Classifications

• • 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
  • A61K36/02—Algae
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

• This invention relates generally to methods and compositions for treating human diseases, disorders, and conditions using a preparation of a phyto-percolate isolated from a complex mixture of fresh water algae and other microorganisms.
• Enzymes have a very important use within biochemical cycles in the human body.
• proteolytic enzymes are responsible for the body's detoxification processes. As humans age and chronic disease processes progress, a deficiency of the proteolytic enzymes that carry out the body's waste detoxification processes may be experienced. This enzymatic deficiency aids in the production of a chronic hyper-inflammatory state, and the disease process becomes much more complex.
• Enzymes are the catalysts that control and direct all metabolic processes. Without adequate enzymes in the body, chaos reigns and the immune system and other metabolic processes become less efficient, making tissue repair slow and poorly replicated.
• Proteolytic enzymes, or proteases are enzymes capable of breaking down proteins by cleaving peptide bonds. They are produced and utilized by every living organism on Earth for protection, nutrient breakdown and assimilation, and waste removal. Many degenerative diseases stem from proteolytic enzyme deficiencies, leading to the inadequate removal of carcinogenic wastes from the body.
• the immune system which helps protect us from diseases including cancer, cardiovascular disease, and other immune deficient or deregulated disorders, can become ineffective because of advanced disease state or age.
• Immune deficiency caused by disease state or advancing age can impair benefits received from the use of therapeutic drugs that may be taken for the treatment of these various disorders.
• Therapeutic drugs may lose their effectiveness in a compromised immune system as a disease state progresses due to metabolic dysfunction or poor therapeutic drug assimilation.
• humans experience an increasing accumulation of environmental influences that are believed to have toxic effects on the. human body.
• An observed effect associated with aging is a less accurate tissue repair process, possibly an expression of DNA mutations caused by environmental factors.
• Tissue destruction also activates the body's coagulation, or blood-clotting, mechanism, generating a barrage of intra-vascular thrombi, or blood clots, and blood-thickening fibrin, that can precipitate strokes, heart attacks, pulmonary emboli, kidney damage, and phlebitis.
• the resulting pathological agents secondary to this influence of white cell activity create an ongoing destructive pattern upon local surrounding tissue, the endothelial cells that line the vascular bed, and the epithelial cells lining the intestinal tract. Not only is there destructive activity upon the above-mentioned tissues but also there is oxidative breakdown or pathological activation of the coagulation factors.
• This soluble fibrin not only negatively influences general capillary circulation but also kidney filtration, oxygen exchange within the alveoli of the lungs, and oxygenation of brain tissue. It not only thickens the blood, but is in itself an oxidative free radical, and contributes to the degenerative oxidation process.
• Much of the expressed symptomatology from the production of soluble fibrin is caused by gram-negative bacteria, mycoplasma and Candida albicans, which are allowed to flourish in the immune-compromised environment created by excess wastes and fibrin, and is related to the cellular destruction and by-products of ongoing free radical activity. Fibrinolytic activity, or the process of breaking down fibrin, along with the eradication of the foreign pathological agents by other therapeutic interventions, can lead to increasingly effective immune system and white cell activity, and will greatly accelerate the healing process.
• fibrinolytic agents will increase immune regulation and the effectiveness of white cell activity, improve capillary circulation and nutrient flow to the body's organs, aid in eliminating toxins, and enhance the benefits of other therapeutic agents.
• fibrinolytic agents will reduce the amount of free radical soluble fibrin that accelerates degenerative oxidation, and can increase the body's immune effectiveness in combating cancer growth.
• the invention provides a method for treating or preventing a disorder in a mammal (e.g., human, dog, cat, horse, etc.) by administering to the mammal a therapeutically effective amount of phyto-percolate or derivative thereof.
• a mammal e.g., human, dog, cat, horse, etc.
• the phyto-percolate derivative is a protein having a molecular weight of about 67.5 kDa, a protein having a molecular weight of about 21.0 kDa, or a polysaccharide.
• the phyto-percolate derivative has fibrinolytic enzymatic activity.
• the phyto-percolate derivative may be isolated from the phyto-percolate or it may be produced by any appropriate method known in the art. Suitable methods for producing the phyto-percolate derivative include, for example, recombinantly expressing the derivative (e.g., protein) by a microorganism and synthetically producing a derivative (i.e., chemical (cell-free) synthesis).
• the recombinant microorganism may be one or more of the species present in ATCC Deposit #PTA-5863, or it may be any other appropriate specie.
• a particular dosage is between about 1 and about 8 ounces per day of the phyto-percolate. Particularly noted is a dosage of about 1 to about 4 ounces per day.
• the phyto-percolate that is administered to the human contains between about 10 ppm and about 150 ppm of a phyto-percolate derivative.
• a therapeutically effective amount of one or more of the derivatives is administered to the human.
• the mammal is administered between about 1 mg and 1000 mg of the derivative per day.
• Suitable methods for administration of the phyto- percolate include oral administration.
• Suitable methods for administration of a phyto- percolate derivative include, for example, oral, topical, rectal, or vaginal administration as well as intravenous, intramuscular, and subcutaneous injection.
• Another aspect of this invention is directed to a method of treating an overweight condition or obesity comprising administering to the mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• Another aspect of this invention is directed to a method for treating type I and TI diabetes comprising administering to the mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• Another aspect of this invention is directed to a method for treating an inflammatory disorder comprising administering to the mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof. It is believed that the phyto-percolate and derivatives have broad spectrum anti-inflammatory properties and therefore may be used to reduce or prevent inflammation in a wide range of diseases and disorders.
• Another aspect of this invention is directed to a method for treating a stomach disorder comprising administering to the mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• Stomach disorders amenable to treatment with the phyto-percolate and/or derivatives thereof include, for example, a stomach ulcer and gastric reflux disease.
• the phyto-percolate or derivatives may be used to alleviate side-effects of another primary therapy.
• the phyto-percolate may be administered to reduce the oxidative stress, chemotherapy-induced nausea, chemotherapy-induced liver damage, appetite suppression, hair loss, fingernail and toenail loss and discoloration that result from anti-AIDS therapy and anti-cancer therapy (e.g., chemotherapy and radiation therapy).
• the phyto-percolate or derivatives may be used to reduce the recovery time in mammals (e.g., humans and horses) after periods of stress (e.g., exercise).
• the phyto-percolate or derivatives are administered in order to restore physical energy and mental acuity following periods of stress.
• the phyto-percolate or derivates may also be administered topically directly to the eye (e.g., in the form of eye drops) to treat lesions of the cornea, dry eyes, and similar ocular disorders.
• Another aspect of this invention is directed to a method for treating conditions or disorders associated with infectious disease (e.g., a viral infection) comprising administering to the mammal (e.g., human) a therapeutically effective amount of a phyto- percolate or derivative thereof.
• Infectious disease may be the cause of many of the above and below listed diseases such as pneumonia, all viruses, acariosis, acne, adenovirus, AIDS, amebiasis, anthrax, athlete's food, babesiosis, bartonellosis, Bell's palsy, botulism, candidiasis, carbuncles, Chaga's disease, chicken pox, Chlamydia, coccidiomycosis, coronavirus, cryptococcosis, cytomegalovirus, Dengue fever, echovirus, erysipelas, furuncle, gangrene, Guillan-Barre syndrome, hepatitis, impetigo, influenza, leucopen ⁇ a, Lyme's disease, malaria, martolditis, measles, mumps, mycobacterium.
• diseases such as pneumonia, all viruses, acariosis, acne, adenovirus, AIDS, amebiasis, anth
• Another aspect of this invention is directed to a method for treating diseases related to the heart, blood vessels, renal, liver, and endocrine system comprising administering a therapeutically effective amount of a phyto-percolate or derivative thereof.
• Another aspect of this invention is directed to a method for treating a vasospasm comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• a mammal e.g., human
• Another aspect of this invention is directed to a method for treating heart failure comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• a mammal e.g., human
• Another aspect of this invention is directed to a method for treating cardiac hypertrophy comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• a mammal e.g., human
• Another aspect of this invention is directed to a method for treating dysregulated blood pressure comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• Another aspect of this invention is directed to a method for treating angina comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• Another aspect of this invention is directed to a method for treating peripheral vascular disease comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• a mammal e.g., human
• Another aspect of this invention is directed to a method for treating cerebral diseases and diseases related to the central nervous system that are vascular in origin comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• Another aspect of this invention is directed to a method for treating neuro- degeneration comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• Another aspect of this invention is directed to a method for treating Alzheimer's disease comprising administering to a mammal (e.g., human) a therapeutically effective amount of a compound of a phyto-percolate or derivative thereof.
• Another aspect of this invention is directed to a method for treating depression comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• a mammal e.g., human
• Another aspect of this invention is directed to a method for treating addiction, including drug detoxification and/or substance abuse including nicotine, cocaine and alcohol abuse comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• a mammal e.g., human
• Another aspect of this invention is directed to a method for treating attention deficit disorder and attention deficit hyperactivity disorder comprising administering to a mammal (e.g., human) a therapeutically effective amount of a compound of a phyto-percolate or derivative thereof.
• Another aspect of this invention is directed to a method for treating sleep disorders comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• Another aspect of this invention is directed to a method for treating seasonal affective disorder comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• a mammal e.g., human
• Another aspect of this invention is directed to a method for treating environmental and food allergies comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• a mammal e.g., human
• Another aspect of this invention is directed to a method for treating conditions related to pain or nocioception comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• a mammal e.g., human
• Another aspect of this invention is directed to a method for treating migraine comprising administering to a mammal (e.g., human) a therapeutically effective amount of a compound of a phyto-percolate or derivative thereof.
• Another aspect of this invention is directed to a method for treating disorders related to disruption of circadian rhythms including jet lag comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• Another aspect of this invention is directed to a method for treating diseases related to abnormal gastrointestinal motility, secretion, and/or function comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• a mammal e.g., human
• Another aspect of this invention is directed to a method for treating diarrhea and/or incontinence comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• Another aspect of this invention is directed to a method for treating a gastric ulcer comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• Another aspect of this invention is directed to a method for treating irritable bowel syndrome comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• a mammal e.g., human
• Another aspect of this invention is directed to a method for treating inflammatory bowel disease comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• a mammal e.g., human
• Another aspect of this invention is directed to a method for treating nausea comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• a mammal e.g., human
• Another aspect of this invention is directed to a method for treating sexual dysfunction comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• Another aspect of this invention is directed to a method for altering fertility comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate or derivative thereof.
• Another aspect of this invention is directed to a method for treating conditions or disorders associated with the immune system comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate.
• Immune system deficiency may be the cause of many of the above and below listed diseases such as cancer, emphysema, encephalitis, environmental sensitivity, erysipelas, food poisoning and Reynaud's disease.
• Another aspect of this invention is directed to a method for treating conditions or disorders associated with hormonal imbalances comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate.
• Hormonal imbalances may be the cause of many of the above and below listed diseases such as acne, Addison's disease, endometriosis, Grave's disease, osteoporosis, menstrual and menopausal regulation, glucose, and other metabolic regulation.
• the phyto- percolate and derivatives may be used to improve the general health and overall function of metabolic organs like the kidney, liver, and pancreas. It is believed that the phyto- percolate and derivatives improve the efficiency of those organs and increases their metabolic and endocrine functions.
• Another aspect of this invention is directed to a method for treating conditions or disorders associated with neurological deficiencies comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate.
• Neurological deficiencies may be the cause of many of the above and below listed diseases such as Lou Gehrig's disease, chronic pain, Huntingdon's Chorea, diabetic neuropathy, multiple sclerosis, Myasthenia Gravis, Parkinson's disease, poliomyelitis, senile dementia, nigrostriatal degeneration, stroke, tardive dyskinesia and tinnitus.
• Another aspect of this invention is directed to a method for treating respiratory diseases comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate.
• Another aspect of this invention is directed to a method for treating asthma comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate.
• a mammal e.g., human
• Another aspect of this invention is directed to a method for treating diseases related to abnormal hormone release and utilization comprising administering to a mammal (e.g., human) a therapeutically effective amount of a phyto-percolate.
• a mammal e.g., human
• Another aspect of this invention is directed to a method for treating abnormal insulin release and utilization comprising administering to a mammal (e.g., human) a therapeutically effective amount of a compound of a phyto-percolate.
• a mammal e.g., human
• Another aspect of this invention is directed to a method for treating skin lesions and disorders.
• a method of making the inventive phyto- percolate is disclosed.
• the phyto-percolate is prepared by cultivating a mixture of freshwater algae and bacteria that is augmented by a nutrient blend that is related to the production of fibrinolytic enzymes, proteins, and other molecules, forming a fortified algae culture.
• Added to this fortified algal and bacterial culture is purified fresh water that has been purified by reverse osmosis, distillation and/or deionization.
• the culture is percolated with said purified fresh water and nutrient blend for a predetermined time forming a phyto- percolate that is fibrinolytic and proteinaceous in nature.
• the phyto-percolate is decanted from the fortified algal and bacterial culture and processed. Suitable methods of processing the phyto-percolate include filtration, centrifugation, lyophilization, purification, dilution, and other methods.
• the filtering of the decanted phyto-percolate in one particular embodiment is by micro-filtration where the micro-filtration removes particles larger than about 0.22 ⁇ m.
• this invention provides a substantially pure compound isolated from a phyto-percolate.
• the compound is isolated from the percolate produced by culturing the microorganisms of ATCC Deposit #PTA-5863 or other appropriate species as described herein.
• the compound is a protein having a molecular weight of about 67.5 kDa.
• the invention provides a pharmaceutical formulation comprising a substantially pure compound isolated from a phyto-percolate and a pharmaceutically acceptable excipient.
• a pharmaceutical formulation comprising a substantially pure compound isolated from a phyto-percolate and a pharmaceutically acceptable excipient.
• the term "inflammatory disorder” encompasses a variety of conditions including conditions related to a hyperactive immune system, chronic inflammation, and autoimmune disorders.
• Inflammatory disorders include, for example, acne vulgaris; acute febrile neutrophilic dermatosis; acute respiratory distress syndrome; Addison's disease; adrenocortical insufficiency; adrenogenital ayndrome; allergic conjunctivitis; allergic rhinitis; allergic intraocular inflammatory diseases, ANCA-associated small-vessel vasculitis; angioedema; ankylosing spondylitis; aphthous stomatitis; arthritis, asthma; atherosclerosis; atopic dermatitis; autoimmune disease; autoimmune hemolytic anemia; autoimmune hepatitis; Behcet's disease; Bell's palsy; berylliosis; balanitis circumscripta plasmacellularis; balanoposthitis; bronchial asthma; bullous herpetiformis dermatitis; bullous pemphigoid; carditis; celiac disease; cerebral ischaemia; chronic obstructive
• substantially pure when referring to a protein or other derivative of the phyto-percolate, means the state of a substance that has been separated from the other components of the phyto-percolate.
• a substantially pure derivative is at least 80%, by weight, free from the proteins and other organic molecules of the phyto-percolate.
• the substantially pure derivative is at least 90%, 95%, or 99%, by weight, free from those organic molecules.
• a substantially pure protein derivative may be obtained, for example, by extracting it from a source other than the phyto-percolate.
• a protein derivative for example, may be recombinantly expressed in another microorganism or in a cell-free translation system.
• FIGURE 1 is a flow chart showing a method of preparing a phyto-percolate
• FIGURE 2 is an HPLC chromatogram of the diluted phyto-percolate
• FIGURE 3 is an FTIR spectrum of the diluted phyto-percolate.
• FIGURE 4 is a [ 1 H]-NMR spectrum of the diluted phyto-percolate.
• the present invention provides a phyto-percolate that has therapeutic and other beneficial properties when administered to humans and other animals. Without being bound by any theory, it is believed that at least one of the therapeutically active agents in the phyto-percolate is an enzyme. Methods for preparing the phyto-percolate are also provided. Detailed embodiments of the present invention are disclosed herein, however, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms. Therefore, specific functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriately detailed embodiment.
• a phyto-percolate is derived from a culture comprised of freshwater algae, moss, bacteria, actinomycetes, and fungi. It is believed that the culture is comprised of at least one or more of the following genera:
• a heterotrophic rotifer species exists in the cultures, as well as bacteria that have been identified as Stenotrophomonas maltophilia, Ralstonia pickettii, Ralstonia paucula, Acinetobacter genospecies 11, Acinetobacter junii, Leifsonia aquatica, Riemerella anatipestifer, Variovorax paradoxus, and Streptomyces griseorubens . Without being bound to any particular theory, it is believed that these bacteria may produce proteolytic enzymes that are contributors to the effectiveness of the phyto- percolate. A method of producing phyto-percolate is depicted in FIGURE 1.
• Phyto-percolate cultures of approximately 100-200 ml of dense algal cells in approximately 2.5 gal, or approximately 10 liters, of reverse-osmosis purified sterile water are fed about 1 milliliter (ml) per week of liquid extract of live active yeast, or Baker's yeast, Saccharomyces cerevisiae, which has been prepared from 1.Og dry active yeast added to 50ml warm water, at between about 37° and about 43°C. The mixture is allowed to incubate for 10-30 minutes, or until it slightly foams.
• the cultures are fed in either 1.0 ml weekly doses, or 0.5ml twice-weekly doses. It is contemplated within the scope of the invention that other yeast cultures may be used. It is further contemplated that other organic nutrients or substrates known in the art may be used such as glucose or proteose, or other algal growth media prepared from inorganic nutrients, supplements, and/or vitamins.
• the cultures are grown under full-spectrum grow lights at about 25°C, and produce a final unadjusted pH of between about 6.2 to about 7 that fluctuates.
• the cultures are grown in clear glass fishbowl containers having a volume of approximately 2.5 gal with semi-transparent plastic lids, with the exception of about a 3mm hole in the Hd for gas exchange. It is contemplated within the scope of the invention that other culture containers, ingredients, conditions and methods known in the art may be used that allow the cells to grow in a manner in which the phyto-percolate derivatives are expressed.
• Such methods may include larger batch, semi-continuous, continuous or other type culture systems including bireactors or photoreactors, may or may not include aeration or agitation, may or may not include solid, liquid, semi-solid or other form of growth media or substrate, may or may not include the above particular conditions of temperature, contact time or area, or light intensity.
• the cultures are harvested weekly or bi-weekly, between the 5 rd and 10 th day after feeding, by drawing off the top 1.25 gal of phyto- percolate from each 2.5 gal culture. This is referred to as the "raw phyto-percolate.”
• the algal or other cells and yeast food forming the phyto-percolate culture remain in the bottom of the culture container substantially undisturbed while the phyto-percolate is decanted.
• the decanted material is then processed as desired.
• the volume of the container is then optionally returned to original volume. Conveniently this is accomplished with reverse- osmosis purified water at approximately room temperature, about 25°C. It is contemplated within the scope of the invention that other culture and harvest systems, timetables volumes and methods may be used that result in phyto-percolate derivatives.
• the peaks of enzyme concentration in the percolate over the course of several weeks are mapped under various feeding regimens, and serve to dictate the optimal date for harvests.
• the enzyme concentration is analyzed in the cultures and processed phyto-percolate to detect any negative effects of regular harvesting on the algal cultures over time, and is combined with data on the effects of environmental and stress factors such as dark/light, starvation, and/or changes in temperature or pH, which may stimulate or discourage enzyme production.
• Methods for analyzing these parameters include the isolation and homogenization of select cultures to eliminate all variables besides those being tested, and include monitoring of chlorophyll, total protein and enzyme activity, utilizing spectro-photometric methods, to measure the health and enzyme activity of the cultures over the course of an isolated-variable experiment.
• the method for analyzing proteolytic activity is a typical chromogenic assay using Chromogenix substrate from DiaPharma, S-2251: chromogenic substrate for plasmin and streptokinase-activated plasminogen.
• Chromogenic substrates are peptides that react with proteolytic enzymes and proportionally change color as the substrate is lysed by the enzymes. The color change may be measured spectro- photometrically over time and is proportional to the proteolytic activity.
• the synthetic chromogenic assay substrates are designed to have enzyme binding selectivity similar to that of the enzyme's natural substrate. It is believed that the enzymes present in phyto- percolate are selective for substrates including fractionated proteins and fibrin.
• Enzyme activity for samples of described phyto-percolate currently ranges from 15- 50 mU/mL of plasmin-like activity, when phyto-percolate is prepared as described. These values have been found to correlate with clinical observations of reduced pathological fibrin in humans orally consuming phyto-percolate.
• Methods for evaluating in vivo effects of phyto-percolate include peripheral blood observations on wet and dry blood smears, diagnostic and/or analytical blood tests, and various clinical observations and measurements such as body weight. Reductions in excess pathological fibrin and platelet aggregation have been observed, which are secondary to inflammation and tissue destruction. Changes in white blood cell mobility and. number have also been observed. Anti- inflammatory effects of phyto-percolate in vivo have also been monitored with independent blood laboratory studies focusing on chronic inflammatory activity and hyper- coagulant states.
• the phyto-percolate may be produced using a continuous culture format in which the phyto-percolate is substantially continuously removed from the culture and the lost volume is replaced with fresh culture media and/or nutrients. Further, the phyto-percolate may be produced using a bioreactor that is suitable for production on a larger scale than the batch culture method described above.
• the decanted fluid is filtered through a series of depth pref ⁇ lters and sterile membrane filters made of low-protein binding materials.
• suitable final sterilizing filters are provided by Millipore Corp. Durapore brand filters, made of PVDF material. These have been shown to protect the enzyme concentration, and provide a final sterile filtration level of about 0.22 microns, as well as being chemically inert to ozonated water. Ozonated water is used for sterilizing the filter system, as it does not leave a damaging residue like chlorine.
• All filters are 10" cartridge membrane or depth filters of various chemically- inert materials.
• the pref ⁇ lters are housed in cartridge filter housings made of styrene- acrylonitrile (SAN).
• the final filters are housed in polypropylene (PP) housings with Kynar fittings.
• the material is harvested and filtered using Tygon tubing, peristaltic pumps and 55 gallon containers or other containers that have been pre-sterilized with ozonated water.
• the phyto-percolate passes through a filtration regimen comprised of two pre- filters in SAN housings of pore size l ⁇ m (nominal), made of pleated cellulose/polyester.
• filters are manufactured by Cole-Parmer, Vernon Hills, Illinois, USA, catalog number EW-29830-20. It is contemplated within the scope of the invention that other filters know in the art may be used in this step as pre-filters, that are chemically inert.
• the phyto-percolate is again filtered using a second stage pre-filter made of polypropylene in a polypropylene housing, with a nominal pore size of about 0.5um.
• this finishing filter is manufactured by Millipore Corporation,
• a final filter set consists of sterile membrane filters in PP housing having progressively smaller pore sizes of 0.45um and 0.22 ⁇ m (absolute).
• These finishing filters' membranes are made of hydrophilic extremely- low protein-binding PVDF.
• these finishing filters are manufactured by Millipore Corporation, Durapore ® brand, Catalog #'s CVHIOlTPE and CVDIOlTPE.
• Filtration by size exclusion removes approximately >99.9% of contaminants such as bacteria, yeast and mold spores, and algal cells. It is also believed to preserve enzymatic activity if filter materials are made of low-protein-binding, chemically-inert materials.
• the resulting liquid, the phyto-percolate is substantially comprised of water, active enzymes, proteins and sugars.
• the phyto-percolate, after passing through the finishing filter is then usefully stored in sealed sterile 55 gal HDPE drums at between 21° and 27°C until bottling. Samples are taken from each batch immediately after filtering to test for enzyme efficacy and contamination and for standardization. It is contemplated within the scope of the invention that other methods of sampling and testing may be used.
• the phyto-percolate is processed and bottled under sanitary conditions known in the art using ozone sterilization. It is believed that this step avoids enzyme degradation associated with the use of chlorine or heat sterilization because ozone leaves no residue if left to dissipate, or if followed by a rinse of sterile water. It is contemplated within the scope of the invention that other methods of filtration and sanitization known in the art may be used that are not unreasonably degrading of the enzymatic or other activity.
• the phyto-percolate is usefully packaged in opaque UV-protectant bottles and shipped with cold packs to reduce product degradation. It is contemplated within the scope of the invention that other methods of packing, bottling, storing, and transporting may be used.
• Phvto-Percolate Characterization It is believed that the raw phyto-percolate, prior to filtration, is a complex mixture of macromolecules. It was expected that the filtration process described above reduced the molecular complexity of the phyto-percolate filtrate. We performed several physico- chemical tests to determine the composition of the filtrate. In each case, the phyto- percolate filtrate was lyophilized, redissolved in ddE ⁇ O, and refiltered to remove any undissolved particulate matter.
• a sample of the lyophilized phyto-percolate was subjected to isocratic reverse phase HPLC, on a size-exclusion chromatography column (TSK-GEL Super SW Series; Tosoh Biosciences, Montgomeryville, PA), under non-denaturing conditions. Proteins were identified using a micro flow cell UV detector at 280 nm. As shown in FIGURE 2, a major protein species of 67.5 kDa was identified (retention time 18.747 minutes). The 67.5 kDa peak contributed about 90% of the total signal measured at 280 nm. Also detected were peaks at retention times of 21.544 minutes (21.0 kDa) and 23.957 minutes.
• phyto-percolate derivatives The major components of the phyto-percolate (the 67.5 kDa protein, 21.0 kDa protein, and the polysaccharide identified at 23.957 minutes) are referred to herein as phyto-percolate derivatives and may contribute to the biological and therapeutic efficacy of the phyto- percolate.
• FIGURE 3 shows a spectrum that is characteristic of a dissolved protein sample.
• a 21 day weight loss study using twelve mature (12 month old) Sprague-Dawley rats was performed. Each animal was orally administered 10 ml/kg of undiluted and unf ⁇ ltered phyto-percolate (i.e., raw phyto-percolate) for 14 days, followed by non-dosing for 7 days. Each animal was weighted daily and observed for signs of toxicity. As shown in more detail in Table 1, the rats lost an average of 33 grams (6.3%) of body weight over the initial 14 day dosing period. They immediately began to regain lost body weight upon cessation of phyto-percolate administration. By the 21 day time point (7 days of non- dosing), the rats had lost an average of 25 grams (4.7%) of initial body weight (i.e., gained an average of 8 grams since phyto-percolate cessation).
• test animals were observed for adverse reactions immediately after each dose and at 4 and 24 hours subsequent. Daily observation for adverse reactions was continued during the 7 day non-dosing period. Specifically, clinical observations for adverse reactions were made for respiration, motor activity, convulsions, reflexes, ocular signs, salivation, piloerection, analgesia, muscle tone, gastrointestinal effects, and skin/dermal alterations. Gastrointestinal effects were the only observed adverse reaction. Soft to loose stool was observed in all test animals. No other adverse reaction was observed.
• a single-center, prospective, randomized, triple-masked, placebo-controlled parallel-group-design pilot clinical trial of the phyto-percolate was performed using two different batches of the phyto-percolate. This trial was conducted in accordance with FDA regulations and under a protocol approved by an Institutional Review Board (IRB).
• IRS Institutional Review Board
• Subjects Primary inclusion criteria were men and women having a body mass index (BMI) of 25-40 m/kg 2 , 18-70 years old (inclusive), and desirous of losing weight.
• Major exclusion criteria were moderate to severe co-morbid disease (e.g., cancer); history of stroke, transient ischemic attack (TIA), or similar conditions; uncontrolled hypertension, insulin-dependent diabetes, renal disease, moderately severe cardiac disease, lupus, alcohol abuse, and current or recent use of certain medications including medications and/or supplements for weight loss, glucose management, or arthritis. Women were excluded if they were pregnant, nursing, or actively trying to become pregnant.
• Protocol Patients were assigned to self-administer one ounce of filtered phyto- percolate or placebo three times each day (t.i.d.) on an empty stomach at least 30 minutes before a meal. Subjects were asked to participate in a reduced carbohydrate diet and light exercise program and complete a one-day-per-week Food Log and a daily Exercise Log for the duration of the clinical trial. Patients were evaluated during a baseline examination and then again at 2-week, 4-week, and 6-week visits. Evaluations included measurement of body weight, arm and waist circumference, and body fat measurements.
• Glucose Control Study At the baseline examination and at the 4-week and 6-week visits, patients' fasting (12 hour) blood glucose was measured and then their blood glucose was measured one hour after a glucose challenge (25 grams of jelly beans; 90.4% carbohydrate). The difference between the glucose challenge reading and the baseline reading in a single visit is an indicator of the patient's ability to regulate serum glucose levels.
• Test Materials The patients in the treatment groups were assigned one of two different lots (Batch 1 and Batch 2) of phyto-percolate prepared as described above. The placebo product was similar in appearance (color, viscosity, and odor) to the diluted phyto- percolate. All test materials were dispensed in unlabeled blue bottles with instructions to refrigerate after opening.
• Test subjects demonstrated an average of 2.6x (156%) and 1.7x (69%) improved glucose control at 4 weeks and 6 weeks, respectively, when compared to the placebo group. Furthermore, 6 of the 22 test subjects met the clinically important criterion of > 50% control over baseline. Three of these six demonstrated complete control of the glucose challenge, defined as > 85% glucose control over baseline.
• Cyclooxygenase-2 (COX-2) is a key regulator of the inflammatory cascade. COX- 2 inhibitors are believed to reduce inflammation by blocking prostaglandin production. In view of the adverse effects associated with mixed COX inhibitors (aspirin, ibuprofen, and naproxen) and the presently available COX-2-specific inhibitors (valdecoxib, celecoxib, rofecoxib), there is a need for improved anti-inflammatory therapies with fewer side effects. Three separate preparations of the phyto-percolate were screened, using an in vitro assay, for COX-2 inhibition. Riendeau et al., Can. J. Physiol. Pharmacol. 75: 1088-1095, 1997; Warner et al., Proc.
• this assay measured to conversion of 0.3 ⁇ M arachidonic acid to PGE 2 by human recombinant insect Sf21 cells expression human COX-2.
• the incubation buffer contained 100 mM Tris-HCl (pH 7.7), 1 mM glutathione, 1 ⁇ M hematin, and 500 ⁇ M phenol.
• PGE 2 was quantified using an enzyme-linked immunoassay (EIA).
• Sample 1 was a sample of diluted phyto-percolate concentrated approximately 100- fold by drying under N 2 .
• Sample 2 was prepared by drying a 4800 ⁇ l sample of diluted phyto-percolate under N 2 and reconstituting it in 96 ⁇ l of ddHzO just prior to assay.
• Sample 3 was prepared by lyophilizing a 4800 ⁇ l sample of diluted phyto-percolate and reconstituting it in 96 ⁇ l of ddH 2 O just prior to assay.
• the concentrations of phyto- percolate used, 10OX, 10X, and IX refer to 10 ⁇ l, 1 ⁇ l, and 0.1 ⁇ l of sample, respectively, in a final assay volume of 100 ⁇ l.
• Rofecoxib was used as a positive control for COX-2 inhibition. Each sample was assayed in at least three concentrations and the assays were performed in duplicate.
• the carrageenan-induced paw edema assay was used as an in vivo indicator of the anti-inflammatory effects of the phyto-percolate.
• Carrageenan induces local inflammation and edema when injected into the paw pad of a rat (Di Rosa et al., 1971).
• the development of paw edema is believed to be biphasic (Vinegar et al., 1969).
• the initial phase is attributable to the local release of histamine and serotonin (Crunkhon et al., 1971) and the second phase is caused by prostaglandin release as a result of COX activation.
• the second phase is measured as an increase in paw volume and has been demonstrated to be responsive to steroidal and non-steroidal anti-inflammatory agents.
• Paw volume was measured at 0, 2, 4, 6, 8, and 20 hours after treatment using a plesthysmometer to measure volume displacement. Each treatment group is compared to control.
• serum was drawn by cardiac puncture for baseline (pre-inoculation) values.
• the treatment group received diluted phyto-percolate for their drinking water, which was allowed ad libitum, while the control group received filtered water.
• Serum was drawn by cardiac puncture, as above, every thirty (30) days until the termination of the study.
• the treatment group After 60 days of treatment with the diluted phyto-percolate, the treatment group had an average 30% increase in IgA levels, 50% increase in IgG levels, and a 40% reduction in C-reactive protein (C-RP) levels, relative to the untreated group (Table 11). No significant differences in body weight, average daily food consumption, or average daily liquid consumption were detected between the groups.
• C-RP C-reactive protein
• the phyto-percolate dosage will vary with the severity of the disease, the
• the phyto-percolate is orally administered as a liquid. As described in several of the foregoing examples, the phyto-percolate is diluted in filtered
• the concentration of phyto-percolate may be altered.
• the protein species may be present in the orally administered liquid in concentrations including about 100 ppm, 250 ppm, 500 ppm,
• the protein fraction is isolated from the phyto-percolate and formulated for parentera administration (e.g., intravenous, intramuscular, and subcutaneous injection, topical, rectal or vaginal administration or other).
• parentera administration e.g., intravenous, intramuscular, and subcutaneous injection, topical, rectal or vaginal administration or other.
• the dosage of diluted phyto-percolate will vary from about one
• phyto-percolate function from food-stimulated gastrointestinal activities.
• a 50-70 Ib. child is dosed at about three to four ounces per day, generally dosing on an empty stomach, during an acute infection.
• phyto-percolate can be used to enhance the well being and performance of animals.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Medicinal Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Veterinary Medicine (AREA)
• Chemical & Material Sciences (AREA)
• Public Health (AREA)
• General Health & Medical Sciences (AREA)
• Animal Behavior & Ethology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Organic Chemistry (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Immunology (AREA)
• Diabetes (AREA)
• Obesity (AREA)
• Hematology (AREA)
• Virology (AREA)
• Oncology (AREA)
• Natural Medicines & Medicinal Plants (AREA)
• Biotechnology (AREA)
• Rheumatology (AREA)
• Communicable Diseases (AREA)
• Orthopedic Medicine & Surgery (AREA)
• Alternative & Traditional Medicine (AREA)
• AIDS & HIV (AREA)
• Tropical Medicine & Parasitology (AREA)
• Molecular Biology (AREA)
• Pain & Pain Management (AREA)
• Cardiology (AREA)
• Heart & Thoracic Surgery (AREA)
• Physical Education & Sports Medicine (AREA)
• Endocrinology (AREA)
• Emergency Medicine (AREA)
• Child & Adolescent Psychology (AREA)
• Botany (AREA)
• Medical Informatics (AREA)
• Microbiology (AREA)

Priority Applications (4)

Applications Claiming Priority (2)

Publications (2)

Family

ID=38092904

Family Applications (1)

Country Status (5)

Cited By (7)

Families Citing this family (2)

Family Cites Families (2)

• 2006
  • 2006-12-04 CA CA2631773A patent/CA2631773C/en active Active
  • 2006-12-04 EP EP06838974A patent/EP1954800A4/en not_active Withdrawn
  • 2006-12-04 JP JP2008543545A patent/JP2009518312A/ja active Pending
  • 2006-12-04 AU AU2006320264A patent/AU2006320264A1/en not_active Abandoned
  • 2006-12-04 WO PCT/US2006/046320 patent/WO2007065024A2/en not_active Ceased

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events