly, 0.5μl and 1 Oul of a water extract obtained from freeze-powdered whole dπed plant matenals caused a significant (p<0.001) 68% and 73% dissolution/disruption of Aβ 1-40 amyloid fibnls, respectively On the other hand, 0 Olμl of either water extracts from Hypencum perforatum did not cause a significant dissolution/ disruption of pre-formed Aβ 1-40 amyloid fibnls These data demonstrated that Hypeπcum perforatum causes dissolution of pre-formed Alzheimer's disease amyloid fibnls in a dose-dependent manner. Confirmation of the "dissolution effect" of Hypeπcum perforatum on Alzheimer's disease Aβ 1-40 fibnls was demonstrated by Congo red staining assays, whereby a marked reduction of congophi a (1 e red/green birefπngence v» hen viewed under polanzed light, and which represents a dissolution/disruption of the amyloid fibπllar structure) was observed when Aβ amyloid fibnls were treated with Hypeπcum perforatum (from either source) for 2 hours (not shown)
Example 5
Hypericum perforatum Causes a Dissolution/Disruption of Aβ (1-42) Alzheimer's Amyloid Fibrils
The amyloid fibnls of Alzheimer's disease pπmaπly consist of Aβ in a form containing residues 1-40 or 1-42 The longer vanant of Aβ contains two hydrophobic residues which cause substantial fibnl formation almost immediately (Castillo et al, J Neurochem 69 2452-2465, 1997) Aβ 1-42 is also believed to be the predominant form of Aβ existing in Alzheimer's amyloid plaques, whereas Aβ 1-40 is believed to be the predominant form of Aβ existing in Alzheimer's cerebrovascular amyloid deposits (Tamaoka et al, Br Res 679 151-156, 1995, Biochem Biophvs Res Comm 205 834-842, 1994) The next study was therefore implemented to determine whether Hypencum perforatum also causes dissolution/disruption of pre-formed Aβ (1-42) amyloid fibnls and whether this effect was long-lasting
For this study, the method of Thioflavin T fluorometry as descnbed in example 1 was used Bnefly, 60 μl of 25 μM of Aβ (l-42)(Bachem Inc, Torrance, CA, USA, Lot # 516817) in TBS (pH 7 0) either alone, or containing increasing amounts (I e 0 Olμl, 0 lμl, 0,5μl, and 1 Oμl) of Hypeπcum perforatum water extracts were incubated in microcentπfuge tubes at 37°C for 48 hours (in tnphcate)
For this study, the compounds tested included increasing concentrations of a water extract of Hypencum perforatum obtained from freeze-powdered whole dπed plant mateπals (leaves, buds and flowers obtained from The Herbalist, Seattle, WA, U S A ) or from the powdered contents of gelatin-coated capsules containing standardized extract (I e 0 3% hypencin) and obtained commercially (Sundown Herbals, Lot #04228 04-01) Water extracts were prepared as descπbed in example 1
As shown Figure 5, Alzheimer s Aβ (1-42) alone, following a 2 hour incubation at 37°C, demonstrated an initial fluorescence of 1370 +/- 97 fluorescence units Both water extracts of Hypencum perforatum deπved from either whole dπed plant mateπals or from a commercial source (containing 0.3% hypencin) caused a dose-dependent dissolution/disruption of pre-formed Aβ 1-42 fibnls withm a 2 hour incubation penod. For example, 0 5 μl and l.Oμl of the commercial standardized extract of Hypencum perforatum caused a significant (p<0.01) 46% and 72% (p<0.001) dissolution/ disruption of Aβ 1-42 amyloid fibnls, respectively 0.5μl and l.Oul of a water extract obtained from freeze- powdered whole dπed plant mateπals caused a significant (p<0.05) 24% and 55% dissolution/disruption of Aβ 1-42 amyloid fibnls, respectively. On the other hand, O.lμl and O.Olμl of either water extracts from Hypeπcum perforatum did not cause a significant dissolution/ disruption of pre-formed Aβ 1-42 amyloid fibnls. These data demonstrated that Hypeπcum perforatum causes dissolution of pre-formed Alzheimer's disease amyloid fibnls in a dose-dependent manner. Confirmation of the "dissolution effect" of Hypencum perforatum on Alzheimer's disease Aβ 1-42 fibnls was demonstrated by Congo red staining assays, whereby a marked reduction of congophiha (i.e. red/green birefnngence when viewed under polanzed light, and which represents a dissolution/disruption of the amyloid fibnllar structure) was observed when Aβ amyloid fibnls were treated with Hypencum perforatum (from either source) for 2 hours (not shown).
Example 6
Hypericum perforatum Causes Dissolution/Disruption of Islet Amyloid
Fibrils (Amylin)
90% of patients with type II diabetes demonstrate the deposition and accumulation of amyloid fibnls in the islets of Langerhans in the pancreas (Cooper et al, Proc Natl Acad. Sci USA 84.8628-8632, 1987) This amyloid protein involved consists of a 37 amino acid protein known as islet amyloid polypeptide or amylin. Islet amyloid is believed to contπbute to the destruction of the beta-cells of the pancreas, thus eventually leading many patients to become insulin-dependent (le. type I diabetes). Amylin has the ability to also form substantial amyloid fibnls immediately when placed m solution.The next study was therefore implemented to determine whether Hypeπcum perforatum also causes dissolution/disruption of another type of amyloidosis, and whether this effect was also long-lasting.
For this study, the method of Thioflavin T fluorometry as descπbed in Example 5 was used. Bnefly, 60 μl (final volume) of 25 μM of human amylin (Bachem Inc,Torrance, CA, USA, Lot # WL934) in TBS (pH 7 0) was incubated in microcentπfuge tubes at 37°C for 2 days (in tnphcate), either alone, or in the presence of increasing amounts (i.e. O.Olμl, O.lμl, 0.5μl and l.Oμl) of Hypencum perforatum, water extracts
For this study, the compounds tested included increasing concentrations of a water extract of Hypencum perforatum obtained from freeze-powdered whole dned plant mateπals (leaves, buds and flowers obtained from The Herbalist, Seattle, WA, U.S.A.) or from the powdered contents of gelatm-coated capsules containing standardized extract (i.e. 0.3% hypencin) and obtained commercially (Sundown Herbals, Lot #04228 04-01). Water extracts were prepared as descnbed in example 1.
As shown Figure 6, freshly suspended amylin alone, following a 2-hour incubation at 37°C, demonstrated an initial fluorescence of 1367 +/- 12 fluorescence units Both water extracts of Hypencum perforatum deπved from either freeze-powdered whole dπed plant mateπals or from a commercial source (containing 0.3% hypencin) caused a dose-dependent dissolution/disruption of islet amyloid (i.e. amylin or islet amyloid polypeptide) fibnls within a 2 hour incubation peπod. For example, O.lμl, 0.5 μl and l.Oμl of the commercial standardized extract of Hypeπcum perforatum caused a significant (p<0.001) 67%, 91% and 92% dissolution/ disruption of amylm amyloid fibrils, respectively. Similarly, O.lμl, 0.5μl and l.Oul of a water extract obtained from freeze- powdered whole dπed plant matenals caused a significant (p<0.001) 54%, 68% and 73% dissolution/disruption of amylin amyloid fibnls, respectively. These data demonstrated that Hypencum perforatum causes dissolution/ disruption of pre-formed islet amyloid (i.e. amylin) fibnls in a dose-dependent manner. Confirmation of the "dissolution effect" of
Hypeπcum perforatum on amylin fibnls was demonstrated by Congo red staining assays, whereby a marked reduction of congophiha (i.e. red/green birefnngence when viewed under polanzed light, and which represents a dissolution/disruption of the amyloid fibnllar structure) was observed when amylin amyloid fibnls were treated with Hypencum perforatum (from either source) for 2 hours (not shown) This study demonstrated that
Hypeπcum perforatum is capable of causing significant dissolution/disruption of other forms of amyloid (such as islet amyloidosis)
Further Aspects and Utilizations of the Invention
Therapeutic Applications
One embodiment of the present invention is to formulate pπor to administration in a patient, a pharmaceutical formulation compπsing Hypeπcum perforatum (and or its active ingredients) in one or more pharmaceutical acceptable earners, diluents or excipients. In a preferred embodiment, a patient who has Alzheimer's disease, type II diabetes or any other amyloidosis, would orally consume commercially available Hypencum perforatum m pill, tablet, caplet, soft and hard gelatin capsule, lozenge, vegicap, liquid drop, solution, syrup, tea bag, and/or powder form. In another preferred embodiment Hypencum perforatum obtained commercially in any form could be further modulated using suitable earners, excipients and diluents including lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, algmates, tragacanth, gelatin, calcium silicate, microcrystallme cellulose, polyvinylpyrrohdone, cellulose, water syrup, methyl cellulose, methyl and propylhydroxybenzoates, talc, magnesium stearate and mineral oil. The formulations can additionally include lubπcating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweeting agents or flavonng agents The compositions of the invention may be formulated so as to provide quick, sustained or delayed response of the active ingredient after administration to the patient. The compositions are preferably formulated m a unit dosage form, each dosage containing from about 1 to about 1000 mg of Hypencum perforatum (or its active ingredients), more usually about 400 to about 750 mg of Hypeπcum perforatum (or its active ingredients). However, it will be understood that the therapeutic dosage administered will be determined by the physician m the light of the relevant circumstances including the clinical condition to be treated, the organ or tissues affected or suspected to be affected with amyloid accumulation, and the chosen route of administration
Therefore, the above dosage ranges are not intended to limit the scope of the invention in any way. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active matenal calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical earner
The following formulation examples are illustrative only and are not intended to limit the scope of the invention in any way. For each formulation provided as an example, loweπng or raising of the Hypeπcum perforatum (or its active ingredients) concentration will cause a proportional loweπng or raising of the other ingredients as indicated. Hard gelatin capsules may be prepared by using 500mg of Hypeπcum perforatum (or its active ingredients), 400mg of starch, and 20 mg of magnesium stearate. The above ingredients are mixed and filled into hard gelatin capsules in 920mg quantities.
A tablet is prepared by using 500 mg of Hypeπcum perforatum (or its active ingredients), 800 mg of microcrystallme cellulose, 20 mg of fumed silicon dioxide and 10 mg of steaπc acid. The components are blended and compressed to form tablets each weighing 1230mg
An aerosol solution is prepared by using 0.25 active ingredient, 29.75 ethanol, and 70 of propellent 22 (chlorodifluoromethane). The Hypeπcum perforatum (or its active ingredients) is mixed with ethanol The mixture is added to a portion of the Propellent 22, cooled to -30°C, and transferred to a filling device. The required amount is then fed to a stainless steel container and diluted with the remainder of the propellent. The value units (listed above) are then fitted to the container. Such an aerosol form of Hypeπcum perforatum (or its active ingredients) may be useful for the treatment of amyloids involving the brain (such as Alzheimer's disease. Down's syndrome, pπon diseases etc) by using an aerosol or nasal spray. Previous studies have suggested that in these central nervous system amyloidoses the initial form of entry of a possible environmental agent which may be playing a role in pathogenesis may be denved from the outside world through the nasal passages.
Tablets are made by using 120 mg of Hypencum perforatum (or its active ingredients), 90 mg of starch. 70mg of microcrystallme cellulose, 8 mg of polyvmylpynolidone (as 10% in water), 9 mg of sodium carboxymethyl starch, 1 mg of magnesium stearate and 1 mg of talc (total = 300mg). Hypeπcum perforatum (or its active ingredients), starch and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly The solution of polyvmylpynolidone is mixed with the resultant powders which are then passed through a No 14 mesh U.S. sieve. The granules so produced are dπed at
50°C and passed through a No 18 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate and talc, previously passed through a No. 60 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 300 mg Capsules each containing 160mg of medicant are made by using 160mg of
Hypeπcum perforatum (or its active ingredients), 118mg of starch, 118mg of microcrystallme cellulose, and 4 mg of magnesium stearate (total = 400mg). The Hypencum perforatum (or its active ingredients), cellulose, starch and magnesium stearate are blended, passed through a No. 45 mesh U.S sieve, and filled into hard gelatin capsules in 400 mg quantities.
Suppositones each containing 225 mg of Hypeπcum perforatum (or its active ingredients) are made by using 225 mg of Hypeπcum perforatum (or its active ingredients), 2,000mg of saturated fatty acid glyceπdes (total =2,225mg). The Hypencum perforatum (or its active ingredients) are passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glyceπdes previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2 g capacity and allowed to cool.
Suspensions each containing 50 mg of medicant per 5 ml dose are made by using 50mg of Hypeπcum perforatum (or its active ingredients), 50 mg of sodium carboxymethyl cellulose, 1.25ml of syrup, 0 10ml of benzoic acid solution, flavor, color, and puπfied water to total 5 ml. The medicant is passed though a No. 45 mesh U.S. sieve and mixed with the sodium carboxymethyl cellulose and syrup to form a smooth paste. The benzoic acid solution, flavor and color are diluted with some of the water and added, with stirnng. Sufficient water is then added to produce the required volume. An intravenous formulation is prepared by using 250mg of Hypencum perforatum
(or its active ingredients), and lOOOmg of isotonic saline. The solution of the above ingredients is administered intravenously at a rate of 1ml per minute to a subject in need of treatment
In a preferred embodiment the therapeutic compound of the invention can be administered in any pharmaceutically acceptable vehicle. As used herein "pharmaceutically acceptable vehicle" includes, but is not limited to, any and all solvents, stenle liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic oπgin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, dispersion media, coatings, antibacteπal and antifungal agents, isotonic and adsorption delaying agents, and the like which are compatible with the activity of the compound and are physiologically acceptable to the subject. An example of a pharmaceutically acceptable vehicle is buffered normal saline (0.15 molar NaCl). The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active compounds can also be incorporated into the compositions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, πce, fluor, chalk, silica gel. magnesium carbonate, magnesium stearate, sodium stearate. glycerol monostearate. talc, sodium chlonde, dπed sk m milk, glycerol, propylene, glycol, water, ethanol and the hke.These compositions can take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained-release formulations and the like. In the methods of the invention, amyloid formation, deposition, accumulation and/or persistence in a subject is inhibited by administrating Hypencum perforatum (or its active ingredients) in a therapeutic dosage to the subject. The term subject is intended to include living organisms in which amyloidosis can occur. Examples of subjects include humans, monkeys, cows, dogs, sheep, cats, mice. rats, and transgenic species thereof Administration of the compositions of the present invention to a subject to be treated can be earned out using known procedures, at dosages and for penods of time effective to inhibit amyloidosis in the subject An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the amount of amyloid already deposited at the organ or tissue site in the subject, the age, sex and weight of the subject, and the ability of the therapeutic compound to inhibit amyloid formation, deposition, accumulation, persistence, and/or to cause dissolution of pre-formed amyloid in the subject Dosage regimens can therefore be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the needs of the therapeutic situation A non- mitmg example of an effective dose range for Hypencum perforatum (or its active ingredients) is between 400 and lOOOmg/kg of body weight/per day
Different modes of delivery of Hypencum perforatum (or its active ingredients) may be used Accordingly, a prefened route of administration is oral administration Alternatively, Hypencum perforatum (or its active ingredients) may be administered by other suitable routes such as subcutaneous, intravenous, mtrapentoneal, all routes administered by injection. Depending on the route of administration, the active compound may be coated m a matenal to protect the compound from the action of acids and other natural conditions which may inactivate the compound To administer Hypencum perforatum (or its active ingredients), it may be necessary to coat the compound with, or co-administer the compound with, a mateπal to prevent its activation For example, the therapeutic compound may be administered to a subject in an appropπate earner, for example, liposomes or a diluent Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes
The Hypeπcum perforatum (or its active ingredients) may also be administered parenterally or intrapentoneally. Dispersions can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms Pharmaceutical compositions suitable for injectable use include steπle aqueous solutions or dispersions and steπle powders for the preparation of steπle injectable solutions or dispersion. In all cases, the composition must be stenle and must be fluid to the extent that easy use in the synnge exists It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, prabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
Sterile injectable solutions can be prepared by incorporating the therapeutic compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the therapeutic compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the therapeutic agent plus any desired ingredients from a previously sterile-filtered solution thereof.
The Hypericum perforatum (or its active ingredients) for Alzheimer's disease and other central nervous system amyloidoses may be optimized to cross the blood-brain barrier. Methods of introductions include but are not limited to systemic administration, parenteral administration i.e., via an intraperitoneal, intravenous, perioral, subcutaneous, intramuscular, intraarterial, intradermal, intramuscular, intranasal, epidural and oral routes. In a prefened embodiment, Hypericum perforatum (or its active ingredients) may be directly administered to the cerebrospinal fluid by intraventricular injection. In a specific embodiment, it may be desirable to administer Hypericum perforatum (or its active ingredients) locally to the area or tissue in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, by injection, by infusion using a cannulae with osmotic pump, by means of a catheter, by means of a suppository, or by means of an implant.
In yet another embodiment Hypericum perforatum (or its active ingredients) may be delivered in a controlled release system, such as an osmotic pump. In yet another embodiment, a controlled release system can be placed in proximity to the therapeutic target, ie. the brain, thus requiring only a fraction of the systemic dose.
With regards to systems and components above refened to, but not otherwise specified or described in detail herein, the workings and specifications of such systems and components and the manner in which they may be made or assembled or used, both cooperatively with each other and with the other elements of the invention described herein to effect the purposes herein disclosed, are all believed to be well within the knowledge of those skilled in the art. No concerted attempt to repeat here what is generally known to the artisan has therefore been made.
INDUSTRIAL APPLICABILITY
Use of extracts from Hypericum perforatum, and use of the ingredients contained within the various commercial preparations of Hypericum perforatum, benefit human patients with Alzheimer's disease and other amyloidoses due to Hypericum perforatum's newly discovered ability to inhibit amyloid fibril formation, inhibit amyloid fibril growth, inhibit amyloid-proteoglycan interactions, inhibit amyloid-glycosaminoglycan interactions, and cause dissolution and/or disruption of preformed amyloid fibrils. In compliance with the statute, the invention has been described in language more or less specific as to structural features. It is to be understood, however, that the invention is not limited to the specific features shown, since the means and construction show comprise preferred forms of putting the invention into effect. The invention is, therefore, claimed in any of its forms or modifications within the legitimate and valid scope of the appended claims, appropriately interpreted in accordance with the doctrine of equivalents.

Claims

We claim -
1. A pharmacological agent for promoting, maintaining or enhancing in a patient one or more of the mental or cognitive qualities selected from the group of mental or cognitive qualities consisting of mental acuity, mental alertness, cognitive well being, normal bram function, cognitive ability, mental performance, memory, concentration, mental sharpness, mental vitality, mental clarity, short-term memory, normal bram function, and learning, and good bram health, wherein the pharmacological agent comprises a therapeutically effective amount of plant matter from a plant ofthe genus Hypericum, species perforatum.
2. The pharmacological agent of claim 1 wherein the selected mental or cognitive quality is mental alertness.
3. The pharmacological agent of claim 1 wherein the plant matter comprises an extract from the leaves, flowers or buds of Hypericum perforatum. 4. A pharmacological agent for providing, supporting or improving in a patient one or more ofthe mental or cognitive qualities selected from the group of mental or cognitive qualities consisting of nutritional support for age related cognitive or memory decline, normal bram function, cognitive ability, and concentration, wherein the pharmacological agent comprises a therapeutically effective amount of plant matter from a plant of the genus Hypeπcum, species perforatum
5. A pharmacological agent for reducing in a patient one or more of the mental or cognitive effects selected from the group of mental or cognitive effects consistmg of, age associated cognitive or memory decline, mental decline, and likelihood of age related bram or cognitive disorders, wherein the pharmacological agent comprises a therapeutically effective amount of plant matter from a plant ofthe genus Hypericum, species perforatum 37 6 A pharmacological agent for reducing, disrupting, dissolving, inhibiting or preventing m a patient one or more conditions involving the bram selected from the group of conditions involving the bram consistmg of amyloid fibril deposits, amyloid protein deposits, bram associated amyloid fibril deposits, AJ3 bram deposits, bram associated Aβ deposits, bram associated amyloid protein deposits, bram amyloid deposits, amyloid fibril formation and growth, age associated amyloid fibril formation and growth, interaction of amyloid protein with glycosaminoglycans, and interaction of amyloid protein with proteoglycans, wherein the pharmacological agent compπses a therapeutically effective amount of plant matter from a plant of the genus Hypericum, species perforatum
7. The pharmacological agent of claim 1 wherein the selected condition involving the brain is brain amyloid deposits
8 The pharmacological agent of claim 1 where said pharmacological agent is derived from the group comprising commercially available pills, tablets, • aplets, soft and hard gelatm capsules, lozenges, sachets, vegicaps, liquid drops, elixers, suspensions, emulsions, solutions, syrups, tea bags, aerosol (as a solid or in a liquid medium), suppositories, steπle injectable solutions, sterile packaged powders, dried leaves, dried buds, dried flowers, which contain plant matter from a plant ofthe genus Hypericum, species perforatum 9 The pharmacological agent of claim 1 wherein the pharmacological agent is an amyloid inhibitory ingredient selected from the group consisting of flavanoids, xanthones, proanthocyanidins, dianthrones, tannms, monoterpenes, hyperoside, biapigenm, rutm, quercetin, quercitin, isoquercitrin, pseudohypericin, hypericin, hyperforin, procyanidines, amentoflavine, luteolin, pectm, Vitamin A and Vitamin C
10. The pharmacological agent of claim 1 whereby a therapeutically effective dosage is in the range of from about 10 to 1,000 mg/kg of body weight, but more preferably m the range of about 10 to lOOmg/kg of body weight of the patient.
11. The pharmacological agent of claim 1 whereby a therapeutically effective dosage is used to treat a patient with an amyloid disease.
12 The amyloid disease of claim 11, whereby said amyloid disease is selected from the group consisting
Figure imgf000041_0001
syndrome and hereditary cerebral amyloidosis ofthe Dutch type (wherein the specific amyloid is referred to as beta-amyloid protein or AJ3), the amyloid associated with chronic inflammation, various forms of malignancy and Familial Mediterranean Fever (wherein the specific amyloid is referred to as AN amyloid or inflammation-associated amyloidosis), the amyloid associated with multiple myeloma and other B-cell dyscrasias (wherein the specific amyloid is referred to as AL amyloid), the amyloid associated with type II diabetes (wherein the amyloid protein is referred to as amylm or islet amyloid polypeptide), the amyloid associated with pπon diseases including Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, kuru and animal scrapie (wherein the specific amyloid is referred to as PrP amyloid), the amyloid associated with long-term hemodialysis and carpal tunnel syndrome (wherein the specific amyloid is referred to as beta2-mιcroglobulm amyloid), the amyloid associated with senile cardiac amyloid and Familial Amyloidotic Polyneuropathy (wherein the specific amyloid is referred to as transthyretin or prealbumin), and the amyloid associated with endocrine tumors such as medullary carcinoma of the thyroid (wherein the specific amyloid is referred to as variants of procalcitonm)
13. A pharmacological agent for promoting or supporting healthy pancreatic function in a patient, by helping to promote normal insulin function, wherein the pharmacological agent comprises a therapeutically effective amount of plant matter from a plant of the genus Hypericum, species perforatum.
14. A pharmacological agent for reducing, disrupting, dissolving, inhibiting or eliminating or preventing in a patient one or more conditions involving the pancreas selected from the group of conditions involving the pancreas consistmg of amyloid fibril deposits, amyloid protein deposits, pancreas associated amyloid fibπl deposits, amylin deposits, islet amyloid polypeptide deposits, pancreas associated amyloid protein deposits, amyloid fibril formation and growth, pancreas associated amyloid fibril formation and growth, interaction of amyloid protein with glycosaminoglycans, and interaction of amyloid protein with proteoglycans, wherein the pharmacological agents comprises a therapeutically effective amount of plant matter from a plant ofthe genus Hypericum, species perforatum.
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