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EP1699470A2 - Durch elektrostimulation erhaltenes biologisch aktives blutserum - Google Patents

Durch elektrostimulation erhaltenes biologisch aktives blutserum

Info

Publication number
EP1699470A2
EP1699470A2 EP04804108A EP04804108A EP1699470A2 EP 1699470 A2 EP1699470 A2 EP 1699470A2 EP 04804108 A EP04804108 A EP 04804108A EP 04804108 A EP04804108 A EP 04804108A EP 1699470 A2 EP1699470 A2 EP 1699470A2
Authority
EP
European Patent Office
Prior art keywords
serum
blood
blood serum
cell line
pharmaceutical composition
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP04804108A
Other languages
English (en)
French (fr)
Inventor
Vitali A. Shestakov
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Owen Holding Ltd
Original Assignee
Owen Holding Ltd
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 PCT/EP2003/014578 external-priority patent/WO2005058333A1/de
Application filed by Owen Holding Ltd filed Critical Owen Holding Ltd
Priority to EP04804108A priority Critical patent/EP1699470A2/de
Publication of EP1699470A2 publication Critical patent/EP1699470A2/de
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/16—Blood plasma; Blood serum
    • 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
    • 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
    • A61K41/10—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person
    • A61K41/17—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person by ultraviolet [UV] or infrared [IR] light, X-rays or gamma rays
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/141—Feedstock

Definitions

  • the present invention relates to a method for preparing a blood serum product, the blood serum product, and a pharmaceutical composition comprising said blood serum product as well as uses thereof in the treatment of various diseases and conditions including epileptic seizures and apoplexy.
  • blood serum with different biological activity can be obtained which shows mitogenic, somnogenic, opthalmogenic, audio active, thermo active, diatary active, sexually active, anti-hypoxic, anti-alcohol and anti-nicotine activity.
  • EP 1 283 047 A different method is disclosed in EP 1 283 047 and concerns the treatment of animal blood serum by gamma irradiation with the aim to increase the biological activity of the blood serum product.
  • Such peptides as, for example, the parathyroid peptides, gastrin or bombesin foster the development of tumor cells as well as the development of breast, bone and colon cancer (see, for example, Kitazawa S. and Maeda S. (1995) Clin. Orthop. 312: 45-50 and Kaji et al. (1995) Endocrinology 136: 842).
  • An object of the present invention was the development of a novel method for the preparation of a biological active blood serum from animal blood. Surprisingly it was found that the biological active blood serum prepared according to the method of the invention exhibited new therapeutic properties.
  • one aspect of the present invention is a method for producing a biological active blood serum comprising the steps of: a) electrostimulation of a non-human animal b) withdrawal of blood from said animal, c) isolation of serum from said blood, and d) gamma irradiation of said serum.
  • the non-human animal is selected form the group consisting of mammals and birds, preferably from poultry, e.g. chicken, dug, goose, ostrich, and quail.
  • step a) of the method of the present invention is applied to the head, the neck, the body and/or one or more limbs of the animal. Out of those it is preferred that the head of the animal is electrostimulated. In the context of the present invention the terms electrostimulation and electroshock are used interchangeably.
  • the electrostimulation is carried out for a time period of between 1 and 60 seconds, preferably between 1 and 30 seconds, and more preferably between 2 and 10 seconds. It is also preferred that the electrostimulation is carried out with a voltage in the range of between 50 V and 150 V, preferably in the range of between 80 V to 120 V, and more preferably in the range of between 110 V and 120 V. During the performance of the electrostimulation certain currents are preferred and preferably the electrostimulation is carried out with a current in the range of between 0.01 A and 0.4 A, preferably in the range of between 0.02 A and 0.1 A, and more preferably in the range of between 0.04 A and 0.06 A.
  • the electrostimulation is carried out with a frequency in the range of between 10 and 200 Hz, preferably in the range of between 20 to 100 Hz and more preferably in the range of between 45 to 65 Hz.
  • the gamma irradiation is administered with an adsorbed radiation dose of between 10 to 40 kGy, preferably 15 to 35 kGy and more preferably of between 20 and 30 kGy.
  • the gamma radiation source can be any source, however, a preferred source of gamma radiation is selected from the group consisting of 60 Co, 137 Cs, 67 Cu, 67 Ga, m In, 192 Ir, 99m Tc and 170 Tm.
  • the method further comprises the step of incubating said blood prior to step c).
  • the method further comprises the step of lyophilization of said serum prior to step d).
  • the blood is arterial and/or venous blood.
  • Another aspect of the present invention is the biological active blood serum which is producible according to a method of the present invention.
  • a further aspect of the present invention is a pharmaceutical composition
  • a pharmaceutical composition comprising a blood serum according to the present invention and one or more pharmaceutically acceptable diluents; carriers; excipients, including fillers, binders, lubricants, glidants, disintegrants, adsorbents; and/or preservatives.
  • the composition is formulated as a syrup, an infusion or injection solution, a tablet, a capsule, a capslet, lozenge, a liposome, a suppository, a plaster, a band-aid, a retard capsule, a powder, or a slow release formulation.
  • the diluent is water, a buffer, a buffered salt solution or a salt solution and the carrier preferably is selected from the group consisting of cocoa butter and vitebesole.
  • a further aspect of the present invention is the use of a blood serum of the present invention or of a pharmaceutical composition of the present invention for the production of a medicament for the treatment of a disease or condition, which can be affected by an increase of cyclic adenosine monophosphoric acid contents in the brain of the subject requiring treatment.
  • Another aspect of the present invention is the use of a blood serum of the present invention or of a pharmaceutical composition of the present invention for the production of a medicament for the improvement of cognitive and/or learning skills in particular improvement of the long term memory.
  • Another aspect of the present invention is the use of a blood serum of the present invention or of a pharmaceutical composition of the present invention for the production of a medicament for the treatment of seizures, in particular epileptic seizures.
  • a further aspect of the present invention is a use of a blood serum of the present invention or of a pharmaceutical composition of the present invention for the production of a medicament for the treatment of proliferative diseases and apoplexy.
  • the proliferative disease is selected from the group consisting of malignomas of the gastrointestinal or colorectal tract, the liver, the pancreas, the kidney, the bladder, the thyroid, the prostate, the endometrium, the cervix, the ovary, the uterus, the testes, the skin, the oral cavity; melanoma; dysplastic oral mucosa; invasive oral cancers; small cell and non-small cell lung carcinomas; mammary tumors, in particular hormone-dependent breast cancers and hormone independent breast cancers; transitional and squamous cell cancers; neurological malignancies including neuroblas- tomas, gliomas, astrocytomas, osteosarcomas, meningiomas; soft tissue sarcomas; heman- gioamas and endocrinological tumors, in particular pituitary adenomas, pheochromocytomas, paragangliomas, haematological malign
  • the proliferative disease comprises cells similar to the human T cell lymphoma cell line Jurkat, the human B cell lymphoma cell line Raji, the human melanoma cell line Bro, the human cervical cancer cell line HeLa, the human adenocarcinoma cell line MCF-7, the osteosarcoma cell line Mg63, the fibrosarcoma cell line HT1080, the neuroblastoma cell line IMR-32 and the hepatocarci- noma cell line HepG2.
  • the medicament is administered to a subject in need of treatment in an amount ranging from 50 to 150 mg/kg body weight, preferably ranging from 90 to 100 mg/kg body weight.
  • the present inventors have surprisingly found that the stimulation of animals, in particular chickens with electric currents and the further treatment of blood serum obtained from the blood with ⁇ -radiation leads to a significant increase of the biological activity of the blood serum product.
  • the resulting products are capable of positively influencing various body functions, conditions and diseases of a patient.
  • a first aspect of the present invention is a method for producing a biological active blood serum comprising the steps of: a) electrostimulation of a non-human animal, b) withdrawal of blood from said animal, c) isolation of serum from said blood, and d) gamma irradiation of said serum.
  • non-human animal is selected from the group consisting of mammals and birds. Because of their easy availability it is particularly preferred to use farm animals like poultry, e.g. chicken, dug, goose, ostrich and quail.
  • farm animals like poultry, e.g. chicken, dug, goose, ostrich and quail.
  • a particular preferred animal which can be used in the method of the present invention is a chicken.
  • the type of mammal that can be used in the method of the present invention is not particularly restricted and comprises without limitation rodents, e.g. mouse, hamster and rat, cats, dogs, horses, donkeys, sheep, cows, and goats.
  • the electrostimulation leads to the release of certain compounds within the animal which cause and/or contribute to the surprising therapeutic effect of the biological active blood serum of the present invention.
  • the non-human animal can be stimulated in different regions of the body.
  • the electrostimulation is carried out at the head, the neck, the body and/or on one or more of the limbs. It is possible to stimulate the body only at one position or at several positions at once.
  • a particular preferred body part for the electrostimulation is the head of the respective animal. When stimulating birds, in particular chicken it is preferred that the head is electrostimulated.
  • the electrostimulation can be carried out by art known methods, preferably using metal electrodes or water baths as used, for example, during culling of cattle or electrocution of poultry.
  • the electrostimulation is carried out for a time period of between 1 and 60 seconds, preferably between 1 and 30 seconds, more preferably between 2 and 10 seconds, and most preferably between 3 and 4 seconds.
  • the length of a time period will be longer in case that a large animal is electrostimulated and can be shorter in cases were small animals are electrostimulated.
  • a particular preferred time period of the electrostimulation is between 2 and 10 seconds and more preferably between 3 and 4 seconds.
  • the electro stimulation is preferably carried out with a voltage in the range of between 50 Volt and 150 Volt, preferably 80 Volt to 120 Volt and more preferably between 110 Volt and 120 Volt.
  • the ranges for the currents that can be applied are between 0.01 A and 0.4 A, preferably between 0.02 A and 0.1 A, more preferably between 0.04 A and 0.06 A and most preferably about 0.05 A.
  • Voltage, current and application time are preferably chosen to administer energy in the range of between 1 and 1,000 Ws, preferably in the range of 10 to 200 Ws and even more preferably in the range of 15 to 100 Ws.
  • the electrostimulation is carried out with a voltage in the range of between 80 Volt to 120 Volt and more preferably between 110 Volt and 120 Volt. Furthermore, a current in the range of between 0.04 A and 0.06 A, in particular of 0.05 A is preferred in the context of the electrostimulation of birds, in particular of chicken.
  • the electrostimulation of a bird, in particular a chicken is carried out for between 3 and 4 seconds at a voltage of between 80 V and 120 V, in particular 110 V and 120 V.
  • the current is preferably between 0.04 A and 0.06 A and most preferably about 0.05 A.
  • the frequency of the electrostimulation does not appear to be particularly critical but is preferably in the range of between 10 and 200 Hertz, more preferably in the range of between 45 to 65 Hz and most preferably around 50 Hz.
  • the gamma irradiation of the serum during step d) of the method of the present invention can be carried out with any gamma source including X-ray sources and radionuclides.
  • the gamma radiation source is a radionuclide with a defined gamma radiation pattern.
  • Pre- ferred sources for the gamma radiation are selected from the group consisting of Co, Cs, 67 Cu, 67 Ca, m In, 192 Ir, 99m Tc and 170 Tm. Out of those 60 Co, 137 Cs, 192 Ir and 170 Tm are particular preferred with Co being the most preferred gamma radiation source, for us in the method of the present invention.
  • the radiation dose adsorbed by the serum is in the range of between 10 to 40 kGy preferably in the range of between 15 to 35 kGy and more preferably in the range of between 20 and 30 kGy, i.e. 25 ⁇ 5 kGy.
  • the withdrawal of the blood from the animal can be effected by any art known method and includes syringes as well as puncturing of arteries or veins or decapitation in particular in the context of the withdrawal of blood from birds. It is possible to withdraw only a part of the blood or to completely with draw the blood of the animal. The later is preferably used, if a lethal dose of electricity has been applied to the animal.
  • the withdrawn blood can be arterial and/or venous blood.
  • the serum can be isolated from the blood by any known method including filtration, sedimentation and centrifugation. It is, however, preferred that the blood is incubated for between 4 and 72 h at a low temperature, e.g. between 2° and 10°C, preferably between 4-8°C to allow clotting of the blood which leads to the release of additional factors into the blood.
  • the method further comprises the step of incubating the blood after the withdrawal of the blood from the animal and prior to the isolation of the serum from the blood, e.g. for between 4 and 72 h at a low temperature, e.g. between 2° and 10°C, preferably between 4-8°C.
  • the method comprises the further step of lyophilization of the serum prior to the irradiation step d).
  • the lyophilization allows easier handling of the serum during irradiation and optimizes absorption of the radiation by the serum components.
  • a further aspect of the present invention is the biological active blood serum itself, which is producible according to a method of the present invention. It is distinct from prior art blood serums which do not employ the steps of the method of the present invention, which is evidenced by its particular therapeutic effects, which are not exhibited by prior art blood serum products.
  • a further aspect of the present invention is a pharmaceutical composition, comprising a biological active blood serum producible according to the method of the present invention.
  • Such pharmaceutical composition can further comprise one or more pharmaceutically acceptable diluents; carriers; excipients, including fillers, binders, lubricants, glidants, disintegrants, and adsorbents; and/or preservatives.
  • the pharmaceutical composition of the present invention can be administered by various well known routs, including oral and parenteral administration, e.g. intravenous, intramuscular, intranasal, intradermal, subcutaneous and similar administration routes. Parenteral administration and particular intravenous administration is preferred. Depending on the route of administration different pharmaceutical formulations are required and some of those may require that protective coatings are applied to the drug formulation to prevent degradation of the biological active serum in, for example, the digestive tract.
  • the pharmaceutical composition of the present invention is formulated as a syrup, an infusion solution, or injection solution, a tablet, a capsule, a capslet, a lozenge, lipo- some, a suppository, a plaster, a band-aid, a retard capsule, a powder or a slow release formulation.
  • Particular preferred pharmaceutical forms are forms suitable for injectionable use and include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the final solution or dispersion form must be sterile and fluid.
  • a solution or dispersion will include a solvent or dispersion medium, containing, for example, water-buffered aqueous solutions, e.g. biocompatible buffers, ethanol, polyol, such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof, surfactants or vegetable oils.
  • the biological active blood serum of the present invention can also be formulated into liposomes, in particular for parenteral administration. Liposomes provide the advantage of increased half life in the circulation, if compared to the free drug and a prolonged more even release of the enclosed drug.
  • Sterilization of infusion or injection solutions can be accomplished by any number of art recognized techniques including but not limited to addition of preservatives like anti-bacterial or anti-fungal angents, e.g. parabene, chlorobutanol, phenol, sorbic acid or thimersal. Further, isotonic agents, such as sugars or salts, in particular sodium chloride may be incorporated in infusion or injection solutions.
  • preservatives like anti-bacterial or anti-fungal angents, e.g. parabene, chlorobutanol, phenol, sorbic acid or thimersal.
  • isotonic agents such as sugars or salts, in particular sodium chloride may be incorporated in infusion or injection solutions.
  • sterile injectable solutions containing the biological active blood serum is accomplished by incorporating the biological active serum in the required amount in the appropriate solvent with various ingredients enumerated above as required followed by sterilization. To obtain a sterile powder the above solutions are vacuum-dried or freeze-dried as necessary.
  • Preferred diluents of the present invention are water, physiological acceptable buffers, physiological acceptable buffer salt solutions or salt solutions.
  • Preferred carriers of the present invention are cocoa butter and vitebesole.
  • Excipients which can be used with the various pharmaceutical forms of the biological active blood serum can be chosen from the following non-limiting list:
  • binders such as lactose, mannitol, crystaline sorbitol, dibasic phosphates, calcium phosphates, sugars, microcrystalline cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, polyvinyl pyrrolidone and the like;
  • lubricants such as magnesium stearate, talc, calcium stearate, zinc stearate, stearic acid, hydrogenated vegetable oil, leucine, glycerids and sodium stearyl fumarates,
  • disintegrants such as starches, croscaramellose, sodium methyl cellulose, agar, bentonite, alginic acid, carboxymethyl cellulose, polyvinyl pyrrolidone and the like.
  • a further aspect of the present invention is the use of a blood serum or of a pharmaceutical composition of the present invention for the production of a medicament for the treatment of a disease or condition which can be effected by an increase of cyclic adenosine monophos- phoric acid content in the brain of the treated subject.
  • Another aspect of the present invention is the use of the biological active blood serum or of a pharmaceutical composition of the present invention for the production of a medicament for the improvement of nootropic, cognitive and/or learning skills of the treated subject and in particular the improvement of the long-term memory.
  • the usefulness for this indication is based on the discovery that the blood serum or the pharmaceutical composition of the present invention increase the learning abilities of treated subjects.
  • the blood serum or the pharmaceutical composition can be used for the treatment of seizures of any type in particular, however, for the treatment of epileptic seizures.
  • the administration of the biologically active blood serum or of the pharmaceutical composition of the present invention can prevent death that is sometimes associated with severe seizures.
  • the blood, serum or the pharmaceutical composition of this invention can be used for the treatment of nervous diseases, including without limitation, bipolar disorder, depression, anxiety related disorders, epilepsy, Alzheimer's disease, Parkinson's disease, peripheral neurophathy, cerebral amyloid angiopathy, neuro degenerative disorders and spinal cord injury.
  • a further aspect of the present invention is the use of the blood serum or of the pharmaceutical composition of the present invention for the production of a medicament for the treatment of proliferative diseases and apoplexy. It was particular surprising that the biological active blood serum or the pharmaceutical composition of the present invention did show a marked anti-proliferative effect, if tested on a variety of tumor cell lines in vitro.
  • the biological active blood serum or the pharmaceutical composition of the present invention is particular suitable for the treatment of proliferative diseases and prefened proliferative diseases which are treatable according to the use of the present invention are selected from the group consisting of malignomas of the gastrointestinal or colorectal tract, the liver, the pancreas, the kidney, the bladder, the thyroid, the prostate, the endometrium, the cervix, the ovary, the uterus, the testes, the skin, the oral cavity; melanoma; dysplastic oral mucosa; invasive oral cancers; small cell and non-small cell lung carcinomas; mammary tumors, in particular hormone-dependent breast cancers and hormone independent breast cancers; transitional and squamous cell cancers; neurological malignancies including neuroblastomas, gliomas, astrocytomas, osteosarcomas, meningiomas; soft tissue sarcomas; hemangioamas and endo- crinological tumors, in particular
  • the anti-proliferative effect of the biological active blood serum of the present invention or of the pharmaceutical composition of the present invention has been first established for a variety of tumor cell lines it is particular suitable for the treatment of proliferative diseases which comprise cells and/or tumor tissue comprising cells similar to the tumor cell lines used in those experiments.
  • preferred prolifertive diseases treatable with the biological active blood serum or the pharmaceutical composition comprising the biological active serum comprise cells similar to the human T cell lymphoma cell line Jurkat, the human B cell lymphoma cell line Raji, the human melanoma cell line Bro, the human cervical cancer cell line HeLa, the human adenocarcinoma cell line MCF-7, the osteosarcoma cell line Mg63, the fi- brosarcoma cell line HT1080, the neuroblastoma cell line IMR-32 and the hepatocarcinoma cell line HepG2.
  • similar cells are cells, which have the same origin, e.g.
  • T-cell, B cell or neural lineage as the respective cell line and which carry a mutation in the same or a functionally equivalent gene and wherein this mutation contributes to the proliferative activity of the cell, e.g. mutation in p53, pRb, cdc 2, cdk 4, cyclin A, cyclin B, p21 ras , c-fos, c-jun, ⁇ l 07, pi 30 and the like; which carry the same or a functionally similar exogenous gene, e.g.
  • HPV human papilloma virus
  • E 6 or E 7 insertion into the cyclin B promoter by hepatitis B virus and the like; or which have the same chromosomal rearrangement or abnormality , i.e. deletion, chromosomal multiplicity etc.
  • the biological active blood serum is preferred for the treatment of the diseases and conditions for which the blood serum and pharmaceutical composition can be employed. It is, however, understood that depending on the respective condition as well as on the respective patient to be treated, i.e. depending on the severity of the disease or condition, the general health status of the patient, etc., different doses of the biological active blood serum or the pharmaceutical composition are required to elicit a therapeutic effect. The determination of the appropriate dose lies within the discretion of the attending physician. It is contemplated that the dosage of the biologically active blood serum in the therapeutic method of the invention should be in the range of about 0.1 mg to about 200 mg serum per kg body weight.
  • the biologically active blood serum is administered to a subject in need thereof in an amount ranging from 50 to 150 mg/kg body weight, preferably ranging from 90 to 100 mg/kg body weight.
  • the duration of therapy with biologically active blood serum will vary, depending on the severity of the disease being treated and the condition and idiosyncratic response of each individual patient.
  • Fig. 1 Cytotoxic effect of two pharmaceutical preparations on Jurkat cells.
  • the cytotoxic effect on the human T cell lymphoma cell line Jurkat is depicted for two different pharmaceutical preparations comprising biological active serum of the invention various concentrations of the.
  • the viability of the Jurkat cells is depicted on the y-axis in percentages, while the amount of the biological active serum is indicated on the x-axis in mg/ml.
  • Fig.2 Cytotoxic effect of two pharmaceutical preparations on Raji cells.
  • the cytotoxic effect on the human B cell lymphoma cell line Raji depicted for two different pharmaceutical preparations comprising biological active serum of the invention various concentrations of the.
  • the viability of the Raji cells is depicted on the y-axis in percentages, while the amount of the biological active serum is indicated on the x-axis in mg/ml
  • Fig. 3 Cytotoxic effect of two pharmaceutical preparations on Bro B-19 cells.
  • the cytotoxic effect on the human T cell lymphoma cell line Bro B-19 is depicted for two different pharmaceutical preparations comprising biological active serum of the invention various concentrations of the.
  • the viability of the Bro B-19 cells is depicted on the y-axis in percentages, while the amount of the biological active serum is indicated on the x-axis in mg/ml
  • Fig. 4 Cytotoxic effect of two pharmaceutical preparations on HeLa cells.
  • the cytotoxic effect on the human T cell lymphoma cell line HeLa is depicted for two different pharmaceutical preparations comprising biological active serum of the invention various concentrations of the.
  • the viability of the HeLa cells is depicted on the y-axis in percentages, while the amount of the biological active serum is indicated on the x-axis in mg/ml
  • Fig. 5 Cytotoxic effect of two pharmaceutical preparations on MCF-7 cells.
  • the cytotoxic effect on the human T cell lymphoma cell line MCF-7 is depicted for two different pharmaceutical preparations comprising biological active serum of the invention various concentrations of the.
  • the viability of the MCF-7 cells is depicted on the y-axis in percentages, while the amount of the biological active serum is indicated on the x-axis in mg/ml
  • Fig. 6 Cytotoxic effect of two pharmaceutical preparations on IMR-32 cells.
  • the cytotoxic effect on the human T cell lymphoma cell line IMR-32 is depicted for two different pharmaceutical preparations comprising biological active serum of the invention various concentrations of the.
  • the viability of the IMR-32 cells is depicted on the y-axis in percentages, while the amount of the biological active serum is indicated on the x-axis in mg/ml
  • Fig. 7 Cytotoxic effect of two pharmaceutical preparations on HT1080 cells.
  • the cytotoxic effect on the human T cell lymphoma cell line HT1080 is depicted for two different pharmaceutical preparations comprising biological active serum of the invention various concentrations of the.
  • the viability of the HT1080 cells is depicted on the y-axis in percentages, while the amount of the biological active serum is indicated on the x-axis in mg/ml
  • Fig. 8 Cytotoxic effect of two pharmaceutical preparations on HepG2 cells.
  • the cytotoxic effect on the human T cell lymphoma cell line HepG2 is depicted for two different pharmaceutical preparations comprising biological active serum of the invention various concentrations of the.
  • the viability of the HepG2 cells is depicted on the y-axis in percentages, while the amount of the biological active serum is indicated on the x-axis in mg/ml
  • Fig. 9 "Bell" curve of the proliferative activity of cells treated with mitogens
  • the curve of the lymphocytes is assayed on the basis of the amount of DNA biosynthesis in relation to the concentration of the mitogen proliferative activity of.
  • the DNA biosynthesis activity is measured by the amount of incorporated radioactivity and the acid insoluble counts per minute.
  • Fig. 10 Mitogenic activity of the pharmacological composition.
  • Fig. 11 Effect of pharmacological composition on MCF-7 cells.
  • the flasks with the lyophilized serum were treated on a RZ-100-M apparatus with 20-30 kGy, preferably at around 25 kGy using 60 CO as a gamma radiation source.
  • the treated serum was stored at a temperature of between 4-8°C for later use.
  • the below experiments were carried out using male Wistar rats with an average body mass of 280-300 g.
  • the animals were randomly assigned to 4 groups of 10 rats each.
  • the first group was administered with 1.0 ml of a physiological salt solution.
  • the second group was administered a dose of 100 ⁇ 5.0 mg/kg body weight of the blood serum of the present invention within 1.0 ml solution.
  • 30 minutes after the injection the rats were decapitated.
  • 1 ml of a physiological salt solution was administered to the rats of the third group and the rats of the fourth group received biological active serum (100 ⁇ 5 mg/kg body weight) in an amount of 1.0 ml solution.
  • 30 minutes after the injection the animals, which had a weight attached to their tail (10% of the body weight of the rat), were placed in a basin with water (25°C). After the first signs of agony the animals were removed from the water and decapitated.
  • the brain, heart, liver (all being organs, which are exposed to an extensive energetic strain in processes of extreme adaptation) as well as the skeletal muscles (as mainly effected organ) were taken as samples for further analysis.
  • the tissue samples of each rat were weight, cooled with an isotonic NaCl solution and rapidly frozen in liquid nitrogen. The complete time which elapsed between the application "stress" and the final processing of the samples was in the range of between 5 to 6 minutes maximally.
  • the amount of adenosine triphosphoric acid, adenosine diphosphoric acid and adenosine mo- nophosphoric acid in the skeletal muscles of the rats was determined. Nucleotides were separated by means of ionexchange chromatography on columns employing Anionit Dowex 1. The determination of the amount of adenosine triphosphoric acid, adenosine diphosphoric acid and adenosine monophosphoric acid was carried out spectophotometrically at a 256 nm range (spectrophotometer Hitachi-557). The energy potential was determined according to the following formula
  • the determination of the amount of cyclic adenosine monophosphoric acid in the brain, heart and liver was carried out using known radioimmunology analysis with a detection apparatus of Amersham (Great Britain).
  • Table 1 Amount of adenosine triphosphoric acid, adenosine diphosphoric acid and adenosine monophosphoric acid and the energy potential within the tissue of the skeletal muscles with and without the administration of the biological active serum
  • the amounts of adenosine phosphoric acid determined during the active period and 30 minutes after mild stress because of the injection confirm that under the influence of serum a mixing of the amount of adenosine triphosphoric acid and adenosine diphosphoric acid occurs at the upper norm values in the muscle tissue, while at the same time the amount of adenosine monosphoric acid decreases.
  • the serum of the present invention treated with electroshock and ⁇ -radiation increases the energy potential in skeletal muscles of rats, increases the amount of cyclic adenosine monophosphoric acid in brain tissue both during rest phases as well as under extreme physical stress and facilitates the increase of cyclic adenosine monophosphoric acid in heart and liver tissue after physical stress of rats.
  • Example 2 Long-term memory.
  • the biological active serum was injected into the peritoneal cavity 30 minutes prior to the onset of the experiment in a dose of 100 mg/kg body weight. 1 ml of a physiological salt solution was administered to the control animals. On the next day the situation reflex behaviour was tested and afterwards the rats were given a 40 day pause.
  • rats - 60 control rats and 60 rats receiving biological active serum - were "educated”.
  • the rats were previously assigned to three groups as another above, i.e. active, medium activity and passive (20 animals within each group) furthermore they were subdivided for this analysis in "fast” and “slow” divers.
  • zytisin a H-cholino-ceptive antagonist of the brain
  • Biologically active serum was administered in a dose of 100 mg/kg body weight to the animals of the second group (ten rats) and 30 minutes later a zytisin solution was administered with a dose of 1 mg/kg body weight.
  • the third animal group (control — ten rats) received a 1 ml injection of a physiological salt solution.
  • the complex behaviour of the animals was tested according to above-indicated method 48 hours after the injection of the indicated substances.
  • the biotests showed 48 hours after the application of zytisin a marked slowing of the "escape reaction" from the closed room namely a 3-fold slowing in comparison to control animals.
  • brain H-cholino receptors play a role in the restoration of the "escape reaction" in connection with the modelling of a complex behaviour of animals and that the biological active serum prevents the establishment of attenuated learning.
  • the biologically active blood serum therefore, stimulates the formation of long-term memory and exhibits this effect in particular in animals with a slowed down escape reaction from a closed room and out of water. Furthermore, it appears that the H-cholino-ceptive brain mechanisms play an important role in the maintenance of long-term memory and that the effects of substances negatively effecting H-cholino-ceptive brain mechanisms can be antagonized by the biologically active serum of the present invention.
  • Example 3 Epilepsy
  • camphor at a toxic dosage leads to hyperactivation of the motoric area of the central nervous system which in turn causes the development of tonic cramps. Because of this camphor is used besides corazole in animal models for for the generation of cramps. Dependent on the doses of the administered camphor it is possible to cause all aspects of a small and large epileptic seizure including grand mal seizures.
  • the seizures were observed and assessed by experts. The following parameters were assessed. The latency time of the reaction, the type of cramp reaction (tonic-klonic, large and small seizures), the length of the epileptic seizure and the time intervals between them, the loss of normal mobility and the final result (death of the animal or the recovery from the pathological condition). The results are summarized in Table 6.
  • the biological active serum at a dosage of 100 mg/kg was administered in the latency period of epileptic seizures caused by injection of camphor decreased all signs of the seizure activity: the latency time of the seizure activity was increased, the klonic cramps are less severe and without loss of normal mobility and additionally the serum treated with electroshock prevented animals in the model of severe epilepsies from death (all animals survived the administration of 0.5 ml of a 20% camphor solution).
  • Example 4 Effect of the blood serum of the present invention on human cell proliferation.
  • the blood serum of the present invention is a lyophilized chicken blood treated with electroshock of grade II to III and ⁇ -radiation.
  • the biological active serum was used in two formula- tions types: (i) serum resuspended in water at a concentration of 100 mg/ml (trial fraction 1) and (ii) the supernatant of the suspension of 100 mg/ml biological active serum in water after three minute of centrifugation of the suspension at 10.000 x g (trial fraction 2).
  • the cells of the Jurkat and Raji cell line as well as lymphocytes of the human peripheral blood were cultured in plastic tissue culture plates (Nunc or Falcon) in 1640-RPMI medium (Sigma) containing 10% fetal calf serum (FCS, Gibco), 100 units/ml penicilin and 100 ⁇ g/ml streptomycin at a temperature of 37°C, at 5% CO content and 95% humidity.
  • the cell lines Bro, HeLa, MCF-7, Mg63, HT1080, IMR-32 as well as HepG2 were cultivated as above, however, using DMEM medium (Sigma) instead of 1640-RPMI medium.
  • Mononuclear leucocytes were isolated according to the method described by Boyum A. (Isolation of mononuclear cells and granulocytes from human blood, 1968, Cand. J. Lab. Clin. Invest, 120 (97): 9-18).
  • lymphocytes The mononuclear leucocytes (ML) which concentrated at the interface between plasma and separation medium were carefully sucked off with a pipette and collected in a centrifuge tube. Thereafter the cells were washed twice with PBS by spinning the cells at 250 x g for 10 minutes and eventually suspending them in culture medium. This fraction comprised between 10- 30% monocytes and 80-90% lymphocytes which are in the following termed "lymphocytes". One part of the lymphocytes was used for studying the mitogenic activity of the biological active serum of the present invention and another to study proliferation. For this purpose phy- tohemaglutinine was added at a concentration of 20 mg/ml to cell suspension.
  • lymphocytes were incubated in 96 well plates with 200 ⁇ l medium comprising between 200 to 800 x 10 3 cells, To each well different concentration of the biological active serum of the present invention was added. 3 H- thymidin (1 ⁇ Ci/Well, 40 mCi/mmol/1) was added two hours prior to termination of the incubation.
  • 3 H- thymidin (1 ⁇ Ci/Well, 40 mCi/mmol/1) was added two hours prior to termination of the incubation.
  • the cells were harvested on filters with an automatic device for cell harvesting.
  • the acid soluble products were washed away with 5% trifluor acetic acid (H 2 O) and the radioactivity of the substances retained was measured with a scintimeter. The amount of DNA biosynthesis was measured in counts/min.
  • the MTT-test was carried out as described by Mo smarm T. (Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays (1983) J Immunol. Meth. 65: 55-63).
  • test cells were collected in the log. phase (adherent cells were collected when they filled about half of the tissue culture plate). They were placed into growth media in a Gorjaew-chamber, counted and subsequently resuspended in medium at a concentration of 50-100 x 10 /ml. The cell solutions were placed into the 96 well plates after the addition of the various concentrations of the trial fractions in a total amount of 100 ⁇ l. For counting of viable cells 50 ⁇ l of a solution of 3-4,5-dimethyl thiazole-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) in culture medium was added to each of the different 96 well plates after termination of incubation.
  • MTT 3-4,5-dimethyl thiazole-2-yl-2,5 diphenyl tetrazolium bromide
  • MTT solution 1 ml MTT stock solution was mixed with 4 ml culture medium.
  • the preparation of the MTT stock solution was carried out by the dissolving MTT in PBS (PBS comprises 0.01 mmol/1 sodium phosphate buffer, pH 7.4, with 0.15 mmol/1 NaCl) at a concentration of 5 mg MTT/ml followed by filtration through a filter with a pore size of 0.45 ⁇ m.
  • PBS PBS comprises 0.01 mmol/1 sodium phosphate buffer, pH 7.4, with 0.15 mmol/1 NaCl
  • the stock solution was stored at +4°C for up to one month.
  • the tissue culture plates were further incubated for 4 hours in the incubator under identical conditions.
  • a high concentration of the biological active serum of the present invention (2.5-20 mg/ml) had an inhibiting effect on all cell lines, however, the reaction of cells from different tissue types to trial fraction 1 and 2, respectively, is different.
  • the IC 50 dose (concentration of the substance which leads to 50% inhibition of the cells) is substantially different among the cell lines ranging from 2.2 (Jurkat) to > 20 mg/ml (Mg63) for trial substance 1 and from 3.6 (lymphocytes of the peripheral blood) to > 20 mg/ml (Mg63 and HeLa) for the soluble fraction of the blood serum of the present invention (trial fraction 2).
  • the agent had a stimulating effect on some cells.
  • a stimulating effect of the serum of the present invention was observed for Jurkat, Raji, Bro B-19, Mg63, HT1080 and HepG2 cells.
  • the stimulating effect was insignificant, (10, 20, 40%) but present for both forms of the test substance.
  • No stimulation was observed for cells of the cell line HeLa, MCF-7, JMR-32 and for lymphocytes of the peripheral blood.
  • the stimulating effect of the test substance in its first form was observable at a much lower concentration as with the soluble fraction (trial fraction 2).
  • a test substance have a mitogenic activity, i.e. does it have an ability to stimulate lymphocyte proliferation (in this case the increase of the amount of the substance usually leads to an increase of the DNA biosynthesis of the lymphocytes which can be evaluated by the incorporation of 3 H-thymidin)? 2. Does a test substance have a toxic effect (this question is usually determined by the inhibition of lymphocyte proliferation as assayed by the amount of incorporated H-thymidin or using vital dyes of the MTT-type on lymphocytes which were previously stimulated with mitogens)?
  • non activated lymphocytes do not proliferate in culture and that the amount of proliferation only increases with increasing amounts of mitogens added to the culture medium. This is reflected in the increase in radioactivly labelled DNA.
  • the effect of mitogens on lymphocytes in relation to the administered doses cam be depicted by a so called "bell curve" (see Fig. 9).
  • the first section of the bell curve reflects the range of the mitogen concentration wherein an increase of mitogen leads to an increase of proliferation (as measured by DNA biosynthesis) and wherein, therefore, direct relation between mitogen concentration and proliferation exists.
  • the second section of the curve (2) shows a saturation effect wherein a further increase of the mitogen concentration does not lead to a further increase of proliferation, i.e. the mitogen has already elicited its maximal effect. A cytotoxic activity is not yet observed.
  • Section (3) shows the range of the mitogen concentration wherein the mitogen exerts an increasingly cytotoxic effect on the lymphocytes.
  • the subject matter of this experiment was the determination of the effect of the trial fractions on non-activated lymphocytes of human peripheral blood. To this end the correlation of the proliferative activity (i.e. the effect) with the administered doses was determined. 2. The subject matter of this experiment was the determination of the effect of the trial substance on the amount of DNA synthesis of lymphocytes activated by phy- tohemaglotinin (FHA 20 ⁇ g/ml) in a second phase.
  • FHA 20 ⁇ g/ml phy- tohemaglotinin
  • the cytotoxic activity of the serum of the present invention was also investigated using cells of the human milk gland carcinoma cell line MCF-7 that were spread into a monocellular layer. Because of contact inhibition no proliferation was observed for the cells. In such a model the toxic effect of a substance can be determined, i.e. it is possible to differentiate the cytotoxic effect from the cytostatic effect. When using this model it was determined that the serum of the present invention had a cytotoxic effect in a concentration of 2.5 mg/ml independent of the fact of whether the insoluble fraction of the serum of the present invention was removed or not, i.e. independent of whether trial fraction 1 or 2 was used.

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