WO2004108919A1 - Compositions biologiques comprenant des cellules de levure et methodes de traitement du cancer - Google Patents

Compositions biologiques comprenant des cellules de levure et methodes de traitement du cancer Download PDF

Info

Publication number
WO2004108919A1
WO2004108919A1 PCT/GB2004/002466 GB2004002466W WO2004108919A1 WO 2004108919 A1 WO2004108919 A1 WO 2004108919A1 GB 2004002466 W GB2004002466 W GB 2004002466W WO 2004108919 A1 WO2004108919 A1 WO 2004108919A1
Authority
WO
WIPO (PCT)
Prior art keywords
yeast cells
range
culturing
mhz
frequency
Prior art date
Application number
PCT/GB2004/002466
Other languages
English (en)
Inventor
Ling Yuk Cheung
Original Assignee
Ultra Biotech Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US10/460,341 external-priority patent/US6987012B2/en
Priority claimed from US10/460,323 external-priority patent/US6984507B2/en
Priority claimed from US10/460,325 external-priority patent/US7204987B2/en
Priority claimed from US10/460,530 external-priority patent/US7223405B2/en
Priority claimed from US10/460,327 external-priority patent/US6989253B2/en
Priority claimed from US10/460,326 external-priority patent/US7223401B2/en
Priority claimed from US10/460,337 external-priority patent/US7223402B2/en
Priority claimed from US10/460,437 external-priority patent/US7223403B2/en
Priority claimed from US10/460,324 external-priority patent/US7220416B2/en
Priority claimed from US10/460,338 external-priority patent/US7226600B2/en
Priority claimed from US10/460,438 external-priority patent/US7223404B2/en
Priority claimed from US10/460,271 external-priority patent/US7223400B2/en
Priority claimed from US10/460,328 external-priority patent/US7201906B2/en
Priority claimed from US10/460,336 external-priority patent/US6984508B2/en
Priority claimed from US10/460,833 external-priority patent/US7208158B2/en
Priority claimed from US10/460,247 external-priority patent/US7214377B2/en
Priority claimed from US10/460,246 external-priority patent/US7204986B2/en
Priority claimed from US10/460,832 external-priority patent/US7204988B2/en
Application filed by Ultra Biotech Limited filed Critical Ultra Biotech Limited
Publication of WO2004108919A1 publication Critical patent/WO2004108919A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/14—Yeasts or derivatives thereof
    • 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
    • A61K36/06—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0004—Homeopathy; Vitalisation; Resonance; Dynamisation, e.g. esoteric applications; Oxygenation of blood
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention relates to biological compositions comprising yeast cells that can produce a healthful benefit in a subject inflicted with cancer.
  • the invention also relates to methods for manufacturing the biological compositions and methods of use thereof.
  • Cancer is one of the leading causes of death in animals and humans. It is characterised primarily by an increase in the number of abnormal cells derived from a given normal tissue. While surgery, chemotherapeutic agents and radiation are useful in the treatment of cancer, there is a continued need to find better treatment modalities and approaches to manage the disease that are more effective and less toxic and non-invasive, especially when clinical oncologists are giving increased attention to the quality of life of cancer patients.
  • the present invention provides an alternative approach to cancer therapy and management of the disease by using a biological composition comprising yeasts.
  • yeasts and components thereof have been developed to be used as dietary supplement or pharmaceuticals.
  • none of the prior methods uses yeast cells which have been cultured in an electromagnetic field to produce a product that has an anti-cancer effect.
  • United States Patent No. 6,197,295 discloses a selenium-enriched dried yeast product which can be used as dietary supplement.
  • the yeast strain Saccharomyces boulardii sequela PY 31 (ATCC 74366) is cultured in the presence of selenium salts and contains 300 to about 6,000 ppm intracellular selenium. Methods for reducing tumor cell growth by administration of the selenium yeast product in combination with chemotherapeutic agents is also disclosed.
  • United States Patent No. 6,143,731 discloses a dietary additive containing whole -glucans derived from yeast, which when administered to animals and humans, provide a source of fiber in the diet, a fecal bulking agent, a source of short chain fatty acids, reduce cholesterol and LDL, and raises HDL levels.
  • United States Patent No. 5,504,079 discloses a method of stimulating an immune response in a subject utilizing modified yeast glucans which have enhanced immunobiologic activity.
  • the modified glucans are prepared from the cell wall of Saccharomyces yeasts, and can be administered in a variety of routes including, for example, the oral, intravenous, subcutaneous, topical, and intranasal route.
  • United States Patent No. 4,348,483 discloses a process for preparing a chromium yeast product which has a high intracellular chromium content.
  • the process comprises allowing the yeast cells to absorb chromium under a controlled acidic pH and, thereafter inducing the yeast cells to grow by adding nutrients.
  • the yeast cells are dried and used as a dietary supplement.
  • the present invention relates to biological compositions useful for treatment of subjects with cancer.
  • the present invention provides biological compositions comprising live yeast cells which are capable of producing a healthful benefit in subjects with cancer.
  • the invention provides methods of making the biological compositions, and methods of using the biological compositions.
  • the methods of the invention comprise culturing yeast cells in the presence of a series of electromagnetic fields such that the yeast cells becomes metabolically active.
  • the electromagnetic fields used are each defined by one of five frequency ranges and a broad range of field strength.
  • the starting yeast cells are commercially available and/or accessible to the public, such as but not limited to Saccharomyces.
  • the methods for making the biological compositions of the invention may further comprise conditioning the activated yeast cells in plant extracts and the gastric juice of animals, while in the presence of another series of electromagnetic fields.
  • the methods of manufacturing also comprise expanding the number of activated or activated and conditioned yeast cells in large scale cultures in the presence of yet another series of electromagnetic fields, performing quality control measures, and packaging.
  • Pharmaceutical compositions of the invention comprises activated and conditioned yeast cells and one or more pharmaceutically acceptable excipients or carriers. Additional ingredients, such as vitamins, herbs, and/or flavors may be added to the biological compositions to form the oral compositions of the invention. Such additional carriers and ingredients can improve the healthful benefits, pharmacological properties, and organoleptic characteristics of the oral compositions.
  • the activated or activated and conditioned yeast cells may be dried and stored for a period of time.
  • the biological or oral compositions of the invention are ingested by the subject or used as an additive to be incorporated into food to be consumed by the subject. Dietary supplement and nutritional compositions comprising activated and conditioned yeast cells are encompassed by the invention.
  • the subject is a human being.
  • the biological compositions of the invention are used to produce a healthful benefit in a subject with cancer or at high risk of developing cancer.
  • the biological composition of the invention can retard the growth of cancer cells in an animal which received the composition orally.
  • the biological composition can also be used to prolong the time of survival of an animal with cancer.
  • Fig. 1 Activation and conditioning of yeast cells.
  • Fig. 2 Large scale propagation of yeast cells. 5 first container; 6 second container; 7 third container; 8 yeast cell cultures; 9 electromagnetic field source.
  • the present invention relates to biological compositions that can produce a healthful benefit in a subject with cancer.
  • the present invention provides methods for manufacturing the biological compositions as well as methods for using the biological compositions.
  • cancer refers to all types of cancers, or neoplasms or benign or malignant tumors, including but not limited to carcinoma, sarcoma, lymphoma, and leukemia.
  • cancer includes but is not limited to lung cancer, nasopharyngeal cancer, esophageal cancer, stomach cancer, colorectal cancer, pancreatic cancer, liver cancer, testicular cancer, prostate cancer, ovarian cancer, breast cancer, cervical cancer, uterine cancer, kidney cancer, bladder cancer, brain cancer, lymphoma, and leukemia.
  • the invention provides biological compositions that comprise yeasts. Unlike the traditional use of yeasts in the making of food, the yeast cells of the invention are not used as a source of enzymes that acts on the food ingredients. The yeasts are not a primary source of nutrients for the subject. Nor are yeast cells used as a carrier of an active ingredient, such as metal salts. The yeast cells of the invention are live when administered orally or ingested along with food by a subject.
  • the inventor believes that the culture conditions activate and/or amplified the expression of a gene or a set of genes in the yeast cells such that the yeast cells become highly effective in stimulating the animal's immune system, including both specific and non-specific immunological reactions, the results of which are manifested as the overall healthful benefits observed in the treated subject.
  • the healthful benefits provided by using the biological compositions are demonstrated in animal models of human cancers, which show inhibition of tumor growth and prolonged survival time of animals with the disease.
  • the invention provides methods for making the yeast cells in the biological compositions.
  • the starting materials are normal yeast cells which can be readily obtained commercially or from public microorganism deposits.
  • the methods of the invention comprise a set of culture conditions that can be applied reproducibly to activate the yeast cells.
  • the key feature of the culture conditions used in the methods of the invention is a series of alternating electromagnetic fields of defined frequency ranges and field strengths which are applied to the growing yeast cell culture.
  • the method further comprises the step of conditioning the activated live yeast cells to the acidic environment of the stomach of the subject.
  • the electromagnetic fields used in these methods can be created reproducibly at various scales, thus enabling even the large scale manufacturing of the biological compositions of the invention.
  • the invention provides methods for manufacturing a biological composition comprising activated and conditioned yeasts of the invention, and additional ingredients, including but not limited to pharmaceutically acceptable carriers or excipients, vitamins, herbs (including traditional Chinese medicine products), herbal extracts, minerals, amino acids, flavoring agents, coloring agents, and/or preservatives.
  • the biological compositions can be added to food which will be consumed by the subject.
  • many methods may be used to mix the biological or oral compositions of the invention with food while the yeast cells remain viable.
  • the culture broth comprising live yeast cells of the present invention are added directly to food just prior to consumption. Dried powders of the yeasts can also be reconstituted and added directly to food just prior to consumption.
  • the oral compositions of the invention can be consumed directly by a subject or be fed directly to a subject.
  • the subject may drink the culture broth or a fraction thereof that comprises live activated and conditioned yeast cells.
  • Oral compositions comprising dried yeast cells can also be given as a solid dosage form to the subject.
  • the biological or oral compositions of the invention can be used in conjunction or in rotation with other types of treatment modalities such as but not limited to surgery, chemotherapeutic agents, and radiation. Since the biological compositions of the invention are administered orally, the assistance of health professionals in administration of the composition is generally not essential. Described below are the yeast cells of the invention and methods of their preparation, followed by descriptions of using the biological compositions of the invention in a subject suffering from cancer. The examples of using the invention in treatment of eighteen (18) commonly occurring cancers are also provided to further demonstrate the therapeutic benefits of an oral composition of the invention.
  • the activated and conditioned yeast cells in the oral composition are characterized by their ability to (i) suppress the growth of cancer cells in an animal model of human cancer or (ii) prolong the survival of animals with transplanted cancer cells in a model of human cancer as compared to yeast cells which have not been activated and conditioned.
  • the yeast cells of the biological composition are produced by culturing a plurality of yeast cells in an appropriate culture medium in the presence of an alternating electromagnetic field over a period of time.
  • the method comprises a first step of activating the yeast cells and a second step of conditioning the activated yeast cells.
  • the activation process comprises culturing yeast cells in the presence of at least two, three, four or five electromagnetic fields of specific frequencies and field strength.
  • the conditioning process comprises further culturing of the activated yeast cells in a medium comprising plant extracts and extracts from the stomach of an animal, in the presence of at least one electromagnetic field.
  • the activated and conditioned yeast cells can be stored as dried cells after drying the cells under appropriate conditions.
  • the dried activated and conditioned yeast cells can be used later in large scale culturing processes for manufacturing the biological compositions of the invention.
  • the various culturing processes of the invention can be performed either as a batch process or a continuous process.
  • yeasts of the genera of Saccharomyces, Candida, Crebrothecium, Geotrichum, Hansenula, Kloeckera, Lipomyces, Pichia, Rhodosporidium, Rhodotorula, Torulopsis, Trichosporon, and Wickerhamia can be used in the invention.
  • fungi used for food manufacturing are preferred.
  • yeast strains include Saccharomyces sp., AS2.311; Schizosaccharomyces pombe Linder, AS2.214, AS2.248, AS2.249, AS2.255, AS2.257, AS2.259, AS2.260, AS2.274, AS2.994, AS2.1043, AS2.1149, AS2.1178, IFFI 1056; Saccharomyces sake Yabe, ACCC2045; Saccharomyces uvarum Beijer, LFFI 1023, LFFI 1032, LFFI 1036, IFFI 1044, LFFI 1072, LFFI 1205, LFFI 1207; Saccharomyces rouxii Boutroux, AS2.178, AS2.180, AS2.370, AS2.371; Saccharomyces cerevisiae Hansen Var.
  • yeast strains useful for the invention can be obtained from private or public laboratory cultures, or publicly accessible culture deposits, such as the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209 and the China General Microbiological Culture Collection Center (CGMCC), China Committee for Culture Collection of Microorganisms, Institute of Microbiology, Chinese Academy of Sciences, Haidian, P.O. Box 2714, Beijing, 100080, China.
  • CGMCC General Microbiological Culture Collection Center
  • Non-limiting examples of using yeast cells of the invention are provided below.
  • the yeast cells of the invention do not comprise an enhanced level of selenium or chromium relative to that found in naturally occurring yeast cells.
  • the biological compositions do not comprise cells of Saccharomyces boulardii (for example, ATCC Accession No. 74366) or cells of a particular strain of Saccharomyces cerevisiae (strain Hansen CBS 5926) that is also commonly referred to as Saccharomyces boulardii.
  • the preparation of the yeast cells of the invention is not limited to starting with a pure strain of yeast.
  • the yeast cells in the biological compositions may be produced by culturing a mixture of yeast cells of different species or strains.
  • the constituents of a mixture of yeast cells can be determined by standard yeast identification techniques well known in the art.
  • an electromagnetic field useful in the invention can be generated by various means well known in the art.
  • An electromagnetic field of a desired frequency and a desired field strength is generated by an electromagnetic wave source (3) which comprises one or more signal generators that are capable of generating electromagnetic waves, preferably sinusoidal waves, and preferably in the frequency range of 1,500 to 15,000 MHz and most preferably 7,800 to 12,900 MHz.
  • signal generators are well known in the art.
  • Signal generators capable of generating signal with a narrower frequency range can also be used. If desirable, a signal amplifier can also be used to increase the output signal, and thus the strength of the EM field.
  • the electromagnetic field can be applied to the culture by a variety of means including placing the yeast cells in close proximity to a signal emitter connected to a source of electromagnetic waves.
  • the signal generator is connected to the signal emitter by cables such as coaxial cables that can transmit signals up to greater than or equal to 30 GHz.
  • the yeast cells are placed in a container which is made of material that is not an electric conductor, such as but not limited to plastic, resin, glass, and ceramic.
  • the electromagnetic field is applied by signal emitters in the form of electrodes (4) that are submerged in a culture of yeast cells (1).
  • one of the electrodes is a metal plate which is placed on the bottom of a non-conducting container (2), and the other electrode comprises a plurality of wires or tubes so configured inside the container such that the energy of the electromagnetic field can be evenly distributed in the culture.
  • the electrodes are preferably made of copper.
  • the number of electrodes used depends on both the volume of the culture and the diameter of the electrode. For example, for a culture having a volume of 10 liter or less, two or three electrodes having a diameter of between 0.5 to 2.0 mm can be used. For a culture volume of 10 to 100 liter of culture, the electrodes can have a diameter of 3.0 to 5.0 mm. For a culture volume of 100 to 1,000 liter, the electrodes can have a diameter of 6.0 to 15.0 mm. For a culture having a volume greater than 1,000 liter, the electrodes can have a diameter of between 20.0 to 25.0 mm.
  • the method for producing activated yeast cells of the invention comprises culturing yeast cells in the presence of at least two, three, four or five alternating electromagnetic (EM) fields.
  • EM alternating electromagnetic
  • the culture process can be initiated by inoculating 1,000 ml of medium with an inoculum of a selected yeast strain (such as one of those described above) such that the starting cell density of the culture is greater than about 10 5 cells per ml.
  • the starting culture can be used to seed larger scale culture.
  • the culture is maintained initially at 28°C to 32°C for 22 to 30 hours prior to exposure to the EM field(s), typically at 30°C for 28 hours.
  • the culturing process may preferably be conducted under conditions in which the concentration of dissolved oxygen is between 0.025 to 0.08 mol/m 3 , preferably 0.04 mol/m 3 .
  • the oxygen level can be controlled by any conventional means known in the art, including but not limited to stirring and/or bubbling.
  • the culture is most preferably carried out in a liquid medium which contains sources of nutrients assimilable by the yeast cells, such as sucrose or glucose, vitamin H, vitamin B 6 , vitamin B ⁇ 2 , fetal calf serum, peptone, and inorganic ions such as K + , Na + , Mg 2+ , Ca 2+ , Cl " , CO 3 2" , PO 4 3" , and SO 4 2" .
  • sources of nutrients assimilable by the yeast cells such as sucrose or glucose, vitamin H, vitamin B 6 , vitamin B ⁇ 2 , fetal calf serum, peptone, and inorganic ions such as K + , Na + , Mg 2+ , Ca 2+ , Cl " , CO 3 2" , PO 4 3" , and SO 4 2" .
  • carbohydrates such as sugars, for example, sucrose, glucose, fructose, dextrose, maltose, xylose, and the like and starches, can be used
  • the exact quantity of the carbohydrate source or sources utilized in the medium depends in part upon the other ingredients of the medium but, in general, the amount of carbohydrate usually varies between about 0.1 % and 5% by weight of the medium and is preferably between about 0.2% and 2%. These carbon sources can be used individually, or several such carbon sources may be combined in the medium.
  • the inorganic salts which can be incorporated in the culture media are the customary salts capable of yielding sodium, calcium, phosphate, sulfate, carbonate, and like ions.
  • Non-limiting examples of nutrient inorganic salts are KH 2 PO 4 , (NH 4 ) 2 HPO 4 , CaCO 3 , MgSO 4 , NaCI, and CaSO 4 .
  • media containing ingredients other than the ones listed above can also be used in culturing the same or different strains of yeast cells.
  • the culturing medium is heated to 45°C and cooled before adding nutrients such as vitamin A, vitamin B 2 , vitamin B 3 , vitamin B 6 , vitamin B 12 , vitamin C, vitamin D, vitamin H, bovine calf serum and/or fetal calf serum.
  • composition of the media provided herein is not intended to be limiting.
  • the process can be scaled up or down according to needs.
  • Various modifications of the culture medium may be made by those skilled in the art, in view of practical and economic considerations, such as the scale of culture and local supply of media components.
  • a series of at least two, three, four or five EM fields are applied to the culture of yeast cells, each having a different frequency within a stated range, and a different field strength within a stated range.
  • the EM fields can be applied in any order and by any means known in the art, such as the apparatus described above. Although any of the following two, three or four EM fields can be applied, preferably, all five EM fields are applied.
  • the frequency is in the range of 7,821 to 10,170
  • the yeast culture is exposed to this first EM field at 30 ⁇ 2°C for about 5 to 36 hours.
  • the frequency is in the range of 7,993 to 11,530 MHz and the field strength is in the range of 190 to 330 mV/cm.
  • the yeast culture is exposed to this second EM field at 30 ⁇ 2°C for about 4 to 36 hours.
  • the frequency is in the range of 9,907 to 12,285 MHz and the field strength is in the range of 230 to 430 mV/cm.
  • the yeast culture is exposed to this third EM field at 30 ⁇ 2°C for about 10 to 34 hours.
  • the frequency is in the range of 11,141 to 12,842 MHz and the field strength is in the range of 220 to 450 mV/cm.
  • the yeast culture is exposed to this fourth EM field at 30 ⁇ 2°C for about 4 to 34 hours.
  • the frequency is in the range of 12,031 to 12,900 MHz and the field strength is in the range of 260 to 450 mV/cm.
  • the yeast culture is exposed to this fifth EM field at 30 ⁇ 2°C for about 5 to 34 hours.
  • the yeast cells can be cultured by exposure to two, three or four of the above-mentioned EM fields in a different order.
  • the yeast cells can remain in the same container and use the same set of electromagnetic wave generator and emitters when switching from one EM field to another EM field.
  • the cell density of the culture at the end of the activation process is typically greater than about 10 6 to 10 9 cells per ml (estimated by hematocytometer).
  • the activated yeast cells may be recovered from the culture by various methods known in the art, and stored at a temperature below about 0°C to 4°C.
  • the activated yeast cells recovered from the liquid culture may be dried and stored in powder form.
  • the powder form of the yeast cells comprises greater than about 10 to 10 yeast cells per gram.
  • performance of the activated yeast cells can be optimized by culturing the activated yeast cells in the presence of an extract from the stomach (e.g., the gastric juice) of an animal with physiology similar to the subject to which the biological composition will be administered.
  • the inclusion of this additional conditioning process allows the activated yeast cells to adapt to and endure the acidic environment of the subject's stomach.
  • the method for conditioning activated yeast cells of the invention comprises culturing yeast cells in such materials in the presence of at least one EM field.
  • the culture process can be initiated by inoculating 1,000 ml of a conditioning medium with about 10 gram of dried activated yeasts containing about 10 10 cells per gram (as prepared by the methods described above).
  • An equivalent number of yeast cells in culture preferably greater than 10 6 to 10 9 cells per ml, more preferably at 10 8 cells per ml, can also be used as an inoculum.
  • the conditioning medium comprises per 1,000 ml about 700 ml of gastric juice of an animal and about 300 ml of wild hawthorn juice.
  • the process can be scaled up or down according to needs.
  • the gastric juice of an animal can be obtained from the stomach content of a freshly slaughtered animal. Although not essential, the animal is preferably kept under a clean environment, and fed a standard diet, preferably germ- free. For example, the content of the stomach of a 120-day old pig is mixed with 2,000 ml of distilled water, and allowed to settle without stirring for 6 hours. The clear liquid above is collected for use as the gastric juice used in the conditioning process.
  • the gastric juice of a pig can be used to condition yeast cells for use in a variety of mammals, including humans. Other methods that can be used to collect the gastric juice include centrifugation or filtration of the mixture to remove debris and/or microorganisms.
  • the gastric juice so obtained can be stored at 4°C. Preferably, the collection procedures and storage are carried out under sterile conditions.
  • the wild hawthorn juice is an extract of wild hawthorn fruits prepared by slicing the fruits and drying the slices in air, preferably to less than 8% moisture (commercial dryer can be used if necessary), crushing the dried fruits to less than 20 mesh, and mixing 1,500 ml of water per 500 gram of the crushed wild hawthorn. The mixture is then allowed to settle without stirring for 6 hours, and the clear liquid above is collected for use as the wild hawthorn juice used in the conditioning process. Other methods that can be used to collect the hawthorn juice include centrifugation or filtration of the mixture. Preferably, the collection procedures and storage are carried out under sterile conditions.
  • the first EM field has a frequency in the range of 11,141 to 12,842 MHz and a field strength in the range of 230 to 440 mV/cm.
  • the temperature is maintained at 28°C to 32°C, and typically at 30°C.
  • the yeast culture is exposed to this first EM field for about 4 to 50 hours.
  • the second EM field has a frequency in the range of 12,031 to 12,900
  • the temperature is maintained at 28°C to 32°C, and typically at 30°C.
  • the yeast culture is exposed to this second EM field for 10 to 52 hours.
  • the activated yeast cells are conditioned by culturing in both of the above-mentioned EM fields.
  • the yeast cells are conditioned in the two different EM fields in a different order.
  • a series of EM fields having field characteristics within the ranges stated above can be applied to condition the yeast cells.
  • the yeast cells can remain in the same container and use the same set of electromagnetic wave generator and emitters when switching from one EM field to another EM field.
  • the cell density of the culture at the end of the activation and conditioning process is typically greater than about 10 7 to 10 10 cells per ml (estimated by hematocytometer).
  • the activated and conditioned yeast cells may be recovered from the culture by various methods known in the art, and stored at a temperature below about 0°C to 4°C.
  • the activated and conditioned yeast cells can be used directly in a biological composition or used as a starter culture for large scale manufacturing.
  • the activated and conditioned yeast cells recovered from the liquid culture may be dried and stored in powder form.
  • the powder form of the activated and conditioned yeast cells comprises greater than about 10 to 10 yeast cells per gram.
  • the present invention also encompasses methods of manufacturing of the biological compositions of the invention at a large scale.
  • the activated and conditioned yeast cells as prepared above are propagated on a large scale to make the biological compositions of the invention.
  • the method comprises culturing the yeast cells in the presence of one or more EM fields for a period of time, diluting the growing yeast cells with fresh medium, and repeating the process.
  • the method can be carried out as a batch process or a continuous process.
  • a set of three containers (5, 6, 7) each comprising a set of electrodes for generating an electromagnetic field as described above are set up each with 1,000 liters of a culture medium. See Figure 2.
  • the culture medium comprises nutrients assimilable by the yeast cells as shown in Table 2.
  • the wild hawthorn juice is an extract of fresh wild hawthorn fruits prepared by washing the fruits clean, drying the fruits in air or using a commercial dryer to less than 8% moisture, crushing the dried fruits to less than 20 mesh, and mixing the crushed wild hawthorn with water at a ratio of 400 liters of water per 100 kg of crushed fruits.
  • the mixture is then stined continuously for 12 hours while the temperature is maintained at 28°C to 30°C.
  • the mixture is then centrifuged at 1,000 rpm to collect the supernatant which is used as described above.
  • the procedures are carried out under sterile conditions.
  • the jujube juice is an extract of fresh jujube fruits prepared by washing the fruits clean, drying the fruits to less than 8% moisture, crushing the dried fruits to less than 20 mesh, and mixing the crushed jujube with water at a ratio of 400 liters of water per 100 kg of crushed fruits.
  • the mixture is then stined continuously for 12 hours while the temperature is maintained at 28°C to 30°C.
  • the mixture is then centrifuged at 1,000 rpm to collect the supernatant which is used as described above.
  • the procedures are carried out under sterile conditions.
  • the wu wei zi juice is an extract of fresh berries of Schisandra chinensis plant prepared by washing the berries, drying the fruits to less than 8% moisture, crushing the dried berries to less than 20 mesh, and mixing the crushed berries with water at a ratio of 400 liters of water per 100 kg of crushed benies.
  • the mixture is then stined continuously for 12 hours while the temperature is maintained at 28°C to 30°C.
  • the mixture is then centrifuged at 1,000 rpm to collect the supernatant which is used as described above.
  • the procedures are carried out under sterile conditions.
  • the soybean juice is prepared by washing the soybeans, drying the soybeans to less than 8% moisture, crushing the soybeans to less than 20 mesh, and mixing the crushed soybeans with water. For 30 kg of soybeans, 130 liters of water is used. The mixture is then stined continuously for 12 hours while the temperature is maintained at 28°C to 30°C. The mixture is then centrifuged at 1,000 rpm to collect the supernatant which is used as described above. Preferably, the procedures are carried out under sterile conditions.
  • the first container is inoculated with activated or activated and conditioned yeast cells as prepared by the methods as set forth above.
  • About 1,000 gram of dried yeast powder are added to 1,000 liter of culture medium.
  • Each gram of the dried yeast powder comprises about 10 10 yeast cells.
  • an equivalent number of yeast cells in a liquid medium can also be used, preferably greater than about 10 6 to 10 9 cells per ml, more preferably about 10 7 cells per ml.
  • the yeast cells in the first container (5) are then subjected to a series of two EM fields.
  • the frequency is in the range of 11,141 to 12,842 MHz and the field strength is in the range of 170 to 450 mV/cm.
  • the yeast culture is exposed to this first EM field for about 4 to 25 hours.
  • the yeast cells are then subjected to a second EM field having a frequency in the range of 12,031 to 12,900 MHz and a field strength in the range of 120 to 450 mV/cm.
  • the yeast culture is exposed to this second EM field for about 4 to 24 hours.
  • the yeast cells from the first container are then transfened to the second container which contains about 1,000 liter of the culture medium.
  • the first yeast culture is diluted by about 50% with fresh culture medium.
  • the yeast cells are again subjected to a series of two EM fields. The frequencies used in the second container are similar to those used in the first container.
  • the yeast cells from the second container are then transfened to the third container which contains yet another 1,000 liter of the culture medium. Again, the second yeast culture is diluted by about 50% with fresh culture medium.
  • the yeast cells are again subjected to a series of two EM fields.
  • the frequencies used in the third container are similar to those used in the first and second container.
  • the yeast cell culture resulting from the end of this stage can be used directly as an oral composition of the invention, or used to form other compositions encompassed by the invention. '
  • the cell density of the culture at the end of the large scale manufacturing process is typically greater than about 10 8 to 10 10 cells per ml (estimated by hematocytometer).
  • the concentration of yeast cells in the medium can be concentrated or diluted accordingly.
  • the concentration of yeast cells in the medium is in the range of 10 3 to 10 10 cells per ml.
  • the concentration of yeast cells in the medium is in the range of 10 3 to 10 6 cells per ml.
  • the concentration of yeast cells in the medium is greater than 10 6 to 10 10 cells per ml.
  • the concentration of yeast cells in the medium is in the range of 10 6 to 5 x 10 8 cells per ml.
  • yeast cell culture can be added to the yeast cell culture.
  • various downstream and packaging process be carried out below room temperature, and preferably at 0°C to 4°C.
  • the yeast cell culture can be packaged in liquid containers.
  • the activated and conditioned yeast cells can be dried as follows.
  • the yeast cell culture is first centrifuged under 75 to 100 g for 10 to 20 minutes to remove the supernatant.
  • the residue which may contain up to 85% moisture is dried in a first dryer at a temperature not exceeding 60 ⁇ 2°C for a period of 5 minutes so that yeast cells quickly became dormant.
  • the yeast cells were then sent to a second dryer and dried at a temperature not exceeding 65 ⁇ 2°C for a period of about 8 minutes to further remove at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of water.
  • the yeast cells may be dried to remove at least 88% of water so the dried yeast cells may contain up to 12% moisture.
  • the dried yeast cells can be packaged by standard pharmaceutical methods in various solid dosage form, each containing a predetermined amount of the dried material.
  • the dried material comprises about 10 5 to 10 11 cells per gram.
  • the dried material comprises about 10 8 to 5 x 10 10 cells per gram.
  • the dried material comprises about 5 x 10 8 cells per gram.
  • compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers.
  • the present invention further provides methods of use of the biological compositions of the invention.
  • the biological composition is used as a medicament for treatment of cancer.
  • the biological composition is used as a dietary supplement, health food, or health drink.
  • the methods comprise administering an effective amount of the biological composition to a subject in need.
  • the biological composition may be administered orally, in liquid or solid form, or enterally through a feeding tube.
  • an effective amount means an amount sufficient to provide a therapeutic or healthful benefit in the context of cancer.
  • the biological composition can produce a healthful benefit in a subject suffering from cancer.
  • the subject is preferably a mammal such as a non-primate (e.g., a cow, pig, horse, cat, dog, rat, mouse, rabbit, etc.) or a primate (e.g., a monkey, chimpanzee, human, etc.).
  • a non-primate e.g., a cow, pig, horse, cat, dog, rat, mouse, rabbit, etc.
  • a primate e.g., a monkey, chimpanzee, human, etc.
  • the subject is non-human.
  • the subject is a human being.
  • the subject in need is one who is diagnosed with cancer with or without metastasis, at any stage of the disease (e.g., TNM staging by the American Joint Committee for Cancer (AJCC) published in 1988, or other staging system acceptable in the art).
  • the subject may be a cancer patient who is receiving concurrently other treatment modalities against the cancer.
  • the subject can be a cancer patient who had undergone a regimen of treatment (e.g., chemotherapy and/or radiation) and whose cancer is regressing.
  • the subject may be a cancer patient who had undergone a regimen of treatment (e.g., surgery) and who appears to be clinically free of the cancer.
  • the biological composition of the invention can be administered adjunctively with any of the treatment modalities, such as but not limited to chemotherapy, radiation, and/or surgery.
  • the biological composition can be used in combination with one or more chemotherapeutic or immunotherapeutic agents, such as anastrozole (Arimidex®), amsacrine (AMSA), L-asparaginase (Elspar®), bleomycin, bleomycin sulfate (Blenoxane®), busulfan (Myleran®), carboplatin (Paraplatin®), carmustine (BCNU®, Gliadel®, BiCNU®), celecoxib (Celebrex®), cetuximab (IMC-C225 or ErbituxTM)), cisplatin (platinum analogs, Platinol®), chlorambucil (Leukeran®), cladribine (2-chlorodeoxyadenosine; "2- CDA”; Leustatin®), cyclophosphamide
  • the subject may be one who has not yet been diagnosed with cancer but are predisposed to or at high risk of developing as a result of genetic factors and or environmental factors.
  • the subject may also be one who displays characteristics that are associated with a high risk of cancer such as nodules detected by computer tomographic scanning or suspect cells in biopsy and/or body fluids.
  • the therapeutic and healthful benefits range from inhibiting or retarding the growth of the cancer and/or the spread of the cancer to other parts of the body (i.e., metastasis), palliating the symptoms of the cancer, improving the probability of survival of the subject with the cancer, prolonging the life expectancy of the subject, improving the quality of life of the subject, and/or reducing the probability of relapse after a successful course of treatment (e.g., surgery, chemotherapy or radiation).
  • the invention provides a method for retarding the growth of cancer cells in a subject, such as a human, comprising administering orally to the subject a biological composition of the invention.
  • the invention also provide a method for prolonging the time of survival of a subject inflicted with cancer preferably a human patient, comprising administering orally to the subject a biological composition of the invention.
  • the effective dose will vary with the subject treated.
  • the effective dose for the subject will also vary with the condition to be treated and the severity of the condition to be treated.
  • the dose, and perhaps the dose frequency, will also vary according to the age, body weight, and response of the individual subject.
  • the total daily dose range of activated and conditioned yeast cells for a subject inflicted with cancer is from about 10 5 to 10 11 cells per day; preferably, about 10 8 to 5 x 10 10 cells per day; more preferably, about 2 x 10 9 cells per day in powder form or 9 in x lO to 1 x 10 cells per day in liquid preparations, administered in single or divided doses orally.
  • the length of time for a course of treatment should be at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 7 weeks, at least 10 weeks, at least 13 weeks, at least 15 weeks, at least 20 weeks, at least 6 months, or at least 1 year. It may be necessary to use dosages outside these ranges in some cases as will be apparent to those skilled in the art.
  • the oral compositions can be administered for a period of time until the symptoms and/or infection of the patients by the bacteria and viruses are under control, or when the disease has regressed partially or completely.
  • the total daily dose range should be from about 10 5 to 10 11 cells per day; preferably, about 5 x 10 7 to 5 x 10 9 cells per day.
  • the oral compositions can be administered as a dietary supplement for as long as 6 months, or in accordance with recommended length of use under the Dietary Supplement Health and Education Act (DSHEA) or other government or industry guidelines. Further, it is noted that the nutritionist, dietician, clinician or treating physician will know how and when to interrupt, adjust, or terminate use of the biological composition as a medicament or dietary supplement in conjunction with individual patient response.
  • DHEA Dietary Supplement Health and Education Act
  • the effect of the biological compositions of the invention on development and progression of cancer can be monitored by any methods known to one skilled in the art, including but not limited to measuring: a) changes in the size and morphology of the tumor using imaging techniques such as a computed tomographic (CT) scan or a sonogram; and b) changes in levels of biological markers of risk for cancer.
  • imaging techniques such as a computed tomographic (CT) scan or a sonogram
  • the biological compositions of the present invention comprise activated and conditioned live yeast cells prepared as described above, as active ingredient, and can optionally contain a pharmaceutically acceptable carrier or excipient, and/or other ingredients provided that these ingredients do not kill or inhibit the yeast cells.
  • Other ingredients that can be incorporated into the biological compositions of the present invention may include, but are not limited to, herbs (including traditional Chinese medicine products), herbal extracts, vitamins, amino acids, metal salts, metal chelates, coloring agents, flavor enhancers, preservatives, and the like.
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, or tablets, each containing a predetermined amount of activated and conditioned yeast cells, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion.
  • compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
  • Such products can be used as pharmaceuticals or dietary supplements, depending on the dosage and circumstances of its use.
  • the oral compositions of the present invention may additionally include binding agents (e.g., pregelatinized maize starch, polyvinylpynolidone or hydroxypropyl methylcellulose); binders or fillers (e.g., lactose, pentosan, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinized maize starch, polyvinylpynolidone or hydroxypropyl methylcellulose
  • binders or fillers e.g., lactose, pentosan, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch
  • Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use.
  • the temperature of the liquid used to reconstitute the dried product should be less than 65°C.
  • Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils
  • preservatives e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid
  • the preparations can also be made to
  • the oral composition can be produced by diluting or concentrating the yeast culture medium produced by the method set forth herein.
  • the oral composition is a cell suspension containing about 10 3 to 10 6 cells per ml.
  • the oral composition is a cell suspension containing greater than about 10 6 to 10 10 cells per ml.
  • the oral composition is a cell suspension containing about 10 6 to 5 x 10 8 cells per ml.
  • the oral composition can be formulated as a health drink and packaged in liquid containers, each containing a predetermined amount of the liquid yeast culture.
  • oral compositions packaged in liquid containers each comprising about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 15 ml, 20 ml, 30 ml, 40 ml, 50 ml, 75 ml, 100 ml, 150 ml, 200 ml, 250 ml, 500 ml, 750 ml, or 1,000 ml of the live yeast cells.
  • the number of container to be taken each day to obtain the total daily dose in a subject depends on the number of activated and conditioned yeast cells contained within each container.
  • a container may comprise 50 ml of liquid with 10 7 cells per ml and when a total daily dose of about 2 x 10 9 cells per day is desired, a subject can drink 4 containers per day to obtain the desired total daily dose.
  • the composition is a capsule.
  • the capsules can be formulated by any commercially available methods.
  • the composition is a capsule containing 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1.0 gram, 1.25 gram, 1.5 gram, or 2.0 gram of live yeast cells in powder form.
  • the powder in the capsule comprises about 10 5 to about 10 ⁇ cells per gram; more preferably, about 10 8 to 5 x 10 cells per gram; and most preferably, about 5 x 10 cells per gram.
  • the number of capsule to be taken each day to obtain the total daily dose in a subject depends on the number of activated and conditioned yeast cells contained within each capsule.
  • a capsule may comprise about 500 mg of powder with 5 x 10 8 cells per gram.
  • a subject can take two capsules at a time for four times per day.
  • the biological compositions comprising activated and conditioned yeast cells can be added directly to foods so that an effective amount of yeast cells is ingested during normal meals. Any methods known to those skilled in the art may be used to add to or incorporate the biological compositions into natural or processed foods, provided that the activated and conditioned yeast cells remain viable.
  • the nutritional compositions of the invention are made and stored under conditions, such as temperature, from about 0°C to 4°C.
  • the term "food” broadly refers to any kind of material, liquid or solid, that is used for nourishing an animal, and for sustaining normal or accelerated growth of an animal including humans.
  • the invention can be further defined by reference to the following prefened embodiments.
  • the use of the cunent invention for treatment of eighteen (18) commonly occurring cancers is set forth in detail below. However, it is to be understood that these embodiments are provided for illustrative purpose only and thus should not be interpreted to limit the scope of the cunent invention.
  • yeast cell strains were used to prepare the biological compositions that were administered to the animals used in the respective human cancer type models.
  • the following electromagnetic (EM) fields can be used to activate the yeast cells to prepare the biological compositions of the invention.
  • the yeast cells as set forth above in Table 3 were cultured in a liquid medium comprising one or more of sucrose or glucose (10-30 g), soluble starch (10 g), mannitol (10 g), vitamin A (40- 60 ⁇ g), vitamin B 2 (40-50 ⁇ g), vitamin B 3 (40-60 ⁇ g), vitamin B 6 (30-80 ⁇ g), vitamin B ⁇ 2 (30-60 ⁇ g), vitamin C (40-50 ⁇ g), vitamin D (20 ⁇ g), vitamin H (20-70 ⁇ g), bovine calf serum (30-35 ml), fetal calf serum (25-45 ml), KH 2 PO 4 (0.20 g), MgSO 4 .7H 2 O (0.20-0.25 g), NaCI (0.20-0.30 g), CaSO 4 .2H 2 O (0.20-0.30 g), CaCO 3 .5H 2 O (3.0-
  • the following electromagnetic (EM) fields can be used to condition the activated yeast cells in the biological compositions of the invention.
  • the activated yeast cells were conditioned by culturing in a liquid medium comprising wild hawthorn juice and gastric juice of a mammal, and subjected to at least one of the two EM fields as shown below in Table 5 (numbers shown in parentheses are for the prefened embodiments) by following the general procedures set forth in the Detailed Description of the Invention.
  • the invention provides a method for preparing a biological composition useful for producing a healthful benefit in a subject suffering from lung cancer.
  • lung cancer includes but is not limited to squamous cell carcinoma, small cell carcinoma, adenocarcinoma, large cell carcinoma, carcinoid tumor, and mesothelioma.
  • the biological composition can ameliorate, reduce, manage, or eliminate the symptoms associated with lung cancer, such as cough, shortness of breath, wheezing, chest pain, hemoptysis (bloody, coughed-up sputum), loss of appetite, weight loss, pneumonia (inflammation of the lungs), weakness, chills, swallowing difficulties, speech difficulties or changes (e.g., hoarseness), finger/nail abnormalities (e.g., "clubbing,” or overgrowth of the fingertip tissue), skin paleness or bluish discoloration, muscle contractions or atrophy (shrinkage), joint pain or swelling, facial swelling or paralysis, eyelid drooping, bone pain/tenderness, and breast development in men.
  • lung cancer such as cough, shortness of breath, wheezing, chest pain, hemoptysis (bloody, coughed-up sputum), loss of appetite, weight loss, pneumonia (inflammation of the lungs), weakness, chills, swallowing difficulties, speech difficulties or changes (e.
  • the biological composition useful for treatment of lung cancer comprises activated and conditioned Saccharomyces cerevisiae Hansen strain IFFI1345 yeast cells cultured in a series of alternating electromagnetic (EM) fields.
  • the method for preparing said biological composition comprises in any order the steps of:
  • the first liquid medium comprises 20 g of sucrose or glucose, 40 ⁇ g of vitamin B 2 , 30 ⁇ g of vitamin B 6 , 30 ⁇ g of vitamin B 12 , 0.20 g of KH 2 PO 4 , 0.22 g of MgSO 4 .7H 2 O, 0.30 g of NaCI, 0.30 g of CaSO 4 .2H 2 0, 4.0 g of CaCO 3 .5H 2 0, 2.5 g of peptone, and 1,000 ml of autoclaved water.
  • the second liquid medium comprises wild hawthorn juice and gastric juice of a mammal.
  • the number of activated and conditioned yeast cells can be further expanded by:
  • mV/cm preferably at three fields strengths, e.g., in the order of 422 mV/cm, 341 mV/cm, and 234 mV/cm; and (i) culturing the yeast cells in a third liquid medium in a ninth electromagnetic field or series of electromagnetic fields having a frequency at 12,755 MHz and a field strength in the range of 145 to
  • the third liquid medium comprises wild hawthorn juice, jujube juice, wu wei zi juice, and soybean juice.
  • the invention provides a method for preparing a biological composition useful for producing a healthful benefit in a subject suffering from nasopharyngeal cancer.
  • nasopharyngeal cancer includes but is not limited to squamous cell carcinoma, nonkeratinizing carcinomas, undifferentiated carcinomas and keratinizing carcinoma.
  • the biological compositions can ameliorate, reduce, manage, or eliminate the symptoms of the cancer, improving the probability of survival of the subject with the cancer, prolonging the life expectancy of the subject, improving the quality of life of the subj ect, and/or reducing the probability of relapse after a successful course of treatment (e.g., surgery, chemotherapy or radiation).
  • the symptoms associated with nasopharyngeal cancer include neck mass, hearing loss, ipsilateral serous otitis, hearing loss, nasal obstruction, frank epistaxis, purulent or bloody rhinonhea, and facial neuropathy or facial nerve palsies.
  • the biological composition useful for treatment of nasopharyngeal cancer comprises activated and conditioned Saccharomyces carlsbergensis Hansen strain AS2.116 yeast cells cultured in a series of alternating electromagnetic (EM) fields.
  • EM electromagnetic
  • the method for preparing said biological composition comprises in any order the steps of: (a) culturing the yeast cells in a first liquid medium in a first electromagnetic field having a frequency at 9,963 MHz and a field strength of 246 mV/cm;
  • the first liquid medium comprises 20 g of sucrose or glucose, 40 ⁇ g of vitamin B 6 , 40 ⁇ g of vitamin B 12 , 70 ⁇ g of vitamin H, 25 ml of fetal calf serum, 0.20 g of KH 2 PO 4 , 0.25 g of MgSO 4 .7H 2 O, 0.30 g of NaCI, 0.20 g of CaSO 4 .2H 2 0, 4.0 g of CaCO 3 .5H 2 O, 2.5 g of peptone, and 1,000 ml of autoclaved water.
  • the second liquid medium comprises wild hawthorn juice and gastric juice of a mammal.
  • the number of activated and conditioned yeast cells can be further expanded by:
  • the third liquid medium comprises wild hawthorn juice, jujube juice, wu wei zi juice, and soybean juice.
  • the invention provides a method for preparing a biological composition useful for producing a healthful benefit in a subject suffering from esophageal cancer.
  • esophageal cancer includes but is not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, carcinosarcoma, pseudosarcoma, sarcomas, melanoma, plasmacytoma, verrucous carcinoma and oat cell carcinoma.
  • the biological compositions can ameliorate, reduce, manage, or eliminate the symptoms associated with esophageal cancer include dysphagia, difficulty swallowing solids or liquids, regurgitation of food, heartburn, vomiting blood and chest plan unrelated to eating.
  • the biological composition useful for treatment of esophageal cancer comprises activated and conditioned are Saccharomyces cerevisiae Hansen strain AS2.375 yeast cells cultured in a series of alternating electromagnetic (EM) fields.
  • EM electromagnetic
  • the first liquid medium comprises 10 g of sucrose or glucose, 10 g of soluble starch, 50 ⁇ g of vitamin B 2 , 50 ⁇ g of vitamin B 6 , 20 ⁇ g of vitamin B ⁇ 2 , 35 ml of fetal calf serum, 0.20 g of KH 2 PO 4 , 0.25 g of MgSO 4 .7H 2 O, 0.30 g of NaCI, 0.20 g of CaSO .2H 2 O, 4.0 g of CaCO 5H 2 O, 2.5 g of peptone, and 1,000 ml of autoclaved water.
  • the second liquid medium comprises wild hawthorn juice and gastric juice of a mammal.
  • the number of activated and conditioned yeast cells can be further expanded by:
  • 340 mV/cm preferably at three fields strengths, e.g., in the order of 322 mV/cm, 332 mV/cm, and 211 mV/cm; and (i) culturing the yeast cells in a third liquid medium in a ninth electromagnetic field or series of electromagnetic fields having a frequency at 12,652 MHz and a field strength in the range of 230 to
  • the third liquid medium comprises wild hawthorn juice, jujube juice, wu wei zi juice, and soybean juice.
  • the invention provides a method for preparing a biological composition useful for producing a healthful benefit in a subject suffering from stomach cancer.
  • tissue cancer includes but is not limited to malignant cancer such as adenocarcinoma, papillary adenocarcinoma, tubular adenocarcinoma, mucinous adenocarcinoma, signet ring cell carcinoma, adenosquamous carcinoma, carcinoid tumor, mixed carcinoid- adenocarcinoma, small cell carcinoma (M80413), and undifferentiated carcinoma.
  • the biological compositions can ameliorate, reduce, manage, or eliminate the symptoms associated with stomach cancer include loss of appetite, difficulty in swallowing, vague fullness, vomiting blood, abdominal pain, belching, breath odor, excessive gas and flatus, heartburn, weight loss, and a decline in general health.
  • the biological composition useful for treatment of stomach cancer comprises activated and conditioned are Saccharomyces cerevisiae Hansen strain AS2.14 yeast cells cultured in a series of alternating electromagnetic (EM) fields.
  • the method for preparing said biological composition comprises in any order the steps of: (a) culturing the yeast cells in a first liquid medium in a first electromagnetic field having a frequency at 7,987 MHz and a field strength of 286 mV/cm;
  • the first liquid medium comprises 20 g of sucrose or glucose, 40 ⁇ g of vitamin B , 30 ⁇ g of vitamin B 6 , 50 ⁇ g of vitamin C, 25 ml of fetal calf serum, 0.20 g of KH 2 PO 4 , 0.25 g of MgSO 4 .7H 2 O, 0.30 g of NaCI, 0.20 g of CaSO 4 .2H 2 0, 4.0 g of CaCO 3 .5H 2 O, 2.5 g of peptone, and 1,000 ml of autoclaved water.
  • the second liquid medium comprises wild hawthorn juice and gastric juice of a mammal.
  • the number of activated and conditioned yeast cells can be further expanded by:
  • yeast cells in a third liquid medium in a ninth electromagnetic field or series of electromagnetic fields having a frequency at 12,171 MHz and a field strength in the range of 230 to 410 mV/cm, preferably at three fields strengths, e.g., in the order of
  • the third liquid medium comprises wild hawthorn juice, jujube juice, wu wei zi juice, and soybean juice.
  • the invention provides a method for preparing a biological composition useful for producing a healthful benefit in a subject suffering from colorectal cancer.
  • colonal cancer includes but is not limited to squamous cell (epidermoid) carcinomas, cloacogenic (basaloid transitional cell) tumors, and adenocarcinomas.
  • the biological compositions can ameliorate, reduce, manage, or eliminate the symptoms associated with colorectal cancer include constipation, blood in the stool, unexplained anemia, abdominal pain and tenderness in the lower abdomen, intestinal obstruction, weight loss with no known reason, stools nanower than usual, constant tiredness, and anal lump.
  • the biological composition useful for treatment of colorectal cancer comprises activated and conditioned are Saccharomyces cerevisiae Hansen strain AS2.1396 yeast cells cultured in a series of alternating electromagnetic (EM) fields.
  • the method for preparing said biological composition comprises in any order the steps of: (a) culturing the yeast cells in a first liquid medium in a first electromagnetic field having a frequency at 7,948 MHz and a field strength of 231 mV/cm; (b) culturing the yeast cells in a first liquid medium in a second electromagnetic field having a frequency at 9,135 MHz and a field strength of 221 mV/cm;
  • the first liquid medium comprises 20 g of sucrose or glucose, 50 ⁇ g of vitamin B 6 , 50 ⁇ g of vitamin B ⁇ 2 , 30 ml of fetal calf serum, 60 ⁇ g of vitamin A, 0.20 g of KH 2 PO 4 , 0.25 g of MgSO 4 .7H 2 O, 0.30 g of NaCI, 0.20 g of CaSO .2H 2 0, 4.0 g of CaCO 3 .5H 2 0, 2.5 g of peptone, and 1,000 ml of autoclaved water.
  • the second liquid medium comprises wild hawthorn juice and gastric juice of a mammal.
  • the number of activated and conditioned yeast cells can be further expanded by: (h) culturing the yeast cells in a third liquid medium in an eighth electromagnetic field or series of electromagnetic fields having a frequency at 11,148 MHz and a field strength in the range of 180 to 300 mV/cm, preferably at three fields strengths, e.g., in the order of 286 mV/cm, 292 mV/cm, and 188 mV/cm; and (i) culturing the yeast cells in a third liquid medium in a ninth electromagnetic field or series of electromagnetic fields having a frequency at 12,778 MHz and a field strength in the range of 230 to 350 mV/cm, preferably at three fields strengths, e.g., in the order of 322 mV/cm, 332 mV/cm, and 236 mV/cm.
  • the third liquid medium comprises wild hawthorn juice, jujube juice, wu wei zi juice, and soybean juice.
  • the invention provides a method for preparing a biological composition useful for producing a healthful benefit in a subject suffering from pancreatic cancer.
  • pancreatic cancer includes but is not limited to adenocarcinomas, acinar cell carcinoma, cystadenocarcinoma (mucinous), adenosquamous carcinoma, solid microglandular carcinoma, carcinoid, sarcoma, and malignant lymphoma.
  • the biological compositions can ameliorate, reduce, manage, or eliminate the symptoms associated with pancreatic cancer include abdominal pain, unexpected weight loss, nausea, loss of appetite, weight loss, digestive problems, jaundice, or yellowing of the skin, restlessness, loss of energy, irritability, sweating, tremor, drowsiness and severe confusion.
  • the biological composition useful for treatment of pancreatic cancer comprises activated and conditioned are Saccharomyces cerevisiae Hansen strain IFFI 1413 yeast cells cultured in a series of alternating electromagnetic (EM) fields.
  • EM alternating electromagnetic
  • the first liquid medium comprises 20 g of sucrose or glucose, 50 ⁇ g of vitamin B 3 , 30 ⁇ g of vitamin B 12 , 20 ⁇ g of vitamin H, 40 ml of fetal calf serum, 0.20 g of KH 2 PO 4 , 0.25 g of MgSO 4 .7H 2 O, 0.30 g of NaCI, 0.20 g of CaSO 4 .2H 2 0, 4.0 g of CaCO 3 .5H 2 0, 2.5 g of peptone, and 1,000 ml of autoclaved water.
  • the second liquid medium comprises wild hawthorn juice and gastric juice of a mammal.
  • the number of activated and conditioned yeast cells can be further expanded by:
  • 340 mV/cm preferably at three fields strengths, e.g., in the order of 328 mV/cm, 334 mV/cm, and 238 mV/cm; and (i) culturing the yeast cells in a third liquid medium in a ninth electromagnetic field or series of electromagnetic fields having a frequency at 12,764 MHz and a field strength in the range of 250 to
  • 370 mV/cm preferably at three fields strengths, e.g., in the order of 338 mV/cm, 362 mV/cm, and 263 mV/cm.
  • the third liquid medium comprises wild hawthorn juice, jujube juice, wu wei zi juice, and soybean juice.
  • the invention provides a method for preparing a biological composition useful for producing a healthful benefit in a subject suffering from liver cancer.
  • liver cancer includes but is not limited to hepatocellular carcinoma, cholangiocarcinoma, mixed hepatocellular cholangiocarcinoma, angiosarcoma, fibrolamellar, cystadenoma, and epithelioid hemangioendothelioma.
  • the biological compositions can ameliorate, reduce, manage, or eliminate the symptoms associated with liver cancer include general malaise as well as pain and tenderness, unexplained weight loss, persistent lack of appetite, fever of unknown origin, limb weakness, sensory loss, persistent abdominal pain, immature feeling of fullness, swelling of the abdominal are with or without breathing difficulties, sudden jaundice, and liver enlargement or a mass that can be felt in the liver area.
  • the biological composition useful for treatment of liver cancer comprises activated and conditioned are Saccharomyces cerevisiae Hansen strain AS2.503 yeast cells cultured in a series of alternating electromagnetic (EM) fields.
  • the method for preparing said biological composition comprises in any order the steps of: (a) culturing the yeast cells in a first liquid medium in a first electromagnetic field having a at 7,976 MHz and a field strength of 335 mV/cm;
  • the first liquid medium comprises 30 g of sucrose or glucose, 40 ⁇ g of vitamin B 6 , 30 ⁇ g of vitamin H, 40 ⁇ g of vitamin A, 30 ml of bovine calf serum, 0.20 g of KH 2 PO 4 , 0.20 g of MgSO 4 .7H 2 O, 0.20 g of NaCI, 0.20 g of CaSO 4 .2H 2 0, 3.0 g of CaCO 3 .5H 2 0, 2.5 g of peptone, and 1,000 ml of autoclaved water.
  • the second liquid medium comprises wild hawthorn juice and gastric juice of a mammal.
  • the number of activated and conditioned yeast cells can be further expanded by:
  • the third liquid medium comprises wild hawthorn juice, jujube juice, wu wei zi juice, and soybean juice.
  • the invention provides a method for preparing a biological composition useful for producing a healthful benefit in a subject suffering from testicular cancer.
  • testicular cancer includes but is not limited to malignant cancer such as seminomas, nonseminomas, choriocarcinoma, embryonal carcinoma, immature teratoma, yolk sac tumors, Leydig and sertoli cell tumors, PNET, leiomyosarcoma, rhabdomyosarcoma, and mesothelioma.
  • the biological compositions can ameliorate, reduce, manage, or eliminate the symptoms associated with testicular cancer include a painless lump, a hardening or a change (increase or decrease) in size of the testicle, a feeling of heaviness or a sudden collection of fluid in the scrotum, a dull ache in the lower abdomen or in the groin, or pain or discomfort in the scrotum or testicle.
  • the biological composition useful for treatment of testicular cancer comprises activated and conditioned are Saccharomyces carlsbergensis Hansen strain AS2.116 yeast cells cultured in a series of alternating electromagnetic (EM) fields.
  • the method for preparing said biological composition comprises in any order the steps of:
  • the first liquid medium comprises 20 g of sucrose or glucose, 60 ⁇ g of vitamin B ⁇ 2 , 60 ⁇ g of vitamin B 3 , 60 ⁇ g of vitamin H, 50 ⁇ g of vitamin B 6 , 30 ml of fetal calf serum, 0.20 g of KH 2 PO 4 , 0.25 g of MgS0 4 .7H 2 O, 0.30 g of NaCI, 0.20 g of CaSO 4 .2H 2 0, 4.0 g of CaCO 3 .5H 2 O, 2.5 g of peptone, and 1,000 ml of autoclaved water.
  • the second liquid medium comprises wild hawthorn juice and gastric juice of a mammal.
  • the number of activated and conditioned yeast cells can be further expanded by:
  • the third liquid medium comprises wild hawthorn juice, jujube juice, wu wei zi juice, and soybean juice.
  • the invention provides a method for preparing a biological composition useful for producing a healthful benefit in a subject suffering from prostate cancer.
  • the term "prostate cancer” includes but is not limited to adenocarcinomas.
  • the biological compositions can ameliorate, reduce, manage, or eliminate the symptoms associated with prostate cancer include frequent urination (especially at night) or an inability to urinate, trouble starting or holding back urine, pain during ejaculation or urination, a weak or interrupted urine flow, blood in the semen or in the urine, or frequent pain or stiffness in the lower back, hips, or upper thighs.
  • the biological composition useful for treatment of prostate cancer comprises activated and conditioned are Saccharomyces carlsbergensis Hansen strain AS2.440 yeast cells cultured in a series of alternating electromagnetic (EM) fields.
  • the method for preparing said biological composition comprises in any order the steps of: (a) culturing the yeast cells in a first liquid medium in a first electromagnetic field having a frequency at 10,164 MHz and a field strength of 239 mV/cm;
  • the first liquid medium comprises 10 g of sucrose or glucose, 10 g of mannitol, 80 ⁇ g of vitamin B 6 , 50 ⁇ g of vitamin B 3 , 60 ⁇ g of vitamin H, 60 ⁇ g of vitamin A, 30 ml of bovine calf serum, 0.20 g of KH 2 PO 4 , 0.25 g of MgSO 4 .7H 2 O, 0.30 g of NaCI, 0.20 g of CaSO 4 .2H 2 0, 4.0 g of CaCO 3 .5H 2 O, 2.5 g of peptone, and 1,000 ml of autoclaved water.
  • the second liquid medium comprises wild hawthorn juice and gastric juice of a mammal.
  • the number of activated and conditioned yeast cells can be further expanded by: (h) culturing the yeast cells in a third liquid medium in an eighth electromagnetic field or series of electromagnetic fields having a frequency at 12,541 MHz and a field strength in the range of 170 to 280 mV/cm, preferably at three fields strengths, e.g., in the order of 260 mV/cm, 267 mV/cm, and 188 mV/cm ; and (i) culturing the yeast cells in a third liquid medium in a ninth electromagnetic field or series of electromagnetic fields having a frequency 12,661 MHz and a field strength in the range of 230 to 340 mV/cm, preferably at three fields strengths, e.g., in the order of 322 mV/cm, 338 mV/cm, and 245 mV/cm .
  • the third liquid medium comprises wild hawthorn juice, jujube juice, wu wei zi juice, and soybean
  • the invention provides a method for preparing a biological composition useful for producing a healthful benefit in a subject suffering from ovarian cancer.
  • ovarian cancer includes but is not limited to serous carcinoma and those arising from the celomic epithelium, specialized stroma, and germ cell layer or unfertilized ovum.
  • the biological compositions can ameliorate, reduce, manage, or eliminate the symptoms associated with ovarian cancer include abdominal pain, abdominal swelling, bloating or dyspepsia, pelvic pressure, weight gain or loss, abnormal menstrual cycles, increased abdominal girth, vaginal bleeding, excessive hair, and increased urinary frequency or urgency.
  • the biological composition useful for treatment of ovarian cancer comprises activated and conditioned are Saccharomyces cerevisiae Hansen strain AS2.502 yeast cells cultured in a series of alternating electromagnetic (EM) fields.
  • the method for preparing said biological composition comprises in any order the steps of:
  • the first liquid medium comprises 20 g of sucrose or glucose, 40 ⁇ g of vitamin B 12 , 30 ⁇ g of vitamin B 6 , 50 ⁇ g of vitamin A, 40 ⁇ g of vitamin C, 35 ml of bovine calf serum, 0.20 g of KH 2 PO 4 , 0.25 g of MgSO 4 .7H 2 O, 0.30 g of NaCI, 0.20 g of CaSO .2H 2 0, 4.0 g of CaCO 3 .5H 2 0, 2.5 g of peptone, and 1,000 ml of autoclaved water.
  • the second liquid medium comprises wild hawthorn juice and gastric juice of a mammal.
  • the number of activated and conditioned yeast cells can be further expanded by:
  • 340 mV/cm preferably at three fields strengths, e.g., in the order of 311 mV/cm, 323 mV/cm, and 236 mV/cm; and (i) culturing the yeast cells in a third liquid medium in a ninth electromagnetic field or series of electromagnetic fields having a frequency 12,786 MHz and a field strength in the range of 220 to 350 mV/cm, preferably at three fields strengths, e.g., in the order of 322 mV/cm, 342 mV/cm, and 234 mV/cm.
  • the third liquid medium comprises wild hawthorn juice, jujube juice, wu wei zi juice, and soybean juice.
  • the invention provides a method for preparing a biological composition useful for producing a healthful benefit in a subject suffering from breast cancer.
  • breast cancer includes but is not limited to malignant epithelial cancer such as adenocarcinomas (ductal carcinomas and lobular carcinomas), squamous cell carcinoma, nonkeratinizing carcinomas, undifferentiated carcinomas, and keratinizing carcinoma, ductal carcinoma in situ (DCIS), infiltrating (or invasive) ductal carcinoma (IDC), infilfrating (or invasive) lobular carcinoma (ILC), inflammatory breast cancer, in situ, lobular carcinoma in situ (LCIS), medullary carcinoma, mucinous carcinoma, phyllodes tumor, tubular carcinoma, and Paget's disease of the nipple.
  • DCIS ductal carcinoma in situ
  • IDC infiltrating (or invasive) ductal carcinoma
  • ILC infilfrating (or invasive) lobular carcinoma
  • LCIS lobular carcinoma in situ
  • the biological compositions can ameliorate, reduce, manage, or eliminate the symptoms associated with breast cancer include breast lump(s), swelling of the skin of the breast, discharge from the nipple, enlargement of the lymph glands of the armpit, nipple erosion or ulceration, diffuse erythema of the breast, and axillary adenopathy.
  • the biological composition useful for treatment of breast cancer comprises activated and conditioned Saccharomyces carlsbergensis Hansen strain AS2.441 yeast cells cultured in a series of alternating electromagnetic (EM) fields.
  • the method for preparing said biological composition comprises in any order the steps of:
  • the first liquid medium comprises 20 g of sucrose or glucose, 30 ⁇ g of vitamin B ⁇ 2 , 50 ⁇ g of vitamin B 3 , 40 ⁇ g of vitamin H, 40 ⁇ g of vitamin C, 35 ml of bovine calf serum, 0.20 g of KH 2 PO 4 , 0.25 g of MgSO 4 .7H 2 O, 0.30 g of NaCI, 0.20 g of CaSO 4 .2H 2 0, 4.0 g of CaCO 3 .5H 2 O, 2.5 g of peptone, and 1 ,000 ml of autoclaved water.
  • the second liquid medium comprises wild hawthorn juice and gastric juice of a mammal.
  • the number of activated and conditioned yeast cells can be further expanded by: (h) culturing the yeast cells in a third liquid medium in an eighth electromagnetic field or series of electromagnetic fields having a frequency at 12,355 MHz and a field strength in the range of 210 to 340 mV/cm, preferably at three fields strengths, e.g., in the order of 312 mV/cm, 322 mV/cm, and 211 mV/cm; and (i) culturing the yeast cells in a third liquid medium in a ninth electromagnetic field or series of electromagnetic fields having a frequency 12,798 MHz and a field strength in the range of 210 to 350 mV/cm, preferably at three fields strengths, e.g., in the order of 317 mV/cm, 348 mV/cm, and 226 mV/cm.
  • the third liquid medium comprises wild hawthorn juice, jujube juice, wu wei zi juice, and soybean juice.
  • the invention provides a method for preparing a biological composition useful for producing a healthful benefit in a subject suffering from cervical cancer.
  • cervical cancer includes but is not limited to adenocarcinomas (ductal carcinomas and lobular carcinomas), squamous cell carcinoma, nonkeratinizing carcinomas, undifferentiated carcinomas, and keratinizing carcinoma.
  • the biological compositions can ameliorate, reduce, manage, or eliminate the symptoms associated with cervical cancer include abnormal bleeding, such as between periods or after intercourse, persistent vaginal discharge, which may be pale, watery, pink, brown, blood streaked, or dark and foul-smelling, and discomfort during intercourse.
  • the biological composition useful for treatment of cervical cancer comprises activated and conditioned Saccharomyces carlsbergensis Hansen strain AS2.444 yeast cells cultured in a series of alternating electromagnetic (EM) fields.
  • the method for preparing said biological composition comprises in any order the steps of:
  • the first liquid medium comprises 20 g of sucrose or glucose, 40 ⁇ g of vitamin B ⁇ 2> 40 ⁇ g of vitamin B 3 , 50 ⁇ g of vitamin H, 50 ⁇ g of vitamin B 6 , 45 ml of fetal calf serum, 0.20 g of KH 2 PO 4 , 0.25 g of MgSO 4 .7H 2 O, 0.30 g of NaCI, 0.20 g of CaSO 4 .2H 2 0, 4.0 g of CaCO 3 .5H 2 O, 2.5 g of peptone, and 1,000 ml of autoclaved water.
  • the second liquid medium comprises wild hawthorn juice and gastric juice of a mammal.
  • the number of activated and conditioned yeast cells can be further expanded by:
  • 330 mV/cm preferably at three fields strengths, e.g., in the order of 305 mV/cm, 315 mV/cm, and 228 mV/cm; and (i) culturing the yeast cells in a third liquid medium in a ninth electromagnetic field or series of elecfromagnetic fields having a frequency 12,792 MHz and a field strength in the range of 240 to 340 mV/cm, preferably at three fields strengths, e.g., in the order of 322 mV/cm, 337 mV/cm, and 255 mV/cm.
  • the third liquid medium comprises wild hawthorn juice, jujube juice, wu wei zi juice, and soybean juice.
  • the invention provides a method for preparing a biological composition useful for producing a healthful benefit in a subject suffering from uterine cancer.
  • uterine cancer includes but is not limited to squamous cell carcinoma, endometrioid carcinoma, adenoacanthoma, adenosquamous carcinoma, papillary serous carcinomas, and clear cell adenocarcinomas.
  • the biological compositions can ameliorate, reduce, manage, or eliminate the symptoms associated with uterine cancer include abnormal vaginal and/or uterine spotting or bleeding, such as between periods or after intercourse, white or clear vaginal discharge, difficult or painful urination, discomfort during intercourse, pain or cramping in the pelvic area, and discomfort during intercourse .
  • the biological composition useful for treatment of uterine cancer comprises activated and conditioned Saccharomyces carlsbergensis Hansen strain AS2.605 yeast cells cultured in a series of alternating electromagnetic (EM) fields.
  • the method for preparing said biological composition comprises in any order the steps of: (a) culturing the yeast cells in a first liquid medium in a first electromagnetic field having a frequency at 10,162 MHz and a field strength of 235 mV/cm;
  • the first liquid medium comprises 20 g of sucrose or glucose, 60 ⁇ g of vitamin B ⁇ 2 , 60 ⁇ g of vitamin B 3 , 60 ⁇ g of vitamin H, 50 ⁇ g of vitamin B 6 , 30 ml of fetal calf serum, 0.20 g of KH 2 PO 4 , 0.25 g of MgSO 4 .7H 2 O, 0.30 g of NaCI, 0.20 g of CaSO 4 .2H 2 0, 4.0 g of CaCO 3 .5H 2 O, 2.5 g of peptone, and 1,000 ml of autoclaved water.
  • the second liquid medium comprises wild hawthorn juice and gastric juice of a mammal.
  • the number of activated and conditioned yeast cells can be further expanded by:
  • the third liquid medium comprises wild hawthorn juice, jujube juice, wu wei zi juice, and soybean juice.
  • the invention provides a method for preparing a biological composition useful for producing a healthful benefit in a subject suffering from kidney cancer.
  • kidney cancer includes but is not limited to adenocarcinomas, hypernephroma, renal cell carcinoma, clear cell cancer, and Grawitz's tumor.
  • the biological compositions can ameliorate, reduce, manage, or eliminate the symptoms associated with kidney cancer include blood in the urine (hematuria), persistent pain in the area between the ribs and the hip not associated with an injury, a mass in the area of the kidneys, high blood pressure, rapid and unexplained weight loss, persistent feeling of fatigue, fever not caused by a cold or flu, swelling of the legs and ankles, anemia, fatigue, and intermittent fever.
  • blood in the urine hematuria
  • persistent pain in the area between the ribs and the hip not associated with an injury a mass in the area of the kidneys
  • high blood pressure rapid and unexplained weight loss
  • persistent feeling of fatigue fever not caused by a cold or flu
  • swelling of the legs and ankles swelling of the legs and ankles
  • anemia fatigue, and intermittent fever.
  • the biological composition useful for treatment of kidney cancer comprises activated and conditioned Saccharomyces carlsbergensis Hansen strain AS2.189 yeast cells cultured in a series of alternating electromagnetic (EM) fields.
  • the method for preparing said biological composition comprises in any order the steps of:
  • the first liquid medium comprises 20 g of sucrose or glucose, 40 ⁇ g of vitamin B 3 , 30 ⁇ g of vitamin B 6 , 60 ⁇ g of vitamin A, 20 ⁇ g of vitamin H, 50 ⁇ g of vitamin C, 30 ml of fetal calf serum, 0.20 g of KH 2 PO 4 , 0.25 g of MgSO 4 .7H 2 O, 0.30 g of NaCI, 0.20 g of CaSO 4 .2H 2 0, 4.0 g of CaCO 3 .5H 2 O, 2.5 g of peptone, and 980 ml of autoclaved water.
  • the second liquid medium comprises wild hawthorn juice and gastric juice of a mammal.
  • the number of activated and conditioned yeast cells can be further expanded by:
  • the third liquid medium comprises wild hawthorn juice, jujube juice, wu wei zi juice, and soybean juice.
  • the invention provides a method for preparing a biological composition useful for producing a healthful benefit in a subject suffering from bladder cancer.
  • bladder cancer includes but is not limited to urothelial carcinoma, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, papillomas, flat urothelial carcinomas, and rhabdomyosarcoma.
  • the biological compositions can ameliorate, reduce, manage, or eliminate the symptoms associated with bladder cancer include microscopic (visible only under a microscope) or gross (visible to the naked eye) hematuria, or blood in the urine, frequent urination, urinary urgency, urinary frequency, painful urinary (dysuria), urinary incontinence, bone pain or tenderness, abdominal pain, anemia, weight loss, and lethargy.
  • the biological composition useful for treatment of bladder cancer comprises activated and conditioned Saccharomyces cerevisiae Hansen strain AS2.4 yeast cells cultured in a series of alternating electromagnetic (EM) fields.
  • the method for preparing said biological composition comprises in any order the steps of:
  • the first liquid medium comprises 20 g of sucrose or glucose, 50 ⁇ g of vitamin B ⁇ 2 , 30 ⁇ g of vitamin B 6 , 20 ⁇ g of vitamin H, 50 ⁇ g of vitamin A, 30 ml of bovine calf serum, 0.20 g of KH 2 PO , 0.25 g of MgSO 4 .7H 2 O, 0.30 g of NaCI, 0.20 g of CaSO 4 .2H 2 0, 4.0 g of CaCO 3 .5H 2 0, 2.5 g of peptone, and 15 1,000 ml of autoclaved water.
  • the second liquid medium comprises wild hawthorn juice and gastric juice of a mammal.
  • the number of activated and conditioned yeast cells can be further expanded by: 0 (h) culturing the yeast cells in a third liquid medium in an eighth electromagnetic field or series of electromagnetic fields having a frequency at 12,665 MHz and a field strength in the range of 170 to 280 mV/cm, preferably at three fields strengths, e.g., in the order of 256 mV/cm, 266 mV/cm, and 188 mV/cm; and 5 (i) culturing the yeast cells in a third liquid medium in a ninth electromagnetic field or series of electromagnetic fields having a frequency 12,897 MHz and a field strength in the range of 230 to 340 mV/cm, preferably at three fields strengths, e.g., in the order of 322 mV/cm, 332 mV/cm, and 247 mV/cm.
  • the third liquid medium comprises wild hawthorn juice, jujube juice, wu wei zi juice
  • the invention provides a method for preparing a biological composition useful for producing a healthful benefit in a subject suffering from brain cancer.
  • the term "brain cancer” includes but is not limited to astrocytoma, meningioma, colloid cyst, ependymoma, metastatic tumors, choroid plexus papilloma, subependymoma, astrocytoma, glioblastoma, lipoma, oligodendroglioma, sarcoma, germ cell tumors, pineal cell tumors, chordoma, pituitary adenoma, craniopharyngioma, chordoma, acoustic schwannoma, glomus jugulare tumor, medullobastoma, hemangioblastoma, glioglastoma multiforme, neurinomas, cerebellar astrocytom
  • the biological compositions can ameliorate, reduce, manage, or eliminate the symptoms associated with brain cancer in adults include recent onset or persistent headache, vomiting, personality and behavior changes,, emotional instability, intellectual decline (e.g., confusion, loss of memory, impaired calculating abilities, and impaired judgment), seizures, reduced level of consciousness, neurologic changes (e.g., vision changes, hearing loss, decreased sensation of a body area, weakness of a body area, speech difficulties, and decreased coordination), fever, weakness, general ill feeling, positive Babinski's reflex, and decerebrate or decorticate posture.
  • Common symptoms associated with brain cancer in infants include bulging fontanelles, separated sutures, opisthotonos, increased head circumference, no red reflex in the eye. Additional symptoms include tongue problems, difficulty swallowing, impaired smell, obesity, uncontrollable movement, absent menstruation, hiccups, hand tremor, facial paralysis, different pupil sizes, eyelid drooping, and breathing problem.
  • the biological composition useful for treatment of brain cancer comprises activated and conditioned Saccharomyces cerevisiae Hansen strain AS2.501 yeast cells cultured in a series of alternating elecfromagnetic (EM) fields.
  • the method for preparing said biological composition comprises in any order the steps of:
  • the first liquid medium comprises 20 g of sucrose or glucose, 60 ⁇ g of vitamin B ⁇ 2 , 60 ⁇ g of vitamin B 3 , 60 ⁇ g of vitamin H, 50 ⁇ g of vitamin B 6 , 30 ml of fetal calf serum, 0.20 g of KH 2 PO 4 , 0.25 g of MgSO 4 .7H 2 0, 0.30 g of NaCI, 0.20 g of CaSO .2H 2 0, 4.0 g of CaCO 3 .5H 2 0, 2.5 g of peptone, and 1,000 ml of autoclaved water.
  • the second liquid medium comprises wild hawthorn juice and gastric juice of a mammal.
  • the number of activated and conditioned yeast cells can be further expanded by:
  • the third liquid medium comprises wild hawthorn juice, jujube juice, wu wei zi juice, and soybean juice.
  • the invention provides a method for preparing a biological composition useful for producing a healthful benefit in a subject suffering from lymphoma cancer.
  • lymphoma includes but is not limited to Hodgkin's disease and non-Hodgkin's disease.
  • Rosenberg SA Kaplan HS, eds. "Malignant Lymphomas: Etiology, Immunology, Pathology, Treatment” New York, NY, Academic Press 1982, which is incorporated herein by reference in its entirety.
  • Classic Hodgkin's disease is divided into four subtypes: (1) nodular sclerosis Hodgkin's disease (NSHD); (2) mixed cellularity Hodgkin's disease (MCHD); (3) lymphocyte depletion Hodgkin's disease (LDHD); and (4) lymphocyte-rich classic Hodgkin's disease (cLRHD).
  • NSD nodular sclerosis Hodgkin's disease
  • MCHD mixed cellularity Hodgkin's disease
  • LDHD lymphocyte depletion Hodgkin's disease
  • cLRHD lymphocyte-rich classic Hodgkin's disease
  • Non-Hodgkin's lymphoma includes but is not limited to (1) slow-growing lymphoma and lymphoid leukemia (e.g., chronic lymphocytic leukemia, small lymphocytic leukemia, lymphoplasmacytoid lymphoma, follicle center lymphoma, follicular small cleaved cell, follicular mixed cell, marginal zone B- cell lymphoma, hairy cell leukemia, plasmacytoma, myeloma, large granular lymphocyte leukemia, mycosis fungoides, sezary syndrome); (2) moderately aggressive lymphomas and lymphoid leukemia (e.g., prolymphocytic leukemia, mantle cell lymphoma, follicle center lymphoma, follicular small cleaved cell, follicle center lymphoma, chronic lymphocytic leukemia/prolymphocytic leukemia, angiocentric lymphoma,
  • the biological composition can ameliorate, reduce, manage, or eliminate the symptoms associated with lymphoma include painless swelling in one or more of the lymph nodes of the neck, collarbone region, armpits, or groin., chest pain, coughing, fatigue, shortness of breath, fever, drenching night sweats, weight loss, fatigue, appetite loss, red patches on the skin, and severely itchy skin, often affecting the legs/feet.
  • the biological composition useful for treatment of lymphoma cancer comprises activated and conditioned Saccharomyces cerevisiae Hansen strain AS2.562 yeast cells cultured in a series of alternating electromagnetic (EM) fields.
  • the method of preparing said biological composition comprises in any order the steps of:
  • the first liquid medium comprises 20 g of sucrose or glucose, 60 ⁇ g of vitamin H, 40 ⁇ g of vitamin B 6 , 40 ⁇ g of vitamin B 12 , 0.20 g of KH 2 PO , 0.20 g of MgSO 4 .7H 2 O, 0.20 g of NaCI, 0.20 g of CaSO 4 .2H 2 O, 3.0 g of CaCO 3 .5H 2 0, 2.5 g of peptone, and 1,000 ml of autoclaved water.
  • the second liquid medium comprises wild hawthorn juice and gastric juice of a mammal.
  • the number of activated and conditioned yeast cells can be further expanded by: (h) culturing the yeast cells in a third liquid medium in an eighth electromagnetic field or series of electromagnetic fields having a frequency at 12,837 MHz and a field strength in the range of 230 to 450 mV/cm, preferably at three fields strengths, e.g., in the order of 442 mV/cm, 341 mV/cm, and 244 mV/cm; and (i) culturing the yeast cells in a third liquid medium in a ninth electromagnetic field or series of electromagnetic fields having a frequency at 12,862 MHz and a field strength in the range of 120 to 380 mV/cm, preferably at three fields strengths, e.g., in the order of 350 mV/cm, 224 mV/cm, and 137 mV/cm.
  • the third liquid medium comprises wild hawthorn juice, jujube juice, wu wei zi juice., and soybean juice.
  • the invention provides a method for preparing a biological composition useful for producing a healthful benefit in a subject suffering from leukemia.
  • leukemia includes but is not limited to acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and hairy cell leukemia.
  • the biological compositions can ameliorate, reduce, manage, or eliminate the symptoms associated with leukemia include a weakened immune system, infections, fevers, decrease in red blood cells and platelets, weakness, fatigue, loss of appetite, loss of weight, swollen or tender lymph nodes, liver, or spleen, easy bleeding or bruising, tiny red spots (called petechiae) under the skin, swollen or bleeding gums, sweating (especially at night), bone or joint pain, headaches, vomiting, confusion, loss of muscle control, and seizures.
  • the biological composition useful for treatment of leukemia comprises activated and conditioned Saccharomyces cerevisiae Hansen strain AS2.11 yeast cells cultured in a series of alternating electromagnetic (EM) fields.
  • the method for preparing said biological composition comprises in any order the steps of:
  • the first liquid medium comprises 20 g of sucrose or glucose, 40 ⁇ g of vitamin B ⁇ 2 , 30 ⁇ g of vitamin B 6 , 20 ⁇ g of vitamin D, 20 ⁇ g of vitamin H, 30 ml of bovine calf serum, 0.25 g of MgSO .7H 2 O, 0.30 g of NaCI, 0.20 g of CaSO 4 .2H 2 0, 4.0 g of CaCO 3 .5H 2 0, 2.5 g of peptone, and 1,000 ml of autoclaved water.
  • the second liquid medium comprises wild hawthorn juice and gastric juice of a mammal.
  • the number of activated and conditioned yeast cells can be further expanded by: (h) culturing the yeast cells in a third liquid medium in an eighth electromagnetic field or series of electromagnetic fields having a frequency at 12,565 MHz and a field strength in the range of 170 to 290 mV/cm, preferably at three fields strengths, e.g., in the order of 270 mV/cm, 277 mV/cm, and 184 mV/cm; and
  • the third liquid medium comprises wild hawthorn juice, jujube juice, wu wei zi juice, and soybean juice.
  • the biological compositions used in the following animal studies comprised 10 8 per ml of activated and conditioned yeast cells that were prepared by the methods described above.
  • the following example illustrates the benefit of a biological composition of the invention comprising activated and conditioned S. cerevisiae IFFI1345 cells.
  • the Lewis lung cancer model was used to study the growth of tumors in animals and the survival time thereof after tumor inj ection and treatment.
  • the animals used to generate the Lewis lung cancer cells for the experiments were C57/B1 mice, 6 to 8 weeks old.
  • Lewis lung carcinoma (obtainable from the National Cancer Institute, Bethesda, MD) in a suspension containing about 10 6 viable tumor cells was injected subcutaneously in six animals. The animals were allowed to grow for 21 days. The animals that showed robust growth of the tumor were used as donors.
  • the tumor was removed from a mouse and minced aseptically in 4 ml of Hank's solution.
  • the suspension of tumor cells were injected into a healthy batch of C57/B1 mice that were 6 to 8 weeks old; each mouse receiving 0.2 ml of the tumor cell suspension.
  • the mice injected with tumor cells were divided into 3 experimental groups often mice per group and one control group.
  • mice received 0.3 ml of the biological composition once per day.
  • group B the mice received 0.5 ml of the biological composition once per day.
  • group C the mice received 0.5 ml of physiological saline once per day.
  • group D which did not receive tumor cells, was given 0.3 ml of physiological saline per day.
  • mice received the biological compositions or saline on the same day as the tumor cells were transplanted.
  • the mice in group D also started receiving saline on the same day as the other three groups.
  • the biological composition or saline were administered orally by a feeding tube for 24 consecutive days.
  • the mice were sacrificed and the weight of the lungs as well as the weight of the tumor were determined by standard techniques.
  • Table 7 shows the differences in the weight of the lungs and tumors of the mice in the various treatment and control groups.
  • mice bearing Lewis cancer cells that received 0.5 ml of the biological composition of the invention showed the least deviation in the weight of lungs as compared to healthy mice not injected tumor cells (group D).
  • the mice of group B also had less tumor mass as compared to mice that did not receive treatment (group C) as well as the mice in group A (0.3 ml per day).
  • the animals used to generate the Lewis lung cancer cells for the experiments were C57/B1 mice, 6 weeks old.
  • Lewis lung carcinoma (obtainable from the National Cancer Institute, Bethesda, MD) in a suspension containing about 10 8 viable tumor cells was injected subcutaneously in five animals. The animals were allowed to grow for 15 days. The animals that showed robust growth of the tumor were used as donors.
  • the tumor was removed from a mouse and minced aseptically in 4 ml of Hank's solution to form a suspension of tumor cells. 0.1 ml of the cell suspension were injected intramuscularly into each mouse. Healthy C57/B1 mice that were 6 weeks old were used.
  • mice injected with tumor cells were divided into 3 experimental groups and one control group often mice per group.
  • the groups were in triplicates, i.e., using a total of 120 mice for the four treatments.
  • group A the mice received 0.3 ml of the biological composition once per day.
  • group B the mice received 0.5 ml of the biological composition once per day.
  • group C the mice received 0.5 ml of physiological saline once per day.
  • the activated and conditioned yeast cells were at a concentration of 10 8 per ml of the biological composition.
  • mice received the biological compositions or saline 15 days after the tumor cells were transplanted.
  • the mice in group D also started receiving saline on the same day as the other three groups.
  • the biological composition or saline were administered orally by a feeding tube for 60 consecutive days. The mice were observed over a year from the day of tumor inoculation.
  • mice were fed the biological composition or saline for 90 days.
  • Table 8 shows the number of mice in the various treatment and control group that survived the tumor injection over a period of 12 months. Each of the 30 mice in each group received 60 consecutive days of either saline or biological composition of the invention.
  • mice receiving the different treatments for 90 consecutive days instead of 60 days were treated with mice receiving the different treatments for 90 consecutive days instead of 60 days.
  • Table 9 shows the number of mice in the various treatment and control group that survived the tumor injection over a period of 12 months.
  • mice died of other causes unrelated to the lung cancer.
  • the following example illustrates the benefit of a biological composition of the invention comprising activated and conditioned Saccharomyces carlsbergensis Hansen strain AS2.116 cells.
  • the Wistar rat model and a mouse model of human nasopharyngeal cancer were used to study the growth of the tumors in animals and the survival time thereof after tumor injection and treatment.
  • the animals used to generate the nasopharyngeal cancer cells for the experiments were Wistar rats, male and female, 190 to 210 gram in body weight. The rats were starved for 24 hours before the surgery.
  • nasopharyngeal cancer samples (obtainable from Cancer Institute, The Chinese Academy of Medical Sciences, Beijing, China) were carefully selected before, during, and after surgery in order to ensure engraftment occuned at the original position in the rat.
  • the nasopharyngeal cancer samples were obtained from patients who have not received any radiation, chemotherapy or immune enhancement treatment.
  • Nasopharyngeal cancer samples were divided into sections of about 1 mm 3 in size and immediately stored in RPMI-1640 suspension after removal from the human patient.
  • the animals were generally anaesthetized.
  • the barbiturates was first diluted in saline and then injected at 0.3 to 0.4 ml per animal, or 30 mg per kg body weight into the abdomen of the rats.
  • a 1 cm long opening in the body cavity was cut along the right rib of the rats for engraftment of the tumor. Afterwards, the opening was sutured and the rats were put back into the cage.
  • the animals were allowed to grow for three to five weeks.
  • the animals that showed robust growth of the tumor were used as donors.
  • the tumor was removed from a donor rat and minced aseptically in 4 ml of Hank' s solution.
  • the suspension of tumor cells were transplanted into a healthy batch of Wistar rats for experimentation.
  • the rats transplanted with tumor cells were divided into 4 experimental groups often rats per group and one control group.
  • the four experimental groups were triplicated (i.e., using a total of 120 rats in the experimental groups).
  • group AY the rats received 2.0 ml of the biological composition once per day.
  • group NY the rats received 2.0 ml of the untreated yeast cells once per day.
  • group TSPA the rats were injected intravenously with 1.5 mg of thiotepa (TSPA) per kg body weight per day.
  • TSPA thiotepa
  • the rats received 2.0 ml of physiological saline once per day.
  • the rats received the biological compositions, untreated yeast cells, TSPA or saline on the same day as the tumor cells were transplanted.
  • the rats in group CK2 also started receiving saline on the same day as the other four groups.
  • the biological compositions, untreated yeast cells and saline were administered orally by a feeding tube and the TSPA by intravenous injection for 21 consecutive days.
  • the rats were sacrificed and the weight of the rats as well as the weight of the tumor were determined by standard techniques.
  • Table 10 shows the differences in the weight of the rats and tumors of the rats in the various treatment and control groups.
  • the rats bearing nasopharyngeal cancer cells that received 2.0 ml of the biological composition of the invention showed the least deviation in weight as compared to healthy rats not injected tumor cells (group CK2).
  • the rats in group AY also had less tumor mass as compared to rats that did not receive treatment (group CK1) as well as the rats in group NY (2.0 ml of untreated yeast cells per day) and the rats in group TSPA (1.5 mg of thiotepa per kg body weight per day).
  • the animals were prepared in a similar manner as described immediately above.
  • the rats transplanted with tumor cells were divided into 4 experimental groups often rats per group and one control group.
  • the four experimental groups were triplicated (i.e., using a total of 120 rats in the experimental groups).
  • group 2AY the rats received 2.0 ml of the biological composition once per day.
  • group 2NY the rats received 2.0 ml of the untreated yeast cells once per day.
  • group 2TSPA the rats were injected intravenously 1.5 mg of thiotepa (TSPA) per kg body weight per day.
  • hi group 2CK1 the rats received 2.0 ml of physiological saline once per day.
  • a fifth group of rats, group 2CK2, which did not receive tumor cells was given 2.0 ml of physiological saline per day.
  • the rats received the biological compositions, untreated yeast cells,
  • the rats in group 2CK2 also started receiving saline on the same day as the other four groups.
  • the biological compositions, untreated yeast cells and saline were administered orally by a feeding tube and the TSPA by intravenous injection for 21 consecutive days.
  • the rats were observed over 6 months from the day of tumor inoculation and survival was recorded.
  • the weight of the rats and the weight of the tumor were determined by standard techniques.
  • Table 11 shows the number of rats in the various treatment and confrol group that survived the tumor injection over a period of 6 months.
  • Each of the 30 rats in each group received 21 consecutive days of either unfreated yeast cells, TSPA, saline or biological compositions of the invention.
  • Table 6 shows the weight of the rats that survived and the weight of their tumors in the various treatment and control groups.
  • the rats bearing nasopharyngeal cancer cells that received 2.0 ml of the biological composition of the invention survived for more than 6 months and the tumor never reoccuned.
  • group 2NY 2.0 ml of untreated yeast cells per day
  • group 2TSPA 1.5 mg of thiotepa per kg body weight per day
  • group 2CK1 2.0 ml of saline per day
  • the rats bearing nasopharyngeal cancer cells that received 2.0 ml of the biological composition of the invention showed the least deviation in the weight of rats as compared to healthy rats not injected tumor cells (group 2CK2).
  • the animals used to generate the nasopharyngeal cancer cells for the experiments were nasopharyngeal cancer mice (obtainable from the Chinese Academy of Military Medical Sciences, Beijing, China), 6 to 8 weeks old, male and female, 18 to 20 gram in body weight. Highly differentiated nasopharyngeal cancer
  • mice injected with tumor cells were divided into 4 experimental groups often mice per group and one control group.
  • the four experimental groups 0 were triplicated (i.e., using a total of 120 mice in the experimental groups).
  • group AY the mice received 0.3 ml of the biological composition once per day.
  • group NY the mice received 0.3 ml of the untreated yeast cells once per day.
  • CTX the mice were injected subcutaneously with 30 mg of cyclophosphamide (CTX) per kg body weight per day.
  • group CK1 the mice received 0.3 ml of physiological 5 saline once per day.
  • mice received the biological compositions, untreated yeast cells, CTX or saline on the same day as the tumor cells were transplanted.
  • the mice in group CK2 also started receiving saline on the same day as the other four groups.
  • the 0 biological compositions, unfreated yeast cells and saline were administered orally by a feeding tube and the CTX by intravenous injection for 30 consecutive days.
  • the mice were sacrificed and the weight of the mice as well as the weight of the tumor were determined by standard techniques. The results are shown below. Table 13
  • mice were prepared in a similar manner as described immediately above.
  • the mice injected with tumor cells were divided into 4 experimental groups often mice per group and one confrol group.
  • the four experimental groups were triplicated (i.e., using a total of 120 mice in the experimental groups).
  • group 2AY the mice received 0.3 ml of the biological composition once per day.
  • group 2NY the mice received 0.3 ml of the unfreated yeast cells once per day.
  • hi group 2CTX the mice were injected intravenously with 30 mg of cyclophosphamide (CTX) per kg body weight per day.
  • CTX cyclophosphamide
  • group 2CK1 the mice received 0.3 ml of physiological saline once per day.
  • a fifth group of mice, group 2CK2, which did not receive tumor cells were given 0.3 ml of physiological saline per day.
  • the mice received the biological compositions, untreated yeast cells,
  • the mice in group 2CK2 also started receiving saline on the same day as the other four groups.
  • the biological compositions, untreated yeast cells and saline were administered orally by a feeding tube and the CTX by intravenous injection for 30 consecutive days. The mice were observed over 6 months from the day of tumor inoculation and survival was recorded. The weight of the mice and the weight of the tumor were determined by standard techniques.
  • the following example illustrates the benefit of a biological composition of the invention comprising activated and conditioned Saccharomyces cerevisiae Hansen sfrain AS2.375 cells.
  • the Wistar rat model and a mouse model of human esophageal cancer were used to study the growth of the tumors in animals and the survival time thereof after tumor injection and freatment.
  • N-methyl-N-benzylnifrosamine is a well known effective trigger of esophageal cancer in Wistar rats. Accordingly, this experiment used MBNA in setting up a Wistar rat model of human esophageal cancer.
  • MBNA N-methyl-N-benzylnifrosamine
  • the animals used in the experiments were Wistar rats (obtainable from the Chinese Academy of Military Medical Sciences, Beijing, China), male and female, with an average body weight of about 150 to 180 gram.
  • the rats were given water containing 100 ppm MBNA and fed a diet containing MBNA such that each rat received 0.75 mg MBNA (obtainable from Beijing Chemical Reagent Company,
  • the rats that were fed with MBNA for 30 days were divided into 4 experimental groups often rats per group and one control group.
  • the four experimental groups were triplicated (i.e., using a total of 120 rats in the experimental groups).
  • group AY the rats received 1.5 ml of the biological composition in two doses per day.
  • group NY the rats received 1.5 ml of the untreated yeast cells in two doses per day.
  • group VDS the rats were injected intravenously with 3.0 mg of vindesine (VDS) per kg body weight once a week for four weeks.
  • group CK1 the rats received 1.5 ml of physiological saline in two doses per day.
  • the biological compositions, untreated yeast cells and saline were administered orally by a feeding tube and the VDS by intravenous injection for 30 consecutive days. On the 31st day from tumor inoculation, the rats were sacrificed.
  • the experiment was carried out in a manner similar to that described immediately above, except that the survival time over a period of over 6 months was observed.
  • mice Numerous animal studies have reported the use of murine models in the study of human esophageal cancer. There is almost a 100% success rate for transplanting esophageal tumor SGA-73 cells in mice. Detailed description of the esophageal tumor cell line SGA-73 can be found in Ding R. et al., Esophageal Cancer Experimental Research, Ren Ming Wei Shen Publisher, 1980, p. 36, which is incorporated herein by reference in its entirety. The animals used for the experiments were TA1 mice (obtainable from the Chinese Academy of Military Medical Sciences, Beijing, China), 6 to 8 weeks old, male and female, with an average body weight of about 18 to 22 gram.
  • the esophageal cancer cell line SGA-73 (obtainable from the Beijing Institute of Chinese Medicine and Pharmacology, Beijing, China) in a suspension containing about 10 7 viable tumor cells (about 0.2 ml culture suspension) was injected subcutaneously into the animals.
  • the experiment was carried out in a manner similar to that described in the Tumor Growth in Mouse Model section for the nasopharyngel cancer.
  • the following example illustrates the benefit of a biological composition of the invention comprising activated and conditioned Saccharomyces cerevisiae Hansen strain AS2.14 cells.
  • the Wistar rat model and a mouse model of human stomach cancer were used to study the growth of the tumors in animals and the survival time thereof after tumor injection and treatment. Tumor Growth in Wistar Rat Model
  • the stomach cancer cell line (obtainable from the First Military Medical University of China, Guangzhou, China) was generated in 1991 from a Wistar rat stomach cancer which was induced by subcutaneous injection of DMH. The rats were injected subcutaneously with 2.5 x 10 6 stomach cancer cells per animal.
  • the experiment was carried out in a manner similar to that described above in the Tumor Growth in Wistar Rat Model for the nasopharyngeal cancer, except that a dosage of 0.8 ml per day was used for groups AY, NY, CK1 and CK2, and a group ADM was introduced in lieu of group TSPA, wherein the rats were injected intravenously with 10 5 units of doxorubicin (adriamycin, ADM) per kg body weight per day.
  • Methylcholantbrene is a well-known effective trigger of stomach cancer in mice. Accordingly, this experiment used MC in designing TA2 mouse model of human stomach cancer. Detailed description of the use of methylcholanthrene to induce stomach cancer can be found in Wang X.H. et al, 1984, Chin Med J (Engl) 97(3):215-22, which is incorporated herein by reference in its entirety.
  • the animals used for the experiments were TA2 mice, 6 to 8 weeks old (obtainable from the Second Military Medical University, Shanghai, China). Both male and females with an average weight of about 18 to 20 gram were used.
  • the stomach cancer cell line S784 (obtainable from MC-treated TA2 mouse provided by the Tianjian Medical University, China) in a suspension containing about 1.2 x 10 7 viable tumor cells (about 0.2 ml cell culture) was injected subcutaneously into the animals. The mice injected with tumor cells were kept for 5 days.
  • the experiment was canied in a manner similar to that described in section of Tumor Growth in Mouse Model for the nasopharyngeal cancer, except that a group ADM was used in place of group CTX, wherein the mice were injected intravenously with 10 5 units of doxorubicin (adriamycin, ADM) per kg body weight per day.
  • the following example illustrates the benefit of a biological composition of the invention comprising activated and conditioned Saccharomyces cerevisiae Hansen strain AS2.1396 cells.
  • the Wistar rat model and a mouse model of human colorectal cancer were used to study the growth of the tumors in animals and the survival time thereof after tumor injection and treatment. Tumor Growth in Wistar Rat Model
  • dimethyl hydrazine is a well known effective trigger of colorectal cancer in Wistar rats. Accordingly, this experiment used DMH in setting up a Wistar rat model of human colorectal cancer. Detailed description of the use of dimethyl hydrazine to induce colorectal cancer can be found in Madarnas P. et al, 1992, Anticancer Res. 12(1): 113-7, which is incorporated herein by reference in its entirety.
  • a DMH mixture was prepared by mixing 400 mg DMH with 100 ml saline and 37 mg ethylene diamine tefraacetate (EDTA). The pH of the mixture was then adjusted to 6.5 using 0.1 N NaOH.
  • EDTA ethylene diamine tefraacetate
  • the animals used in the experiments were Wistar rats (obtained from the Chinese Academy of Military Medical Sciences, Beijing, China), male and female, with an average body weight of about 180 to 200 gram.
  • the rats were injected subcutaneously with 21 mg of DMH mixture (as prepared above) per kg body weight once a week for 21 weeks.
  • the experiment was carried out in a manner similar to that described in the section of Tumor Growth in Wistar Rat Model for the nasopharyngeal cancer, except that a group AM was used in lieu of Group TSPA, wherein the rats were inj ected subcutaneously with 10 5 units of amycin (AM) per kg body weight per day. The animals were treated for 30 days and was sacrificed on the 31 st day from tumor inoculation.
  • a group AM was used in lieu of Group TSPA, wherein the rats were inj ected subcutaneously with 10 5 units of amycin (AM) per kg body weight per day.
  • the animals were treated for 30 days and was sacrificed on the 31 st day from tumor inoculation.
  • the experiment was carried out in a similar manner as described immediately above, except that the rats were observed for a period of over 6 months from the day of tumor inoculation and the survival time was recorded.
  • the experiment was carried out in a manner similar to that described in the section of Tumor Growth in Mouse Model for the nasopharyngeal cancer, except that a group AM was used in lieu of Group CTX, wherein the mice were injected subcutaneously with 10 5 units of amycin (AM) per kg body weight per day.
  • a group AM was used in lieu of Group CTX, wherein the mice were injected subcutaneously with 10 5 units of amycin (AM) per kg body weight per day.
  • the following example illustrates the benefit of a biological composition of the invention comprising activated and conditioned Saccharomyces cerevisiae Hansen strain IFFI 1413 cells.
  • Two mouse models of human pancreatic cancer were used to study the growth of the tumors in animals and the survival time thereof after tumor injection and treatment.
  • pancreatic cancer cells were BALB/c mice, both male and female with an average body weight of about 18 to 20 gram (obtainable from the Chinese Academy of Military Medical Sciences, Beijing, China).
  • the pancreatic tumor cells were isolated from clinical biopsy samples in 1987 (obtainable from the Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China).
  • pancreatic cancer tumor cells about 0.2 ml culture suspension
  • the experiment was carried out in a manner similar to that described in the section of Tumor Growth in Mouse Model for the nasopharyngeal cancer, except that a group VDS was used in lieu of Group CTX, wherein the mice were injected intravenously with 3 mg of vindesine (VDS) per kg body weight once a week for four weeks.
  • VDS vindesine
  • the transplantable mouse pancreatic cancer cell line MPC-83 (obtainable from Kun-Ming Medical University, Kun-Ming, China) in a suspension containing about 1.2 x 10 7 viable tumor cells (about 0.2 ml culture suspension) was injected subcutaneously into the animals.
  • the experiment was carried out in a manner similar to that described in the section of Tumor Growth in Mouse Model for the nasopharyngeal cancer, except that a group MMC was used in lieu of Group CTX, wherein the mice were injected intravenously with 10 5 units of mitomycin C (MMC) per kg body weight per day.
  • MMC mitomycin C
  • mice were observed for a period of over 6 months from the day of tumor inoculation and the survival time was recorded.
  • the dosage was increased to 0.5 ml per day for 2AY, 2NY, 2CK1 and 2CK2 groups.
  • the dosage was increased to 1.5 x 10 5 units of mitomycin C (MMC) per kg body weight per day.
  • the following example illustrates the benefit of a biological composition of the invention comprising activated and conditioned Saccharomyces cerevisiae Hansen sfrain AS2.503 cells.
  • Two mouse models of human liver cancer were used to study the growth of the tumors in animals and the survival time thereof after tumor injection and treatment.
  • the animals used to generate the liver cancer cells for the experiments were Swiss mice about 5 weeks old (obtainable from the Chinese Academy of
  • mice Both male and female Swiss mice with an average weight of about 18 to 20 gram were used in the following experiment. The mice were starved for 24 hours before the surgery. Liver tumor tissues were obtained from the Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China. Animal preparation was carried out in a manner similar to that described above in the nasopharyngeal cancer section. The experimental was performed in a manner similar to that described in the section of Tumor Growth in Mouse Model for the nasopharyngeal cancer.
  • mice were observed for a period of over 6 months from the day of tumor inoculation and their survival time was recorded.
  • Murine transplantable liver tumor type H-22 closely resembles the human liver cancer pathology.
  • Detailed description of the murine ascites hepatoma cell line H-22 can be found in Ling M.Y., 1991, Zhonghua Zhong Liu Za Zhi 13(l):13-5, which is incorporated herein by reference in its entirety.
  • the expression of the alphafetoprotein (AFP) gene is associated with liver cancer, and there is an association between changes in the liver tumor and the level of AFP protein in blood plasma. More information on the use of AFP in the diagnosis of liver cancer can be found in Shi X. et al., 1998, Zhonghua Zhong Liu Za Zhi 20(6):437- 439, which is incorporated herein by reference in its entirety.
  • the animals used to generate the liver cancer cells for the experiments were C 3 H mice, 6 to 8 weeks old (obtainable from the Chinese Academy of Military Medical Sciences, Beijing, China). Both male and females were used. The mice were starved for 24 hours before the surgery. Liver tumor H-22 (obtainable from the Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China) were injected into the mice. Ten days after transplantation, the tumors are visible and the level of AFT in plasma is detectable. The animals were allowed to grow for 21 days. The animals that showed robust growth of the tumor were used as donors. The tumor was removed from a mouse and minced aseptically in 4 ml of Hank's solution.
  • the suspension of tumor cells were injected into a healthy batch of C 3 H mice that were 6 to 8 weeks old; each mouse receiving 0.2 ml of the tumor cell suspension.
  • the experiment was carried out in a manner similar to that described in the section of Tumor Growth in Mouse Model for the nasopharyngeal cancer.
  • mice were observed for a period of over 6 months from the day of tumor inoculation and their survival time was recorded.
  • the following example illustrates the benefit of a biological composition of the invention comprising activated and conditioned Saccharomyces cerevisiae Hansen strain AS2.182.
  • the Kun Ming mouse model and the Wistar rat model of human testicular cancer were used to study the growth of the tumors in animals and the survival time thereof after tumor injection and freatment.
  • testicular tumor cell line HTB-104 can be ordered from the American Type Culture Collection.
  • the animals used for the experiments were kun ming mouse (obtainable from the Chinese Academy of Military Medical Sciences, Beijing, China). Only males 20 to 22 gram in body weight, 6 to 7 months old were used.
  • About 1.2 x 10 viable tumor cells of the testicular cancer cell line HTB-104 obtained from the Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China) in about 0.2 ml culture suspension was injected subcutaneously into the animals.
  • testicular tumor cell line CRL-1973 can be ordered from the American Type Culture Collection.
  • the animals used for the experiments were male Wistar rats (obtainable from the Chinese Academy of Military Medical Sciences, Beijing, China). About 2 x 10 7 viable tumor cells of the testicular cancer cell line CRL-1973 (obtainable from the Cancer Institute, Chinese Academy of Medical Sciences,
  • the experiment was carried out in a manner similar to that described in the section of Tumor Growth in Wistar Rat Model for the nasopharyngeal cancer, except the dosages for groups AY, NY, CKl and CK2 were decreased to 0.8 ml, and a group VLB was introduced in lieu of group TSPA, wherein the rats were injected intravenously with 0.4 mg of vinblastine (VLB) per kg body weight per day. Thirty (30) days of consecutive treatment was performed and the rats were sacrificed on the 31 st day from tumor inoculation.
  • VLB vinblastine
  • the following example illustrates the benefit of a biological composition of the invention comprising activated and conditioned Saccharomyces carlsbergensis Hansen strain AS2.440.
  • the nude mouse model of human prostate cancer was used to study the growth of the tumors in animals and the survival time thereof after tumor injection and freatment.
  • the animal preparation was carried out in a manner similar to that described in the section of the nasopharyngeal cancer.
  • the experiment was ca ⁇ ied out in a manner similar to that described in the section of Tumor Growth in Mouse model for the nasopharyngeal cancer, except that a group TSPA was infroduced in lieu of group CTX, wherein the mice were injected intravenously with 1.5 mg of thiotepa (TSPA) per kg body weight per day.
  • TSPA thiotepa
  • the following example illustrates the benefit of a biological composition of the invention comprising activated and conditioned Saccharomyces cerevisiae Hansen sfrain AS2.502 cells.
  • Two mouse models of human ovarian cancer were used to study the growth of the tumors in animals and the survival time thereof after tumor injection and treatment.
  • C 57 BL Mouse Model The animals used to generate the ovarian cancer cells for the experiments were female C 57 BL mouse (obtainable from the Chinese Academy of Military Medical Sciences, Beijing, China), six weeks old, having an average body weight of about 18 to 20 gram. Detailed description of the use of C 57 BL mice can be found in Roby K.F. et al., 2000, Carcinogenesis 21(4):585-91, which is incorporated herein by reference in its entirety.
  • Ovarian tumor cells (cell line obtainable from the Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China) were injected subcutaneously into the C 5 BL mice. The number of tumor cells injected was about 10 6 units per mouse in a 0.2 ml culture suspension.
  • the experiment was carried out in a manner similar to that described in the section of Tumor Growth in Mouse Model for the nasopharyngeal cancer.
  • breast Cancer The following example illustrates the benefit of a biological composition of the invention comprising activated and conditioned Saccharomyces carlsbergensis Hansen strain AS2.441 cells.
  • Two mouse models of human breast cancer were used to study the growth of the tumors in animals and the survival time thereof after tumor inj ection and freatment.
  • the murine transplantable breast cancer cell line Ca615-B was used in the model which resembles human breast cancer pathology. See e.g., In Oncology Basic and Research Protocols (Ed. J. Kao), People's Health Publishing House, Beijing, China, 1998, ⁇ .72.
  • the animals used to generate the breast cancer cells for the experiments were female 615 mouse (obtainable from the Chinese Academy of Military Medical Sciences, Beijing, China), having an average body weight of about 18 to 20 gram.
  • About 10 6 viable tumor cells of the murine breast cancer cell line Ca615-B (obtainable from the Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China) in about 0.2 ml culture suspension were injected subcutaneously into the animals.
  • the experiment was carried out in a manner similar to that described in the section of Tumor Growth in Mouse Model for the nasopharyngeal cancer, except that a group TSPA was infroduced in lieu of CTX group, wherein the mice were injected intravenously with 1.5 mg of thiotepa (TSPA) per kg body weight per day.
  • TSPA thiotepa
  • the murine transplantable breast cancer cell line MA 782/5S-B was used in the model which resembles human breast cancer pathology. See e.g., In Oncology Basic and Research Protocols (Ed. J. Kao), People's Health Publishing House, Beijing, China, 1998, p.73.
  • the animals used to generate the breast cancer cells for the experiments were female mice derived from an outbreed line of mouse (obtainable from the Chinese Academy of Military Medical Sciences, Beijing, China), having an average body weight of about 18 to 20 gram.
  • About 10 6 viable tumor cells of the breast cancer cell line MA782/5S-B (obtainable from the Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China) in about 0.2 ml culture suspension were injected subcutaneously into the animals.
  • the experiment was carried out in a manner similar to that described in section Tumor Growth in Mouse Model for the nasopharyngeal cancer.
  • the following example illustrates the benefit of a biological composition of the invention comprising activated and conditioned Saccharomyces carlsbergensis Hansen strain AS2.444 cells.
  • Two mouse models of human cervical cancer were used to study the growth of the tumors in animals and the survival time thereof after tumor injection and treatment.
  • the cervical cancer cell line U14 was used in the model which closely resembles the human cervical cancer pathology. Detailed description of the use of cervical cancer cell line U14 can be found in Tao G. et al., 2001, Chin Med J. 114(6): 623 -7, which is incorporated herein by reference in its entirety.
  • the animals used to generate the cervical cancer cells for the experiments were female mice from the 615 strain (obtainable from the Chinese Academy of Military Medical Sciences, Beijing, China) having an average body weight of 20 to 22 gram and 4 to 6 months old.
  • About 2 x 10 6 viable tumor cells of the cervical cancer cell line U14 (obtainable from the Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China) in about 0.2 ml culture suspension were injected subcutaneously into the animals.
  • the experiment was carried out in a manner similar to that described in the section of Tumor Growth in Mouse Model for the nasopharyngeal cancer, except that group MMC was used in lie of group CTX, wherein the mice were injected subcutaneously with 0.8 mg of mitomycin C (MMC) per kg body weight per day.
  • group MMC mitomycin C
  • the cervical cancer cell line U27 was used in the model which closely resembles the human cervical cancer pathology. Detailed description of the use of cervical cancer cell line U27 can be found in Qian S.S. et al., 1987, Zhongguo Yi Xue Ke Xue Yuan Xue Bao 9(l):33-7, wliich is incorporated herein by reference in its entirety.
  • the animals used to generate the cervical cancer cells for the experiments were female kun ming mouse (obtainable from the Chinese Academy of Military Medical Sciences, Beijing, China) having an average body weight of 20 to 22 gram and 5 to 6 months old.
  • About 1.2 x 10 7 viable tumor cells of the cervical cancer cell line U27 (obtainable from the Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China) in about 0.2 ml culture suspension were injected subcutaneously into the animals.
  • the experiment was carried out in a manner similar to that described in the section of Tumor Growth in Mouse Model for the nasopharyngeal cancer, except that group VCR was used in lie of group CTX, wherein the mice were injected intravenously with 3 mg of vinblastine (VCR) per kg body weight per day.
  • group VCR was used in lie of group CTX, wherein the mice were injected intravenously with 3 mg of vinblastine (VCR) per kg body weight per day.
  • the following example illustrates the benefit of a biological composition of the invention comprising activated and conditioned Saccharomyces carlsbergensis Hansen strain AS2.605 cells.
  • Two mouse models of human uterine cancer were used to study the growth of the tumors in animals and the survival time thereof after tumor injection and treatment.
  • Murine transplantable uterine cancer type CRL-1622 closely resembles the human uterine cancer pathology.
  • the uterine cancer cell line CRL-1622 can be ordered from the American Type Culture Collection.
  • the animals used to generate the uterine cancer cells for the experiments were female 615 mice (obtainable from the Chinese Academy of Military Medical Sciences, Beijing, China), 4 to 6 months, having an average body weight of about 20 to 22 gram.
  • About 1.2 x 10 7 viable tumor cells of the uterine cancer cell line CRL-1622 (obtainable from the Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China) in about 0.2 ml culture suspension were injected subcutaneously into the animals.
  • the experiment was carried out in a manner similar to that described in the section of Tumor Growth in Mouse Model for the nasopharyngeal cancer, except that group MMC was used in lie of group CTX, wherein the mice were injected subcutaneously with 0.8 mg of mitomycin C (MMC) per kg body weight per day.
  • MMC mitomycin C
  • the animals used to generate the uterine cancer cells for the experiments were female kun ming mouse (obtainable from the Chinese Academy of Military Medical Sciences, Beijing, China), 5 to 6 months, having an average body weight of about 20 to 22 gram.
  • About 1.2 x 10 7 viable tumor cells of the uterine cancer cell line HTB-114 (obtainable from the Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China) in about 0.2 ml culture suspension were injected subcutaneously into the animals.
  • the experiment was carried out in a manner similar to that described in the section of Tumor Growth in Mouse Model for the nasopharyngeal cancer, except that group VCR was used in lie of group CTX, wherein the mice were injected intravenously with 3 mg of vinblastine (VCR) per kg body weight per day.
  • group VCR was used in lie of group CTX, wherein the mice were injected intravenously with 3 mg of vinblastine (VCR) per kg body weight per day.
  • the following example illustrates the benefit of a biological composition of the invention comprising activated and conditioned Saccharomyces carlsbergensis Hansen sfrain AS2.189 cells.
  • a hamster model and the Wistar rat model of human kidney cancer were used to study the growth of the tumors in animals and the survival time thereof after tumor injection and freatment.
  • the animals used to generate the kidney cancer cells for the experiments were Syrian hamsters (obtainable from the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences, Beijing, China), an equal number of male and female, 5 to 6 months old, having an average body weight of 150 to 180 gram.
  • the complex cancer cells of kidney was generated from neutered male Syrian hamsters which were induced by subcutaneous injection of 17-/3- hydroxysteroid.
  • About 2 x 10 6 viable tumor cells of the complex cancer cells of kidney (obtainable from the Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China) in about 0.2 ml culture suspension were injected subcutaneously into the animals.
  • the animals used to generate the kidney cancer cells for the experiments were Wistar rats (obtainable from the Chinese Academy of Military Medical Sciences, Beijing, China) having an average body weight of about 180 to 200 gram.
  • the complex cancer cells of kidney (obtainable from the Shanghai Medical University, Shanghai, China) were generated from a Wistar rat with kidney cancer that was induced by subcutaneous injection of bismaleimidohexane (BHM).
  • BHM bismaleimidohexane
  • About 2.5 x 10 7 viable tumor cells of the complex cancer cells of kidney in about 0.4 ml culture suspension were injected subcutaneously into the animals.
  • the experiment was carried out in a manner similar to that of the section Tumor Growth in Wistar Rat Model for the nasopharyngeal cancer, except that the dosages for groups AY, NY, CKl and CK2 were 0.8 ml once per, and a group VCR was used in lieu of group TSPA, wherein the rats were injected intravenously with 3 mg of vinblastine (VCR) per kg body weight per day. The treatment continued for 30 days and the rats were scarified on the 31 st day.
  • VCR vinblastine
  • Bladder Cancer The following example illustrates the benefit of a biological composition of the invention comprising activated and conditioned Saccharomyces cerevisiae Hansen strain AS2.4 cells.
  • Mouse model and Wistar rat model of bladder cancer were used to study the growth of the tumors in animals and the survival time thereof after tumor injection and treatment.
  • Murine transplantable bladder cancer cell type N-butyl-N-(4-hydroxybutylnitrosamine (BBN) induced T 739 closely resembles the human bladder cancer pathology.
  • BBN-induced T 739 can be found in, e.g., Li L. et al., 2002, Sheng Wu Hua Xue Yu Sheng 34:21-7, which is incorporated herein by reference in its entirety.
  • the BBN-induced T 39 cells (obtainable from the Cancer Institute,
  • the animals used to receive the bladder cancer cells were T739 mouse (obtainable from the Chinese Academy of Military Medical Sciences, Beijing, China) having an average body weight of about 18 to 20 gram. About 10 6 viable tumor cells of the bladder cancer cells T 739 (in about 0.2 ml culture suspension) were injected subcutaneously into the animals.
  • TSPA thiotepa
  • Murine transplantable bladder cancer type N-butyl-N-(4- hydroxybutylnifrosamine (BBN) closely resembles the human bladder cancer pathology.
  • Numerous animal studies have reported the use of murine transplantable bladder cancer animal models. Detailed description of the use of murine transplantable bladder cancer cell type N-butyl-N-(4-hydroxybutyl)nitrosamine can be found in, e.g., Lummen G. et al., 2002, Urol Res. 30:199-203, which is incorporated herein by reference in its entirety.
  • the animals used to generate the bladder cancer cells for the experiments are Wistar rats (obtainable from the Chinese Academy of Military
  • the following example illustrates the benefit of a biological composition of the invention comprising activated and conditioned Saccharomyces cerevisiae Hansen sfrain AS2.501 cells.
  • the Kun Ming mouse model and Wistar rat model of human brain cancer were used to study the growth of the tumors in animals and the survival time thereof after tumor injection and treatment.
  • the kun ming murine brain cancer cell line B22 was used in the model which resembles human brain cancer pathology. See e.g., In Oncology Basic and Research Protocols (Ed. J. Kao), People's Health Publishing House, Beijing, China, 1998, p.78.
  • the animals used to generate the brain cancer cells for the experiments are male kun ming mice (obtainable from the Chinese Academy of Military Medicine Sciences, Beijing, China), 6 to 7 months, having an average body weight of about 20 to 22 gram.
  • About 1.2 x 10 7 viable tumor cells of the kun ming murine brain cancer cell line B22 (obtainable from the Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China) in about 0.2 ml culture suspension were injected subcutaneously into the animals.
  • mice were injected subcutaneously with 0.8 mg of chloroethylnitrosourea (BCNU) per kg body weight per day.
  • BCNU chloroethylnitrosourea
  • Wistar King rat brain cancer cell line WKS-1 was used in the model which resembles human brain cancer pathology. See e.g., In Oncology Basic and Research Protocols (Ed. J. Kao), People's Health Publishing House, Beijing, China, 1998, p.79.
  • the animals used to generate the brain cancer cells for the experiments are Wistar rats (obtainable from the Chinese Academy of Military Medical Sciences, Beijing, China), both males and females, having an average body weight of about 180 to 200 gram.
  • About 2 x 10 7 viable tumor cells of the Wistar King rat brain cancer type WKS-1 (obtainable from the Shanghai Medical University, Shanghai, China) in about 0.4 ml culture suspension were injected subcutaneously into the animals.
  • the experiment was carried out in a manner similar to that in the section of Tumor Growth in Wistar Rat Model for the nasopharyngeal cancer, except that the dosages for groups AY, NY, CKl and CK2 were 0.8 ml once per, and a group VCR was used in lieu of group TSPA, wherein the rats were injected intravenously with 3 mg of vinblastine (VCR) per kg body weight per day. The treatment continued for 30 days and the rats were scarified on the 31 st day.
  • VCR vinblastine
  • the following example illustrates the benefit of a biological composition of the invention comprising activated and conditioned Saccharomyces cerevisiae Hansen strain AS2.562 cells.
  • Two mouse models of lymphoma were used to study the growth of the tumors in animals and the survival time thereof after tumor injection and freatment.
  • mice that developed lymphoma spontaneously and that had been treated by the method as described above were divided into two series of five groups: one series received treatment for 30 days, the other series received freatment for 60 days.
  • each treatment group has a total of 36 mice, h the first series, the mice in group AY received 0.3 ml of the oral composition of the invention per day.
  • the mice in group NY received 0.3 ml per day of a composition comprising at a similar concentration the same sfrain of yeast which had not been activated and conditioned.
  • the mice in group CTX received subcutaneous injection of cyclophosphamide at a dose of 30 mg per kg body weight per day.
  • the mice in group CKl received physiological saline, while the mice in group CK2 received physiological saline but did not receive transplanted lymphoma cells. The experiment was carried out over a 30-day treatment period.
  • mice in group 2AY received 0.5 ml of the oral composition of the invention per day.
  • the mice in group 2NY received 0.5 ml per day of a composition comprising the same strain of yeast which had not been activated and conditioned at a similar concentration.
  • the mice in group 2CTX received subcutaneous injection of cyclophosphamide at a dose of 45 mg per kg body weight per day.
  • the mice in group 2CK1 received physiological saline, while the mice in group 2CK2 received physiological saline but did not receive lymphoma cells. The experiment was ca ⁇ ied out over a 60-day treatment period.
  • mice After the 30-day or 60-day treatment, the mice were fed a normal diet and allowed to live under the same environment. The health and survival of the mice were observed and compared over 180 days.
  • mice The results of the first series of five groups which received treatment for a total of 30 days are shown in Table 4. Each group consisted of 36 mice at the beginning of the experiment (3 repeats). The total number of surviving mice in each group were counted at the end of every month up to 6 months after treatment stopped.
  • mice The results of the second series of five groups which received treatment for a total of 60 days are shown in Table 5.
  • Each group consisted of 36 mice at the beginning of the experiment (3 repeats). The total number of surviving mice in each group were counted at the end of every month up to 6 months after treatment stopped.
  • Athymic Nude Mouse Model A model based on transplantation of Burkitt's lymphoma cell in athymic nude mice was used (Gurtsevitch et al., 1988, Int J Cancer 15;41(l):87-95). The model was set up as described in Shiyan Dongwu Yu Zhongliu Yanjiu (Experimental Animal & Tumor Research), April 2000, by Chinese Medical Technology Publisher. Essentially, seven week old mice were injected intravenously each with 1 x 10 6 Burkitt's lymphoma cells.
  • mice Twenty-one days after the injection of lymphoma cells, the mice were divided into two series of five groups: one series received treatment for 30 days, the other series received treatment for 60 days. In any one experimental series, each treatment group has a total of 36 mice.
  • the mice in group A received saline orally.
  • the mice in group B received subcutaneous injection of cyclophosphamide at a dose of 30 mg per kg body weight per day.
  • Group C received subcutaneous injection of cyclophosphamide at a dose of 45 mg per kg body weight per day.
  • the mice in group D received 0.3 ml of the oral composition per day.
  • group E received 0.5 ml of the oral composition per day. After the 30-day or 60-day freatment, the mice were fed a normal diet and allowed to live under the same environment. The health and survival of the nude mice were observed and compared over 360 days.
  • mice were counted at the end of every month up to 12 months after freatment stopped.
  • Table 102 The results of the first series of five groups which received treatment for a total of 30 days are shown in Table 7. Each group consisted of 36 mice at the beginning of the experiment (3 repeats). The total number of surviving mice in each group were counted at the end of every month up to 12 months after freatment stopped. Table 102
  • the following example illustrates the benefit of a biological composition of the invention comprising activated and conditioned Saccharomyces cerevisiae Hansen strain AS2.11 cells.
  • Two mouse models of human leukemia were used to study the growth of the tumors in animals and the survival time thereof after tumor injection and freatment.
  • Leukemia cell line HL-60 was also used to study the benefit of the biological composition in regulating the changes in cell cycles.
  • Murine transplantable leukemia cell line L6565 closely resembles the human leukemia pathology. See, e.g., Lab Animal and Animal Experiment Techniques, China Chinese Medicine Publisher, 1997, which is incorporated herein by reference in its entirety.
  • the animals used to generate the leukemia cells for the experiments were SWI mice (obtainable from the Chinese Academy of Military Medicine Science, Beijing, China), both males and females, with an average body weight of about 18 to 22 gram.
  • About 10 6 viable tumor cells of the leukemia cell line L6565 (obtainable from the Cancer Institute, Chinese Academy of Medical Sciences, Beijing, Cancer) in about 0.2 ml spleen cell culture suspension were injected subcutaneously into the animals.
  • the experiment was carried out in a manner similar to that described in the section of Tumor Growth in Mouse Model for the nasopharyngeal cancer, except that 21 days of consecutive treatment was performed in lieu of the 30 day treatment regime.
  • T 73 mice to study cancer can be found in, e.g., Niu Q. et al, 2001, Zhonghua Zhong Liu Za Zhi. 23(5):382-4, and Zheng F. et al, 2000, Zhonghua Jie He He Hu Xi Za Zhi. 23(l):34-6, each of which is incorporated herein by reference in its entirety.
  • the animals used to generate the leukemia cells for the experiments were T 39 mouse (obtainable from the Chinese Academy of Military Medicine Sciences, Beijing, China), both males and females, with an average body weight of about 18 to 22 gram.
  • About 10 6 viable tumor cells of the leukemia cell line T 739 in about 0.2 ml culture suspension were injected subcutaneously into the animals.
  • the experiment was carried out in a manner similar to that described in the section of Tumor Growth in Mouse Model for the nasopharyngeal cancer, except that 21 days of consecutive treatment was performed in lieu of the 30 day treatment regime.
  • the leukemia cell line HL-60 is commonly used to establish the efficacy of a treatment. See, e.g., Modern Pharmacology Experimental Protocols, Peking Union Medical College Publisher, 1998.
  • Flow cytometry was used to study the changes in cell cycles. Flow cytometry measures the number of cells in different cell cycles by analyzing the amount of DNA in a single cell. A fluorescent dye, propidium iodide (PI) intercalates with DNA and emits yellow and red regions fluorescence in response to 488 nm excitation. Cells in the GI and GO cell cycle phases will contain a 2n amount of DNA.
  • PI propidium iodide
  • Cells in the G2 and M phases will contain a 4n amount of DNA, while cells in S phase will have a DNA content between 2n and 4n (as the DNA has only partially been replicated).
  • flow cytometry can be used to determine the percentage of live cells in each phase of the cell cycle and the percentage of dead cells. This information, coupled with the doubling time, allows one to calculate the length of each cell cycle phase. More detailed description of the methodologies and applications of flow cytometry can be found in Darzynkiewicz et al, 2001, Clin Lab Med. 21(4):857-73, which is incorporated by reference herein in its entirety.
  • Activated HL-60 leukemia cells (obtainable from the Cancer Institute, Chinese Academy of Military Medical Sciences, Beijing, China) were cultured in RPMI- 1640 suspension and then divided into 3 groups of 10 9 cells per group. The three groups of HL-60 cells were quintuplicated. Detailed description of the cultivation of human leukemia HL-60 cells can be found in, e.g., Saether A.K. et al, 1991, Eur J Cell Biol. 55(2):346-51, which is incorporated by reference herein in its entirety. In the experimental group AY, the biological composition was filtered through 0.22 ⁇ m filter.
  • the activated and conditioned yeast cells (about 5 /mi in width and 10 ⁇ m in length) were collected and added to 10 9 HL-60 leukemia cells per ml to make 5% final concentration by weight.
  • the untreated yeast cells were filtered through 0.22 ⁇ m filter.
  • the untreated yeast cells (about 5 ⁇ m in width and 10 ⁇ m in length) were collected and added to 10 9 HL-60 leukemia cells per ml to make 5% final concentration by weight.
  • 10 9 HL-60 leukemia cells per ml was used.
  • Tables 10 and 11 show the percentages of live leukemia cells in each cell cycle and the percentages of dead leukemia cells in the freatment and control groups after culturing for 24 hours and 72 hours, respectively.
  • the percentage of programmed cell death is significantly greater (8x) when the HL-60 leukemia cells were co-incubated with the activated and conditioned yeast cells of the present invention (24.7% in group AY) than when the HL-60 leukemia cells were co-incubated with the untreated yeast cells (3.2% in group NY) or cultured alone (3.1% in group CK).
  • co- incubation with the activated and conditioned yeast cells of the present invention have already begun to reduce the number of live leukemia cells. A significant number of live leukemia cells in all three groups were at S stage, i.e., when DNA replication occurs (synthesis phase).
  • the percentage of programmed cell death is still significantly greater (8x) when the HL-60 leukemia cells were co-incubated with the activated and conditioned yeast cells of the present invention (85.2% in group AY) than when the HL-60 leukemia cells were co-incubated with the unfreated yeast cells (11.6% in group NY) or cultured alone (11.3% in group CK).
  • the percentage of programmed cell deaths in group AY increased more than three-folds from 24.7% to 85.2%, suggesting that the activated and conditioned yeast cells of the present invention are extremely effective in reducing the number of live leukemia cells.
  • more than half of the live leukemia cells in group AY at 72 hours (56.2%) are now in Go-G! stage, i.e., a temporary or permanent resting period where the cell has reached an end stage of development and will no longer divide.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Botany (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Biomedical Technology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medical Informatics (AREA)
  • Nutrition Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Hematology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)

Abstract

L'invention concerne des compositions biologiques et un complément alimentaire comprenant des cellules de levure pouvant produire un effet bénéfique du point de la santé chez un sujet souffrant du cancer. Les compositions biologiques peuvent être utilisées pour retarder la croissance de cellules cancéreuse et/ou pour prolonger la durée de survie du sujet. L'invention concerne également des procédés de fabrication des compositions biologiques.
PCT/GB2004/002466 2003-06-11 2004-06-10 Compositions biologiques comprenant des cellules de levure et methodes de traitement du cancer WO2004108919A1 (fr)

Applications Claiming Priority (36)

Application Number Priority Date Filing Date Title
US10/460,246 2003-06-11
US10/460,325 US7204987B2 (en) 2003-06-11 2003-06-11 Biological compositions and methods for treatment of prostate cancer
US10/460,530 US7223405B2 (en) 2003-06-11 2003-06-11 Method to prepareompositions comprising yeast treated with electromagnetic energy
US10/460,832 2003-06-11
US10/460,327 US6989253B2 (en) 2003-06-11 2003-06-11 Biological compositions and methods for treatment of testicular cancer
US10/460,326 2003-06-11
US10/460,326 US7223401B2 (en) 2003-06-11 2003-06-11 Method to prepare compositions comprising yeast treated with electromagnetic energy
US10/460,337 US7223402B2 (en) 2003-06-11 2003-06-11 Method to prepare compositions comprising yeast treated with electromagnetic energy
US10/460,438 US7223404B2 (en) 2003-06-11 2003-06-11 Method to prepare compositions comprising yeast treated with electromagnetic energy
US10/460,341 2003-06-11
US10/460,437 US7223403B2 (en) 2003-06-11 2003-06-11 Method to prepare compositions comprising yeast treated with electromagnetic energy
US10/460,832 US7204988B2 (en) 2003-06-11 2003-06-11 Method to prepare compositions comprising yeast treated with electromagnetic energy
US10/460,341 US6987012B2 (en) 2003-06-11 2003-06-11 Biological compositions and methods for treatment of colorectal cancer
US10/460,338 2003-06-11
US10/460,323 2003-06-11
US10/460,271 US7223400B2 (en) 2003-06-11 2003-06-11 Method to prepare compositions comprising yeast treated with electromagnetic energy
US10/460,833 2003-06-11
US10/460,328 US7201906B2 (en) 2003-06-11 2003-06-11 Method to prepare compositions comprising yeast treated with electromagnetic energy
US10/460,437 2003-06-11
US10/460,328 2003-06-11
US10/460,336 US6984508B2 (en) 2003-06-11 2003-06-11 Biological compositions and methods for treatment of cervical cancer
US10/460,833 US7208158B2 (en) 2003-06-11 2003-06-11 Method to prepare compositions comprising yeast treated with electromagnetic energy
US10/460,247 US7214377B2 (en) 2003-06-11 2003-06-11 Method to prepare compositions comprising yeast treated with electromagnetic energy
US10/460,246 US7204986B2 (en) 2003-06-11 2003-06-11 Method to prepare compositions comprising yeast treated with electromagnetic energy
US10/460,438 2003-06-11
US10/460,325 2003-06-11
US10/460,323 US6984507B2 (en) 2003-06-11 2003-06-11 Biological compositions and methods for treatment of lung cancer
US10/460,338 US7226600B2 (en) 2003-06-11 2003-06-11 Method to prepare compositions comprising yeast treated with electromagnetic energy
US10/460,336 2003-06-11
US10/460,337 2003-06-11
US10/460,271 2003-06-11
US10/460,327 2003-06-11
US10/460,530 2003-06-11
US10/460,324 2003-06-11
US10/460,247 2003-06-11
US10/460,324 US7220416B2 (en) 2003-06-11 2003-06-11 Method to prepare compositions comprising yeast treated with electromagnetic energy

Publications (1)

Publication Number Publication Date
WO2004108919A1 true WO2004108919A1 (fr) 2004-12-16

Family

ID=33515039

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2004/002466 WO2004108919A1 (fr) 2003-06-11 2004-06-10 Compositions biologiques comprenant des cellules de levure et methodes de traitement du cancer

Country Status (1)

Country Link
WO (1) WO2004108919A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021071413A1 (fr) * 2019-10-07 2021-04-15 Milmed Unico Ab Levure pour le traitement de l'allergie

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002062983A1 (fr) * 2001-02-08 2002-08-15 Six Forest Bio-Science Institute Limited Levure faisant preuve d'une activite immunoregulatrice des lymphocytes t, son procede de preparation et ses applications
WO2002070683A2 (fr) * 2001-03-01 2002-09-12 Ultra Biotech Limited Compositions biologiques pour le traitement des dechets solides
EP1375652A2 (fr) * 2002-06-28 2004-01-02 Ultra Biotech Limited Composition orale pour des sujets infectés par le VIH

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002062983A1 (fr) * 2001-02-08 2002-08-15 Six Forest Bio-Science Institute Limited Levure faisant preuve d'une activite immunoregulatrice des lymphocytes t, son procede de preparation et ses applications
WO2002070683A2 (fr) * 2001-03-01 2002-09-12 Ultra Biotech Limited Compositions biologiques pour le traitement des dechets solides
EP1375652A2 (fr) * 2002-06-28 2004-01-02 Ultra Biotech Limited Composition orale pour des sujets infectés par le VIH

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BINNINGER D M ET AL: "Effects of 60Hz AC magnetic fields on gene expression following exposure over multiple cell generations using Saccharomyces cerevisiae", BIOELECTROCHEMISTRY AND BIOENERGETICS, XX, XX, vol. 43, no. 1, 1997, pages 83 - 89, XP002223047, ISSN: 0302-4598 *
DATABASE WPI Section Ch Week 200267, Derwent World Patents Index; Class B04, AN 2002-627548, XP002259470 *
PONNE C T ET AL: "Interaction of electromagnetic energy with biological material--relation to food processing", RADIATION PHYSICS AND CHEMISTRY, ELSEVIER SCIENCE PUBLISHERS BV., AMSTERDAM, NL, vol. 45, no. 4, 1 April 1995 (1995-04-01), pages 591 - 607, XP004051598, ISSN: 0969-806X *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021071413A1 (fr) * 2019-10-07 2021-04-15 Milmed Unico Ab Levure pour le traitement de l'allergie

Similar Documents

Publication Publication Date Title
US6989253B2 (en) Biological compositions and methods for treatment of testicular cancer
US7223400B2 (en) Method to prepare compositions comprising yeast treated with electromagnetic energy
US7204988B2 (en) Method to prepare compositions comprising yeast treated with electromagnetic energy
US6984507B2 (en) Biological compositions and methods for treatment of lung cancer
US7223403B2 (en) Method to prepare compositions comprising yeast treated with electromagnetic energy
US7226600B2 (en) Method to prepare compositions comprising yeast treated with electromagnetic energy
US7223404B2 (en) Method to prepare compositions comprising yeast treated with electromagnetic energy
US7208158B2 (en) Method to prepare compositions comprising yeast treated with electromagnetic energy
US7214377B2 (en) Method to prepare compositions comprising yeast treated with electromagnetic energy
US7223402B2 (en) Method to prepare compositions comprising yeast treated with electromagnetic energy
US7223405B2 (en) Method to prepareompositions comprising yeast treated with electromagnetic energy
US7204986B2 (en) Method to prepare compositions comprising yeast treated with electromagnetic energy
US7220416B2 (en) Method to prepare compositions comprising yeast treated with electromagnetic energy
US7201906B2 (en) Method to prepare compositions comprising yeast treated with electromagnetic energy
US7223401B2 (en) Method to prepare compositions comprising yeast treated with electromagnetic energy
US7204987B2 (en) Biological compositions and methods for treatment of prostate cancer
US6987012B2 (en) Biological compositions and methods for treatment of colorectal cancer
US6984508B2 (en) Biological compositions and methods for treatment of cervical cancer
US7256026B2 (en) Oral compositions for white blood cell activation and proliferation
EP1375652A2 (fr) Composition orale pour des sujets infectés par le VIH
EP1394249A1 (fr) Suppléments alimentaires bénéfiques au système gastrointestinal
WO2004108919A1 (fr) Compositions biologiques comprenant des cellules de levure et methodes de traitement du cancer

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase