EP4072569A1 - Method of preparation of the transfer factor medicine, transfer factor medicine, method of testing its biological activity, pharmaceutical form and its use - Google Patents

Method of preparation of the transfer factor medicine, transfer factor medicine, method of testing its biological activity, pharmaceutical form and its use

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Publication number
EP4072569A1
EP4072569A1 EP21721385.9A EP21721385A EP4072569A1 EP 4072569 A1 EP4072569 A1 EP 4072569A1 EP 21721385 A EP21721385 A EP 21721385A EP 4072569 A1 EP4072569 A1 EP 4072569A1
Authority
EP
European Patent Office
Prior art keywords
transfer factor
preparation
medicine
tablet
temperature
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.)
Pending
Application number
EP21721385.9A
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German (de)
French (fr)
Inventor
Juraj Vronka
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AUMED IMMUNOLOGY A.S.
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Aumed AS
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Filing date
Publication date
Application filed by Aumed AS filed Critical Aumed AS
Publication of EP4072569A1 publication Critical patent/EP4072569A1/en
Pending legal-status Critical Current

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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
    • 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
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/02—Reverse osmosis; Hyperfiltration ; Nanofiltration
    • B01D61/027—Nanofiltration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/145—Ultrafiltration
    • B01D61/146—Ultrafiltration comprising multiple ultrafiltration steps
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/58—Multistep processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/06—Organic material
    • B01D71/56—Polyamides, e.g. polyester-amides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/10—Temperature control
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/145—Ultrafiltration

Definitions

  • the invention concerns major modifications in the technology of the transfer factor preparation, the method of testing its biological activity and the technology of preparation of a new pharmaceutical form of the transfer factor, i.e., a lyophilised tablet with a defined composition.
  • the transfer factor may be obtained with use of various methods; dialysis or ultrafiltration of a mixture of (human or animal) peripheral blood leukocytes belong among the most frequently described methods.
  • the state of the art describes the following technological steps: a) Preparation of leukocyte concentrate; b) Dialysis and/or ultrafiltration; c) Concentration with use of lyophilisation; d) Preparation of the raw medicine solution; e) Inter-operational testing; f) Homogenization; g) Pre-filtration; h) tlitrafiitration; i) Sterilising filtration; d) Tnerma1 inact.1.vation; k ⁇ Filling; i ⁇ Lyophi1isat.ion.
  • the currently produced transfer factors are prepared either as liquid products or in the form of powder obtained via lyophiiisation or another form of drying. Their security is ensured by means of several methods; most, frequently with thermal treatment (pasteurization upon a defined temperature and duration) or through various filtration degrees (e.g., sterilizing filtration) and the input raw material tests concerning absence of any pathological micro-organisms.
  • thermal treatment pasteurization upon a defined temperature and duration
  • filtration degrees e.g., sterilizing filtration
  • the procedures which have been described until now result in a liquid or powder form of the transfer factor that may be applied either by means of an injection (subcutaneously or intravenously) or as products that are orally administered (e.g., solutions) in the digestive tract.
  • the invention deals with ail the above disadvantages concerning modifications of the transfer factor production technology and preparation technologies by means of a lyophilised tablet with a defined compos11ion.
  • the means of production comprises the following technological steps: a) Preparation of leukocyte concentrate; b) Dialysis or ultrafiltration; c) Removal of salts and concentration with use of nanoffitration; d) Thermal inactivation; e) Tablet formulation; f) Preparation of a lyophilized tablet.
  • the active substance dialysate or ultra-filtrate obtained using the known method: contains a great amount of ballast in the form of salts.
  • These salts are either of physiological origin ⁇ natural blood components) or artificial origin (anticoagulants added to blood during sampling thereof. Simultaneously, this material contains an undesirable amount of the water content.
  • the invention is mainly based on including the following new steps when preparing the transfer factor: removal of excessive salts and excessive water content, by using the technology of nancfiltration with use of a membrane with a suitable porosity; preparation of a lyophilised tablet comprising the transfer factor as an active substance; application of the active substance orally in the form, of a lyophilised tablet that is not swallowed, but works through the bucoal mueosa; test of biological activity of the preparation based on monitoring the proliferation effect on the tissue culture.
  • Leukocyte concentrates from the individual donors are transported and stored in a frozen form. After thawing at the temperature up to +37 °C, the material obtained from the individual donors is merged to a greater volume. The work must be performed in the production premises with a defined class of environmental cleanness. Subsequently, all membrane components in the leukocyte homogenate are disrupted with use of repeated freezing and thawing or by disturbing the cells with use of ultrasound or a combination of both procedures. al) Preparation of homogenate with use of the freezing - thawing method:
  • Freezing is carried out in denatured ethyl alcohol at the minimum temperature of -35 °C in a rotary freezing equipment. Thawing is conducted in a water bath at the temperature of maximally +37 °C. The entire cycle of freezing and thawing is repeated 6 times. After conclusion of the 6 th cycle, the homogenate maintained in original bottles is placed in a chest freezer at the minimum temperature of -17 °C until further processing. a2) Preparation of homogenate with use of ultra-sound:
  • the leukocyte homogenate is prepared by disintegrating ceils with ultrasound at 20 kHz for 10 minutes at the temperature of ⁇ 2 °C to +8 °C. After this period of disintegration, the obtained homogenate is placed in a chest freezer at the minimum temperature of -17 °C until further processing.
  • a combined leukocyte homogenization procedure is used as an alternative production procedure.
  • the freezing and thawing cycle is repeated 5 times with subsequent disintegration with use of ultrasound at 20 kHz for 3 minutes at the temperature of +2 °C to +8 °C.
  • the obtained homogenate is placed in a chest freezer at the minimum temperature of -17 °C until further processing.
  • the temperatures specified in this production stage should, not fluctuate by more than 30 °C to both sides of the temperature scale and the time should be a matter of minutes.
  • the leukocyte homogenate processing is carried out within a closed system in a dialysis column.
  • the leukocyte homogenate was frozen before further processing ; it must be thawed in a water bath with the temperature of +37 °C.
  • the temperature may be even lower (up to +4 °C). It is beneficial if the temperature does not fluctuate from the defined temperature by more than +33 °C.
  • the homogenate dialysis is conducted as dialysis in a closed system.
  • Dialysis is conducted in a dialysis unit made of an artificial kidney containing a membrane, which can detain particles with the relative molecular weight exceeding 10,000. Dialysis is carried out at reduced temperature of +2 °C to +8 °C. This temperature should not differ by more than 20 °C.
  • the volume of the leukocyte concentrate homogenate processed within a single cycle ranges with an advantage from 1.1 to 2.4 L. The volume should not be larger than tens of litres and smaller than 0.2 litre.
  • the amount of water for the injection needed for dialysis of low- molecular substances in the temperature range of +2 °C to +8 °C ranges from S to 13 L. The amount of water may differ when using different temperatures, but it.should not exceed units of litres.
  • the disintegrated leukocyte concentrate runs with use of a pump through the dialysis unit in a circuit with the flov; rate of 1.2 to 2.0 L/h and water for injections as a dialysis medium through the same dialysis unit in another circuit with the flow rate of 0.4 to 0.5 L/h.
  • the dialysis duration is 24 hours +1 hours. In the case that different circulation speeds are used in the process, the dialysis duration must also be adjusted accordingly. Dialysis is terminated when the dialysate absorption value drops below 0.6 at 260 nm. This value is only recommended and may differ.
  • the obtained dialysate is purged of undesired salts and simultaneously concentrated with use of nanofiltration to the required volume.
  • the obtained dialysate that contains too much salts and water to allow further processing must be desalinated and concentrated in a way that It is directly usable to construct a lyophilised tablet, i.e., without any need of further diluting or concentrating or enriching or any other supplementary treatment of the obtained solution.
  • the concentration methods may have various forms. The used method deals with two technological requirements: a gentle procedure and purifying of the product from salts and undesired excessive water volume. The product is protected; against thermal stress by the entire process cooling.
  • the temperature of nanofiltration should not exceed +30 °C and it is beneficial when the temperature ranges from 14 to 25 °C and the processed volume of fluid is not larger than 50 litres and smaller than 5 litres.
  • the ideal volume ranges around 15 litres.
  • the working pressure at the filter is 20 bar +5 bar. It is always necessary to avoid static counter pressure of the permeate.
  • Filtration is carried out using the system of maximum permeate separation from the filtered sample. The remaining sample is directly diluted with demineralised water in the filtration equipment according to the volume of the collected permeate. Filtration as such proceeds after mixing the diluted fluid. This procedure is repeated 4 times.
  • a thin-film polyamide composite is preferably used for nanofiitration as a membrane with the porosity allowing for permeability of up to 1,000 Da.
  • the subject of patent protection in this production stage comprises removal of excessive salts and water with use of a described nanofiltration method. d) Thermal inactivation
  • the tablet has the following defined composition:
  • Formulation of one tablet represents the following components: Dextranum 40 0.05 g
  • Macrogolum 300 0.005 g (Polyethylenglycoluxn 300)
  • the active substance solution (of the transfer factor) is prepared in advance in a way that one dose is dissolved in 0.25 ml of water in the quality of "water for injections".
  • dextran 40 is added to this solution and dissolved by stirring.
  • To speed up dextran dissolution we may use an ultrasound bath for 1 minute or maintain the mixture slightly heated in a water bath (40 °C).
  • the lyophilizer Before filling the preparation in the blisters, the lyophilizer is set to the plate freezing mode at the defined temperature of - 50 °C. The plates freeze to the required temperature in approximately 2 hours.
  • blisters with the dispensed material are set up on the frozen plates of the lyophilizer. Subsequently, the lyophiiisation equipment is switched to the primary drying (sublimation) mode and the temperature is set to -55 °C and vacuum to 0.3 xnbar to freeze the condenser. After reaching the set condenser temperature of -55 °C the lyophiiisation process is initiated.
  • the lyophiiisation drying process may be set in an automated or semi-automated regime.
  • the semi- automated regime allows for adjusting and switching the individual stages (freezing, primary drying, secondary drying) through manual control if needed. After 2 hours, the lyophilisation process shifts in the secondary drying mode (desorption).
  • the plate temperature is set to -30 °C, vacuum to 0.005 mbar.
  • the temperature of the plates is gradually decreased to -20 °C, -10 °C, 0 °C f +10 c C and +25 °C.
  • the entire lyophilisation process is completed within 24 hours from its initiation at the latest. The real time of lyophilisation completion is based on reaching the described temperature values of the plates and vacuum values.
  • the lyophilisation equipment is aerated and blisters with the dried product are removed and sealed with a cover foil using the blistering equipment.
  • the subject of the invention in this production stage comprises preparation of a lyophiiised tablet.
  • the transfer factor medicine from peripheral blood leukocytes comprising protein components and nucleic acid components the share of which in the medicine is represented by the absorbance index, i.e., a ratio obtained through specfcrophotoxnetric absorbance measuring in the UV zone at 260 ran and 230 ran.
  • This index must not be smaller than 1.7 with an advantage in the range from 1.7 to 2,1.
  • the medicinal product is purified of ballast salts using the nanofiltration method.
  • the molecular weight the individual substances of the medicinal product must not exceed 10,000 Daltons,
  • a lyophiiised tablet comprises one medicinal dose of the product, which represents the amount of biologically active ingredients obtained from approximately 200 million peripheral leukocytes that are capable of normalising the compromised cellular immune system of the recipient.
  • the lyophilised tablet is manufactured through 1yophi1isation of a liquid matrix.
  • the liquid matrix features the osmolality value ranging from 150 to 400 mmol/kg. Activity of biologically active substances may be proven in the lyophilised tablet.
  • the biological activity test is based on monitoring the transfer factor impact on the model cellular line viability after it was first influenced by an immunosuppressive substance, azathioprine.
  • Immortalised T ⁇ lymphocyte cellular line Jurkat CCL--81 is used as a model cellular line.
  • Cellular viability is measured on a flow cytometer with use of a commercial MTT test.
  • MTT measures the metabolic activity of ceils and thus is suitable to determine the cellular viability.
  • the test is based on tetrazoiium MTT salt (3- (4,5 ⁇ dimethylthiazol-2-yl ⁇ -2.5-diphenyi tetrazoiium bromide) reduction to insoluble formazan.
  • the amount of the created formazan is directly proportional to the number of living cells.
  • the Jurkat cellular line is cultivated in a RPMI media enriched with 10% of foetal bovine serum, 2 mM of glutamine and a mixture of penicillin (100 I/ml) and streptomycin (100 ug/ml).
  • penicillin 100 I/ml
  • streptomycin 100 ug/ml
  • azathioprine with the concentration of 5 pg . /mi after the model cellular line suppression.
  • Transfer factor diluted 80 to ISO times is used for the biological activity test. Incubation of the cells takes 72 hours. After this incubation period, 20 m ⁇ of stock MMT solution (5 mg/ml) is added to each hole in the plate and the plate is further incubated for 3 h at 37 °C. After that, supernatant is removed and the created formazan crystals are dissolved in 150 pi of dimethyl sulfoxide. The resulting optical density of solutions is measured at 570 nm using a plate spectrophotometer and evaluated as viability of the ceils.
  • Control group - individual not influenced Jurkat cells
  • Jurkat cells suppressed by azathioprine and subsequently influenced by the transfer factor.
  • the added transfer factor itself does not affect viability of the ceils compared to the control group.
  • the added azathioprine reduces viability of the cells under 50% of the control group value.
  • the transfer factor in combination with azathioprine returns the cellular viability suppressed by azathioprine back to normal by approximately 40%.
  • the subject of the invention in this • production stage comprises an in vitro test of the transfer factor biological activity.
  • the invention is usable for commercial preparation of any transfer factor, both specific or non-specific, prepared from homogenate of the peripheral blood leukocytes.
  • the invention is usable upon manufacture of a human product, as well as in the case of any modifications designated for veterinary use.
  • the main therapeutic effect is facilitated by the medicine penetration exclusively through the oral mucosa.
  • Method of the transfer factor medicine preparation a) Preparation of leukocyte concentrate; b; Dialysis or ultrafiltration; c) Removal of salts and concentration with use of nanofiltration; d ⁇ Thermal inactivation; e ⁇ Tablet formulation; f ⁇ Preparation of a iyophiiized tablet. a) Preparation of leukocyte concentrate
  • Leukocyte concentrates are stored in a frozen condition. Subsequently, at the maximal temperature of +37 °C, the leukocyte concentrate is merged in the working volume. The work must be performed in the production premises with environmental cleanness class "C". Subsequently, all membrane components in the leukocyte homogenate are disrupted with use of repeated freezing and thawing or by disturbing the cells with use of ultrasound or a combination of both procedures, al) Preparation of homogenate with use of the freezing - thawing method:
  • Freezing is carried out in denatured ethyl alcohol at the minimum temperature of -35 °C in a rotary freezing equipment. Thawing is conducted in a water bath at the temperature of maximally +37 °C. The entire cycle of freezing and thawing is repeated 6 times. After conclusion of the 6 tn cycle, the homogenate maintained in original bottles is placed in a chest freezer at the minimum temperature of -17 °C until further processing. a2) Preparation of homogenate with use of ultra-sound:
  • the leukocyte homogenate is prepared by disintegrating cells with ultrasound at 20 kHz for 10 minutes at the temperature of 2 °C to 8 °C. After this period of disintegration, the obtained homogenate is placed in a chest freezer at the minimum temperature of - 17 °C until further processing.
  • the freezing and thawing cycle is repeated 5 times with subsequent disintegration with use of ultrasound at 20 kHz for 3 minutes at the temperature of +2 °C to -tS c C. After this disintegration, the obtained homogenate is placed in a chest freezer at the minimum temperature of -17 °C until further processing.
  • the leukocyte homogenate processing is carried out within a closed system in a dialysis column.
  • the leukocyte homogenate was frozen before further processing, it must be thawed In a water bath with the temperature of + 37 °C.
  • the temperature may be even lower (up to +4 °C ⁇ . It is beneficial if the temperature does not fluctuate from the defined temperature by more than 20 °C.
  • Dialysis is conducted in a dialysis unit made of an artificial kidney containing a membrane, which can detain particles with the relative molecular weight exceeding 10,000. Dialysis is carried out at reduced temperature of +2 “C to +8 a C. This temperature should not differ by more than 20 °C.
  • the volume of the leukocyte concentrate homogenate processed within a single cycle ranges with an advantage from 1.4 to 2.4 L, The volume should not be larger than tens of litres and smaller than 0.2 litre.
  • the amount of water for the injection needed for dialysis of low-molecular substances in the temperature range of ⁇ 2 °C to +8 °C ranges from 8 to 13 L, The amount of water may differ when using different temperatures, but it should not exceed units of litres.
  • the disintegrated leukocyte concentrate runs with use of a pump through the dialysis unit in a circuit with the flow .rate of 1.2 to 2.0 L/h and water for injections as a dialysis medium through the same dialysis unit in another circuit with the flow rate of 0.4 to 0.5 L/h.
  • the dialysis duration is 24 hours +4 hours. In the case that different circulation speeds are used in the process, the dialysis duration must also be adjusted accordingly. Dialysis is terminated when the dialysate absorption value drops below 0.6 at 260 nm. This value is only recommended and may differ.
  • the obtained dialysate is purged of undesired salts and simultaneously concentrated with use of nanofiltration to the required volume.
  • the aim is that the dialysate comprising salts and water in excessive volumes preventing its further processing is desalinated and concentrated in a way that it will be directly usable to construct a lyophilised tablet.
  • the product is protected against thermal stress by the entire process cooling.
  • the temperature of nanofiltration should not.exceed +30 °C and it is beneficial when the temperature ranges from 14 to 25 °C and the processed volume of fluid is not larger than 50 litres and smaller than 5 litres.
  • the ideal volume ranges around 15 litres.
  • the working pressure at the filter is 20 bar +5 bar. It is always necessary to avoid static counter pressure of the permeate.
  • Filtration is carried out using the system of maximum permeate separation from the filtered sample. The remaining sample is directly diluted with demineralised water in the filtration equipment according to the volume of the collected permeate. Filtration as such proceeds after mixing the diluted fluid. This procedure is repeated 4 times.
  • a thin-film polyamide composite is preferably used for nanofiltration as a membrane with the porosity allowing for permeabi1ity of up to 1,000 Da.
  • Thermal activation this technological step is also carried out according to the state of the art.
  • Macrogoium 300 0.6 g
  • the transfer factor solution (30 doses) in 30 mi of purified water (or water for injection) is prepared in advance.
  • the matrix is prepared as follows: 0.18 g of carrageenan iota is dissolved at increased temperature in a water bath (60 °C) in 20 g of purified water (corresponds to the total sample amount of 60 g for preparation of 120 tables). 6.0 g of dextran 40 is added to this solution and dissolved by stirring. To speed up dextran dissolution we may use an ultra-sound bath for 1 minute or maintain the mixture slightly heated in a water bath (40 °C) .
  • the prepared liquid tablet matrix comprising the transfer factor as an active substance shall be &l--spe*»e-ed filled with use of a dosing pipette in the individual blister pockets in 0.5 mi doses.
  • the lyophiiizer Before filling the TFI product in the blisters, the lyophiiizer shall be set in the freezing mode - freezing the plates to the defined temperature of -50 °C. Freezing of the plates will take approximately 2 hours.
  • the lyophiliter is switched to the primary drying mode ⁇ sublimation ⁇ , the values defined for the condenser freezing are set at -55 °C and the vacuum value at 0.3 mfo&r.
  • the iyophilisation process is initiated.
  • the iyophilisation process may be set in an automated or semi- automated regime. If in the automated regime, the set parameters cannot be modified in the course of the iyophilisation process duration.
  • the semi ⁇ automated regime allows for adjusting of and switching between the individual stages ⁇ freezing, primary drying, secondary drying ⁇ through manual control.
  • the transfer factor medicine from peripheral blood leukocytes comprising protein components and nucleic acid components the share of which in the medicine is represented by the absorbance index, i.e., a ratio obtained through spectrophotometric absorbance measuring in the O ' V zone at 260 nm and 2S0 nm.
  • This index must not be smaller than 1,7 with an advantage in the range from 1.7 to 2.1
  • the medicinal product is purified of ballast salts using the nanofiltration method.
  • the molecular weight the individual substances of the medicinal product must not exceed 10,000 Daltons.
  • Transfer factor diluted 80 to 160 times is used for the biological activity test. Incubation of the ceils takes 72 hours. After this incubation period, 20 m ⁇ of stock MMT solution (5 mg/mi; is added to each hole in the plate and the plate is further incubated for 3 h at 37 °C. After that, supernatant is removed and the created formazan crystals are dissolved in 150 m ⁇ of dimethyl sulfoxide. The resulting optical density of solutions is measured at 570 nm using a plate spectrophotometer and evaluated as viability of the ceils. Optical density of the solution was measured at 570 nm using a plate spectrophotometer (Beckman Coulter, USA).
  • Jurkat cells influenced by the transfer factor; Jurkat ceils suppressed by azathioprine and subsequently influenced by the transfer factor.
  • the added transfer factor itself does not affect viability of the cells compared to the control group.
  • the added azathioprine reduces viability of the cells under 50% of the control group value.
  • the transfer factor in combination with azathioprine returns the cellular viability suppressed by azathioprine back to normal by approximately 40%
  • a lyophiiised tablet comprises one medicinal dose of the product, which represents the amount, of biologically active ingredients obtained from approximately 200 million peripheral leukocytes that are capable of normalising the compromised cellular immune system of the recipient.
  • the lyophiiised tablet is manufactured through iyophiiisation of a liquid matrix.
  • the liquid matrix features the osmolality value ranging from 150 to 400 mmol/kg. Activity of biologically active substances may be proven in the lyophiiised tablet.
  • the pharmaceutical form is prepared as follows:
  • the lyophilizer Before filling the preparation in the blisters, the lyophilizer is set to the plate freezing mode at the defined temperature of -50 °C. The plates freeze to the required temperature in approximately 2 hours.
  • blisters with the dispensed material are set up on the frozen plates of the lyophilizer. Subsequently, the lyophilisation equipment is switched to the primary drying (sublimation) mode and the temperature is set to -55 °C and vacuum to 0.3 mbar to freeze the condenser. After reaching the set condenser temperature of -55 °C the lyophilisation process is initiated.
  • the iyophiiisation drying process may be set in an automated or semi-automated regime.
  • the semi- automated regime allows for adjusting and switching the individual stages (freezing, primary drying, secondary drying) through manual control if needed. After 2 hours, the lyophilisation process shifts in the secondary drying mode (desorption).
  • the plate temperature is set. to -30 °C, vacuum to 0.005 mbar.
  • the temperature of the plates is gradually decreased to -20 °C, -10 °C, 0 °C, +10 °C and +25 °C.
  • the entire lyophilisation process is completed within 24 hours from its initiation at the latest.
  • the real time of lyophilisation completion is based on reaching the described temperature values of the plates and vacuum values.
  • the iyophilisation equipment is aerated and blisters with the dried product are removed and sealed with a cover foil using the blistering equipment.
  • the subject of the invention in this production stage comprises preparation of a lyophilised tablet.
  • the transfer factor medicine from peripheral blood leukocytes comprising protein components and nucleic acid components the share of which in the medicine is represented by the absorbance index, i.e., a ratio obtained through spectrophotometric absorbance measuring in the UV zone at 260 nm and 280 nm.
  • This index must not be smaller than 1.7 with an advantage in the range from 1.7 to 2.1.
  • the medicinal product is purified of ballast salts using the nanofiitration method. The molecular weight the individual substances of the medicinal product must not exceed 10,000 Daltons.
  • transfer factor according to this invention Using of the transfer factor according to this invention as a human or veterinary preparation to cure pathological conditions related with cellular immunity disorders, i.e., chronic: infections, septic conditions, atopic eczema, recurring mycoses, psoriasis or immunodeficiencies related with oncologic treatment.
  • pathological conditions related with cellular immunity disorders i.e., chronic: infections, septic conditions, atopic eczema, recurring mycoses, psoriasis or immunodeficiencies related with oncologic treatment.
  • the invention is usable upon commercial preparation of any transfer factor, both specific or non-specific, prepared from the peripheral blood leukocytes.
  • the invention is also usable upon manufacture of a human product, as well as in the case of any medicinal products designated for veterinary use.
  • the invention is usable for commercial preparation of any transfer factor, both specific or non-specific, usable as a part of food supplements.
  • the preparation obtained through the preparation process according to this invention is usable to cure pathological conditions related with the cellular immunity disorders ⁇ chronic: infections, septic conditions, atopic eczema, recurring mycoses, psoriasis or immunodeficiencies related with oncologic treatment).
  • the preparation is also suitable for prophylactic use. Recommended dosing: a lyophilised tablet is inserted in the mouth where it sticks to the mucosa and speedily melts. The tablet is not swallowed.

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Abstract

Method of the transfer factor medicine preparation; transfer factor medicine; biological activity testing methodology; pharmaceutical form and its use where the Method of the medicine preparation from the peripheral blood leukocytes is based on the following individual steps, i.e. from: a) prepared leukocyte concentrate, b) dialysis and/or filtration, c) removal of salts and concentration with use of nanofiltration, d) thermal activation, e) tablet formulation and f) preparation of a lyophilised tablet, while the steps comprising removal of salts from the product and concentration of the product with use of nanofiltration are based on purifying the obtained dialysate of undesired salts and its concentration with use of nanofiltration to the required volume. A thin-film polyamide composite is used for concentration with use of nanofiltration as a membrane with the porosity allowing for permeability of up to 1,000 Da. The transfer factor active substance prepared as a Iyophilised tablet that is Iyophilised directly in the blister pocket where each pocket represents one dose of the preparation; the medicine is made of dialysate or ultrafiltrate of the peripheral blood leukocytes purified of ballast salts and comprising protein substances and nucleic acid substances whose ratio in the medicine, i.e., the absorption,index, is not under 1.7, where the molecular weight of none of the medicine substances is greater than 10,000 Daltons. The in vitro method of testing the biological activity of the transfer factor active substance is conducted through observing the transfer factor impact on the model cellular line viability after it was influenced by an immunosuppressive substance, azathioprine. Using of the transfer factor in the form of a Iyophilised tablet administered in the oral cavity where the Iyophilised tablet immediately adheres to the mucosa, which prevents its swallowing; it speedily melts on the buccal mucosa and it passes directly to the digestive system as a human or veterinary medicine or a food supplement or a prophylactic preparation.

Description

Method of preparation of the Transfer Factor Medicine, Transfer Factor Medicine, Method of testing its biological activity, Pharmaceutical Form and Its Use
Technical field
The invention concerns major modifications in the technology of the transfer factor preparation, the method of testing its biological activity and the technology of preparation of a new pharmaceutical form of the transfer factor, i.e., a lyophilised tablet with a defined composition.
Background art
Since 1950s and 1960s we have been aware of existence of a transfer factor that is capable of transferring immunological reactivity from the donor to the recipient. It concerns a mixture of biologically active low-molecular substances obtained by means of dialysing the leukocyte homogenate.
The transfer factor may be obtained with use of various methods; dialysis or ultrafiltration of a mixture of (human or animal) peripheral blood leukocytes belong among the most frequently described methods.
The state of the art is described for example in documents EP2Q37949 [Sevapharma A.S., CZ] and documents of the same patent family, e.g,, WO2007143957 a C22006377.
The state of the art describes the following technological steps: a) Preparation of leukocyte concentrate; b) Dialysis and/or ultrafiltration; c) Concentration with use of lyophilisation; d) Preparation of the raw medicine solution; e) Inter-operational testing; f) Homogenization; g) Pre-filtration; h) tlitrafiitration; i) Sterilising filtration; d) Tnerma1 inact.1.vation; k} Filling; i} Lyophi1isat.ion.
The currently produced transfer factors are prepared either as liquid products or in the form of powder obtained via lyophiiisation or another form of drying. Their security is ensured by means of several methods; most, frequently with thermal treatment (pasteurization upon a defined temperature and duration) or through various filtration degrees (e.g., sterilizing filtration) and the input raw material tests concerning absence of any pathological micro-organisms. The procedures which have been described until now result in a liquid or powder form of the transfer factor that may be applied either by means of an injection (subcutaneously or intravenously) or as products that are orally administered (e.g., solutions) in the digestive tract.
Biological activity of the currently manufactured transfer factors has been proven through in vivo testing on animal models or via in vitro monitoring of their impact on the cytokine and cellular activity. Products manufactured on the basis of transfer factors show the following disadvantages:
- 'when administered by means of an injection, they are very painful directly proportional to the applied volume;
- parenteral products must be manufactured as sterile products;
- biological effect of the transfer factors administered orally in the digestive tract is reduced due to the aggressive environment of the digestive organs.
The invention deals with ail the above disadvantages concerning modifications of the transfer factor production technology and preparation technologies by means of a lyophilised tablet with a defined compos11ion.
Summary of the invention
The means of production comprises the following technological steps: a) Preparation of leukocyte concentrate; b) Dialysis or ultrafiltration; c) Removal of salts and concentration with use of nanoffitration; d) Thermal inactivation; e) Tablet formulation; f) Preparation of a lyophilized tablet.
Three technological steps: preparation of the leukocyte concentrate {transport, storage, preparation of a homogenate), technology of the active substance preparation (dialysis or ultrafiltration) and thermal inactivation are conducted in line with the state of the art.
In addition to biologically active substances, the active substance (dialysate or ultra-filtrate obtained using the known method: contains a great amount of ballast in the form of salts. These salts are either of physiological origin {natural blood components) or artificial origin (anticoagulants added to blood during sampling thereof. Simultaneously, this material contains an undesirable amount of the water content.
The invention is mainly based on including the following new steps when preparing the transfer factor: removal of excessive salts and excessive water content, by using the technology of nancfiltration with use of a membrane with a suitable porosity; preparation of a lyophilised tablet comprising the transfer factor as an active substance; application of the active substance orally in the form, of a lyophilised tablet that is not swallowed, but works through the bucoal mueosa; test of biological activity of the preparation based on monitoring the proliferation effect on the tissue culture.
Description of individual steps a) Preparation of leukocyte homogenate;
Leukocyte concentrates from the individual donors are transported and stored in a frozen form. After thawing at the temperature up to +37 °C, the material obtained from the individual donors is merged to a greater volume. The work must be performed in the production premises with a defined class of environmental cleanness. Subsequently, all membrane components in the leukocyte homogenate are disrupted with use of repeated freezing and thawing or by disturbing the cells with use of ultrasound or a combination of both procedures. al) Preparation of homogenate with use of the freezing - thawing method:
Freezing is carried out in denatured ethyl alcohol at the minimum temperature of -35 °C in a rotary freezing equipment. Thawing is conducted in a water bath at the temperature of maximally +37 °C. The entire cycle of freezing and thawing is repeated 6 times. After conclusion of the 6th cycle, the homogenate maintained in original bottles is placed in a chest freezer at the minimum temperature of -17 °C until further processing. a2) Preparation of homogenate with use of ultra-sound:
After thawing, the leukocyte homogenate is prepared by disintegrating ceils with ultrasound at 20 kHz for 10 minutes at the temperature of ÷2 °C to +8 °C. After this period of disintegration, the obtained homogenate is placed in a chest freezer at the minimum temperature of -17 °C until further processing. a3; Preparation of homogenate with use of a combined method:
A combined leukocyte homogenization procedure is used as an alternative production procedure. The freezing and thawing cycle is repeated 5 times with subsequent disintegration with use of ultrasound at 20 kHz for 3 minutes at the temperature of +2 °C to +8 °C. After this disintegration,, the obtained homogenate is placed in a chest freezer at the minimum temperature of -17 °C until further processing. The temperatures specified in this production stage should, not fluctuate by more than 30 °C to both sides of the temperature scale and the time should be a matter of minutes.
The leukocyte homogenate processing is carried out within a closed system in a dialysis column. In the case that the leukocyte homogenate was frozen before further processing; it must be thawed in a water bath with the temperature of +37 °C. The temperature may be even lower (up to +4 °C). It is beneficial if the temperature does not fluctuate from the defined temperature by more than +33 °C.
The homogenate dialysis is conducted as dialysis in a closed system.
Dialysis is conducted in a dialysis unit made of an artificial kidney containing a membrane, which can detain particles with the relative molecular weight exceeding 10,000. Dialysis is carried out at reduced temperature of +2 °C to +8 °C. This temperature should not differ by more than 20 °C. The volume of the leukocyte concentrate homogenate processed within a single cycle ranges with an advantage from 1.1 to 2.4 L. The volume should not be larger than tens of litres and smaller than 0.2 litre. The amount of water for the injection needed for dialysis of low- molecular substances in the temperature range of +2 °C to +8 °C ranges from S to 13 L. The amount of water may differ when using different temperatures, but it.should not exceed units of litres.
It. is beneficial if during the dialysis the disintegrated leukocyte concentrate runs with use of a pump through the dialysis unit in a circuit with the flov; rate of 1.2 to 2.0 L/h and water for injections as a dialysis medium through the same dialysis unit in another circuit with the flow rate of 0.4 to 0.5 L/h. Under these circumstances the dialysis duration is 24 hours +1 hours. In the case that different circulation speeds are used in the process, the dialysis duration must also be adjusted accordingly. Dialysis is terminated when the dialysate absorption value drops below 0.6 at 260 nm. This value is only recommended and may differ.
A similar procedure may be used in the case that dialysis is replaced by ultrafiitration. c) Removal of salts and tion with use of nanofiItration
In this production step, the obtained dialysate is purged of undesired salts and simultaneously concentrated with use of nanofiltration to the required volume. During this production stage, the obtained dialysate that contains too much salts and water to allow further processing must be desalinated and concentrated in a way that It is directly usable to construct a lyophilised tablet, i.e., without any need of further diluting or concentrating or enriching or any other supplementary treatment of the obtained solution. The concentration methods may have various forms. The used method deals with two technological requirements: a gentle procedure and purifying of the product from salts and undesired excessive water volume. The product is protected; against thermal stress by the entire process cooling. The temperature of nanofiltration should not exceed +30 °C and it is beneficial when the temperature ranges from 14 to 25 °C and the processed volume of fluid is not larger than 50 litres and smaller than 5 litres. The ideal volume ranges around 15 litres. The working pressure at the filter is 20 bar +5 bar. It is always necessary to avoid static counter pressure of the permeate. Filtration is carried out using the system of maximum permeate separation from the filtered sample. The remaining sample is directly diluted with demineralised water in the filtration equipment according to the volume of the collected permeate. Filtration as such proceeds after mixing the diluted fluid. This procedure is repeated 4 times. A thin-film polyamide composite is preferably used for nanofiitration as a membrane with the porosity allowing for permeability of up to 1,000 Da. The subject of patent protection in this production stage comprises removal of excessive salts and water with use of a described nanofiltration method. d) Thermal inactivation
To ensure security of the product, it is necessary to ensure its inactivation. The transfer factor is inactivated through thermal pasteurization at +60 °C for 10 hours in a water bath. The described procedure ensures that the product is secure and simultaneously so gentle that it does not cause any damage to the [product. This technological step is also carried out according to the state of the art. e) Tablet formulation
As concerns the content of thickeners and fillers, the tablet has the following defined composition:
Formulation of one tablet represents the following components: Dextranum 40 0.05 g
(C6H10O5)n iota carrageenan 0.0015 g
(b-D-galactose-4-sulphate; 3,6-anhydride~a-D-galactose-2 sulphate)
Macrogolum 300 0.005 g (Polyethylenglycoluxn 300)
Saccharinum natricum 0.0003333 g
(Sodium salt 1.1-dioxid-l,2-benzothiazoie-3(2H)-onu; C7H4NNa03S)
Active substance (of the transfer factor) 1 medicinai dose Aqua pur.1.ficata ad 0.5 g
Preparation of tablets:
The active substance solution (of the transfer factor) is prepared in advance in a way that one dose is dissolved in 0.25 ml of water in the quality of "water for injections".
Preparation of the matrix follows:
0,0015 g of carrageenan iota is dissolved at increased temperature in a water bath (60 °C) in 0.17 g of purified water, 0.05 g of dextran 40 is added to this solution and dissolved by stirring. To speed up dextran dissolution we may use an ultrasound bath for 1 minute or maintain the mixture slightly heated in a water bath (40 °C).
After cooling to the laboratory temperature, we add 0.005 g of macrogol 300 and 0.0003 g of sodium saccharin supplemented with purified water to 0.25 g and we stir until ail ingredients are dissolved. Subsequently, the matrix is blended with a ore-made TFI solution. It is dosed in a blister that serves as a primary package for the lyophilised tablets by 0.5 ml in each blister pocket. The subject of the invention in this production stage comprises preparation of the tablet matrix. f) Preparation of a lyophillzed tablet
According to the prescription for one tablet formulation solution of the tablet matrix is prepared and with use of suitable dosing equipment the solution is directly dispensed in the dose of 0.5 ml per each individual blister pocket.
Before filling the preparation in the blisters, the lyophilizer is set to the plate freezing mode at the defined temperature of - 50 °C. The plates freeze to the required temperature in approximately 2 hours.
After reaching the required temperature of -50 °C, blisters with the dispensed material are set up on the frozen plates of the lyophilizer. Subsequently, the lyophiiisation equipment is switched to the primary drying (sublimation) mode and the temperature is set to -55 °C and vacuum to 0.3 xnbar to freeze the condenser. After reaching the set condenser temperature of -55 °C the lyophiiisation process is initiated.
The lyophiiisation drying process may be set in an automated or semi-automated regime.
When in the automated regime during the lyophiiisation process, it is impossible to adjusted the set. parameters; the semi- automated regime allows for adjusting and switching the individual stages (freezing, primary drying, secondary drying) through manual control if needed. After 2 hours, the lyophilisation process shifts in the secondary drying mode (desorption).
The plate temperature is set to -30 °C, vacuum to 0.005 mbar. In the following steps the temperature of the plates is gradually decreased to -20 °C, -10 °C, 0 °Cf +10 cC and +25 °C. The entire lyophilisation process is completed within 24 hours from its initiation at the latest. The real time of lyophilisation completion is based on reaching the described temperature values of the plates and vacuum values. After lyophilisation is completed, the lyophilisation equipment is aerated and blisters with the dried product are removed and sealed with a cover foil using the blistering equipment. The subject of the invention in this production stage comprises preparation of a lyophiiised tablet.
Using the above means of production, we prepare the transfer factor medicine from peripheral blood leukocytes comprising protein components and nucleic acid components the share of which in the medicine is represented by the absorbance index, i.e., a ratio obtained through specfcrophotoxnetric absorbance measuring in the UV zone at 260 ran and 230 ran. This index must not be smaller than 1.7 with an advantage in the range from 1.7 to 2,1. The medicinal product is purified of ballast salts using the nanofiltration method. The molecular weight the individual substances of the medicinal product must not exceed 10,000 Daltons,
Pharmaceutical form, i.e., a lyophiiised tablet. One lyophiiised tablet comprises one medicinal dose of the product, which represents the amount of biologically active ingredients obtained from approximately 200 million peripheral leukocytes that are capable of normalising the compromised cellular immune system of the recipient. The lyophilised tablet is manufactured through 1yophi1isation of a liquid matrix. The liquid matrix features the osmolality value ranging from 150 to 400 mmol/kg. Activity of biologically active substances may be proven in the lyophilised tablet.
Biological activity testing
The biological activity test is based on monitoring the transfer factor impact on the model cellular line viability after it was first influenced by an immunosuppressive substance, azathioprine.
Immortalised T~lymphocyte cellular line Jurkat CCL--81 is used as a model cellular line. Cellular viability is measured on a flow cytometer with use of a commercial MTT test. MTT measures the metabolic activity of ceils and thus is suitable to determine the cellular viability. The test is based on tetrazoiium MTT salt (3- (4,5~dimethylthiazol-2-yl}-2.5-diphenyi tetrazoiium bromide) reduction to insoluble formazan. The amount of the created formazan is directly proportional to the number of living cells.
The Jurkat cellular line is cultivated in a RPMI media enriched with 10% of foetal bovine serum, 2 mM of glutamine and a mixture of penicillin (100 I/ml) and streptomycin (100 ug/ml). We use azathioprine with the concentration of 5 pg./mi after the model cellular line suppression.
Transfer factor diluted 80 to ISO times is used for the biological activity test. Incubation of the cells takes 72 hours. After this incubation period, 20 mΐ of stock MMT solution (5 mg/ml) is added to each hole in the plate and the plate is further incubated for 3 h at 37 °C. After that, supernatant is removed and the created formazan crystals are dissolved in 150 pi of dimethyl sulfoxide. The resulting optical density of solutions is measured at 570 nm using a plate spectrophotometer and evaluated as viability of the ceils.
During the test, it is suitable to use the following comparison groups:
Control group - individual; not influenced Jurkat cells;
Jurkat cells suppressed by azathioprine;
Jurkat cells influenced by the transfer factor;
Jurkat cells suppressed by azathioprine and subsequently influenced by the transfer factor.
Evaluation of results:
The added transfer factor itself does not affect viability of the ceils compared to the control group.
With regard to the control, the added azathioprine reduces viability of the cells under 50% of the control group value.
The transfer factor in combination with azathioprine returns the cellular viability suppressed by azathioprine back to normal by approximately 40%. The subject of the invention in this •production stage comprises an in vitro test of the transfer factor biological activity.
Use
The invention is usable for commercial preparation of any transfer factor, both specific or non-specific, prepared from homogenate of the peripheral blood leukocytes. The invention is usable upon manufacture of a human product, as well as in the case of any modifications designated for veterinary use.
Recommended dosing of the transfer factor based in administration of a lyophilised tablet to the area of the buccal mucosa is also included in the patent cover.
Administration of the transfer factor in this form will prevent the tablet swallowing; it will xaelt on the mucosa in several seconds.
Thus, the main therapeutic effect is facilitated by the medicine penetration exclusively through the oral mucosa.
Examples of the invention implementation
Example 1
Method of the transfer factor medicine preparation a) Preparation of leukocyte concentrate; b; Dialysis or ultrafiltration; c) Removal of salts and concentration with use of nanofiltration; d} Thermal inactivation; e} Tablet formulation; f} Preparation of a iyophiiized tablet. a) Preparation of leukocyte concentrate
Leukocyte concentrates are stored in a frozen condition. Subsequently, at the maximal temperature of +37 °C, the leukocyte concentrate is merged in the working volume. The work must be performed in the production premises with environmental cleanness class "C". Subsequently, all membrane components in the leukocyte homogenate are disrupted with use of repeated freezing and thawing or by disturbing the cells with use of ultrasound or a combination of both procedures, al) Preparation of homogenate with use of the freezing - thawing method:
Freezing is carried out in denatured ethyl alcohol at the minimum temperature of -35 °C in a rotary freezing equipment. Thawing is conducted in a water bath at the temperature of maximally +37 °C. The entire cycle of freezing and thawing is repeated 6 times. After conclusion of the 6tn cycle, the homogenate maintained in original bottles is placed in a chest freezer at the minimum temperature of -17 °C until further processing. a2) Preparation of homogenate with use of ultra-sound:
After thawing, the leukocyte homogenate is prepared by disintegrating cells with ultrasound at 20 kHz for 10 minutes at the temperature of 2 °C to 8 °C. After this period of disintegration, the obtained homogenate is placed in a chest freezer at the minimum temperature of - 17 °C until further processing. a3) Preparation of homogenate with use of a combined method:
As combined leukocyte homogenization procedure is used as an alternative production procedure. The freezing and thawing cycle is repeated 5 times with subsequent disintegration with use of ultrasound at 20 kHz for 3 minutes at the temperature of +2 °C to -tS cC. After this disintegration, the obtained homogenate is placed in a chest freezer at the minimum temperature of -17 °C until further processing.
The temperatures specified in this production stage should not fluctuate by more than 30 °C to both sides of the temperature scale and the time should be a matter of minutes. b) Dialysis
The leukocyte homogenate processing is carried out within a closed system in a dialysis column. In the case that the leukocyte homogenate was frozen before further processing, it must be thawed In a water bath with the temperature of + 37 °C. The temperature may be even lower (up to +4 °C}. It is beneficial if the temperature does not fluctuate from the defined temperature by more than 20 °C.
Dialysis is conducted in a dialysis unit made of an artificial kidney containing a membrane, which can detain particles with the relative molecular weight exceeding 10,000. Dialysis is carried out at reduced temperature of +2 “C to +8 aC. This temperature should not differ by more than 20 °C. The volume of the leukocyte concentrate homogenate processed within a single cycle ranges with an advantage from 1.4 to 2.4 L, The volume should not be larger than tens of litres and smaller than 0.2 litre. The amount of water for the injection needed for dialysis of low-molecular substances in the temperature range of ÷2 °C to +8 °C ranges from 8 to 13 L, The amount of water may differ when using different temperatures, but it should not exceed units of litres. It is beneficial if during the dialysis the disintegrated leukocyte concentrate runs with use of a pump through the dialysis unit in a circuit with the flow .rate of 1.2 to 2.0 L/h and water for injections as a dialysis medium through the same dialysis unit in another circuit with the flow rate of 0.4 to 0.5 L/h. Under these circumstances the dialysis duration is 24 hours +4 hours. In the case that different circulation speeds are used in the process, the dialysis duration must also be adjusted accordingly. Dialysis is terminated when the dialysate absorption value drops below 0.6 at 260 nm. This value is only recommended and may differ.
A similar procedure may be used in the case that dialysis is replaced by ultrafiltration. c5 Removal of salts arid concentration with use of nanofiltration
In this production step, the obtained dialysate is purged of undesired salts and simultaneously concentrated with use of nanofiltration to the required volume. The aim is that the dialysate comprising salts and water in excessive volumes preventing its further processing is desalinated and concentrated in a way that it will be directly usable to construct a lyophilised tablet. The product is protected against thermal stress by the entire process cooling. The temperature of nanofiltration should not.exceed +30 °C and it is beneficial when the temperature ranges from 14 to 25 °C and the processed volume of fluid is not larger than 50 litres and smaller than 5 litres. The ideal volume ranges around 15 litres. The working pressure at the filter is 20 bar +5 bar. It is always necessary to avoid static counter pressure of the permeate. Filtration is carried out using the system of maximum permeate separation from the filtered sample. The remaining sample is directly diluted with demineralised water in the filtration equipment according to the volume of the collected permeate. Filtration as such proceeds after mixing the diluted fluid. This procedure is repeated 4 times. A thin-film polyamide composite is preferably used for nanofiltration as a membrane with the porosity allowing for permeabi1ity of up to 1,000 Da. d) Thermal activation - this technological step is also carried out according to the state of the art.
To ensure security of the product, it is necessary to ensure its inactivation. The transfer factor is inactivated through thermal pasteurization at +60 °C for 10 hours in a water bath. The described procedure ensures that the product is secure and simultaneously so gentle that it does not cause any damage to the product. e) Tablet formulation Batch size (120 tablets)
Dextranum 40 6.0 g
Iota carrageenan 0.18 g
Macrogoium 300 0.6 g
Saccharinum natricuin 0.04 g
TFI 30 doses
Aqua purificata ad 60.0 g
The transfer factor solution (30 doses) in 30 mi of purified water (or water for injection) is prepared in advance. Subsequently, the matrix is prepared as follows: 0.18 g of carrageenan iota is dissolved at increased temperature in a water bath (60 °C) in 20 g of purified water (corresponds to the total sample amount of 60 g for preparation of 120 tables). 6.0 g of dextran 40 is added to this solution and dissolved by stirring. To speed up dextran dissolution we may use an ultra-sound bath for 1 minute or maintain the mixture slightly heated in a water bath (40 °C) .
After cooling to the laboratory temperature, we add 0.6 g of macxogol 300 and 0.04 g of sodium saccharin supplemented with purified water to 30.0 g and we stir until all ingredients are dissolved. The created matrix is mixed with the transfer factor solution f) Preparation of a lyophilized tablet
The prepared liquid tablet matrix comprising the transfer factor as an active substance shall be &l--spe*»e-ed filled with use of a dosing pipette in the individual blister pockets in 0.5 mi doses. Before filling the TFI product in the blisters, the lyophiiizer shall be set in the freezing mode - freezing the plates to the defined temperature of -50 °C. Freezing of the plates will take approximately 2 hours.
To reach the required temperature, blisters with the dispensed tablet matrix are set on the frozen plates, the lyophiliter is switched to the primary drying mode {sublimation}, the values defined for the condenser freezing are set at -55 °C and the vacuum value at 0.3 mfo&r. After reaching the set condenser temperature, the iyophilisation process is initiated. The iyophilisation process may be set in an automated or semi- automated regime. If in the automated regime, the set parameters cannot be modified in the course of the iyophilisation process duration. The semi~automated regime allows for adjusting of and switching between the individual stages {freezing, primary drying, secondary drying} through manual control.
Approximately after 2 hours, lyophilisation moves to the secondary drying regime {desorption}, temperature of the plates is set to -30 °C and vacuum to 0.005 mbar. Subsequently, the temperature of the plates is gradually decreased to -20 °C,
10 °C, 0 °C, +10 °C and +25 °C.
The entire lyophilisation process is completed within 24 hours from its initiation at the latest; the lyophilisation time depends on reaching the values set for the plates and vacuum. After lyophilisation is completed, the lyophilisation equipment is aerated and blisters with the lyophilizate are removed and sealed with a cover foil using the blistering equipment. Used lyophilisation equipment: LY0BE7A 15
Example 2
The transfer factor medicine
Using the above means of production, we prepare the transfer factor medicine from peripheral blood leukocytes comprising protein components and nucleic acid components the share of which in the medicine is represented by the absorbance index, i.e., a ratio obtained through spectrophotometric absorbance measuring in the O'V zone at 260 nm and 2S0 nm. This index must not be smaller than 1,7 with an advantage in the range from 1.7 to 2.1, The medicinal product is purified of ballast salts using the nanofiltration method. The molecular weight the individual substances of the medicinal product must not exceed 10,000 Daltons. Example 3
Medicine biological activity texting methodology according to the described invention Biological activity testing
We used flow cytometer Guava EasyCyte by Luminex, USA and a commercial MTT test by Sigma-Aldrich, USA to measure viability. To determine the cellular line viability, we used the Guava- ViaCount kit by Luminex, USA, which distinguishes living cells from dead cells based on the principle of different permeability of the cellular membrane for DNA-binding markers. The Jurkat cellular line was cultivated in a RPMI media enriched with 10% of the foetal bovine serum, 2 mM of glutamine and a mixture of penicillin (1001/ml; and streptomycin (100 ug/mi;,
Transfer factor diluted 80 to 160 times is used for the biological activity test. Incubation of the ceils takes 72 hours. After this incubation period, 20 mΐ of stock MMT solution (5 mg/mi; is added to each hole in the plate and the plate is further incubated for 3 h at 37 °C. After that, supernatant is removed and the created formazan crystals are dissolved in 150 mΐ of dimethyl sulfoxide. The resulting optical density of solutions is measured at 570 nm using a plate spectrophotometer and evaluated as viability of the ceils. Optical density of the solution was measured at 570 nm using a plate spectrophotometer (Beckman Coulter, USA).
During the test, it is suitable to use the following comparison groups:
Control group - individual, not influenced Jurkat cells;
Jurkat cells suppressed by azat'nioprine;
Jurkat cells influenced by the transfer factor; Jurkat ceils suppressed by azathioprine and subsequently influenced by the transfer factor.
Evaluation of results:
The added transfer factor itself does not affect viability of the cells compared to the control group.
With regard to the control, the added azathioprine reduces viability of the cells under 50% of the control group value.
The transfer factor in combination with azathioprine returns the cellular viability suppressed by azathioprine back to normal by approximately 40%,
Example 4
Pharmaceutical form
Pharmaceutical form, i.e., a lyophiiised tablet. One iyophilised tablet comprises one medicinal dose of the product, which represents the amount, of biologically active ingredients obtained from approximately 200 million peripheral leukocytes that are capable of normalising the compromised cellular immune system of the recipient. The lyophiiised tablet is manufactured through iyophiiisation of a liquid matrix. The liquid matrix features the osmolality value ranging from 150 to 400 mmol/kg. Activity of biologically active substances may be proven in the lyophiiised tablet.
The pharmaceutical form is prepared as follows:
According to the prescription for one tablet formulation solution of the tablet matrix is prepared and with use of suitable dosing equipment the solution is directly d-i-sp-ens-ed filled in the dose of 0.5 ml per each individual blister pocket.
Before filling the preparation in the blisters, the lyophilizer is set to the plate freezing mode at the defined temperature of -50 °C. The plates freeze to the required temperature in approximately 2 hours.
After reaching the required temperature of -50 °C, blisters with the dispensed material are set up on the frozen plates of the lyophilizer. Subsequently, the lyophilisation equipment is switched to the primary drying (sublimation) mode and the temperature is set to -55 °C and vacuum to 0.3 mbar to freeze the condenser. After reaching the set condenser temperature of -55 °C the lyophilisation process is initiated.
The iyophiiisation drying process may be set in an automated or semi-automated regime.
When in the automated regime during the lyophilisation process, it is impossible to adjusted the set parameters; the semi- automated regime allows for adjusting and switching the individual stages (freezing, primary drying, secondary drying) through manual control if needed. After 2 hours, the lyophilisation process shifts in the secondary drying mode (desorption).
The plate temperature is set. to -30 °C, vacuum to 0.005 mbar. In the following steps the temperature of the plates is gradually decreased to -20 °C, -10 °C, 0 °C, +10 °C and +25 °C. The entire lyophilisation process is completed within 24 hours from its initiation at the latest. The real time of lyophilisation completion is based on reaching the described temperature values of the plates and vacuum values. After lyophilisation is completed, the iyophilisation equipment is aerated and blisters with the dried product are removed and sealed with a cover foil using the blistering equipment. The subject of the invention in this production stage comprises preparation of a lyophilised tablet.
Using the above means of production, we prepare the transfer factor medicine from peripheral blood leukocytes comprising protein components and nucleic acid components the share of which in the medicine is represented by the absorbance index, i.e., a ratio obtained through spectrophotometric absorbance measuring in the UV zone at 260 nm and 280 nm. This index must not be smaller than 1.7 with an advantage in the range from 1.7 to 2.1. The medicinal product is purified of ballast salts using the nanofiitration method. The molecular weight the individual substances of the medicinal product must not exceed 10,000 Daltons.
Example S
Usage of the pharmaceutical form a) Using of the transfer factor according to this invention as the pharmaceutical form of a lyophilised tablet administered in the oral cavity where the lyophilised tablet immediately adheres to the mucosa,- which prevents its swallowing; it speedily melts on the buccal mucosa and therefore, it does not pass directly to the digestive system. b) Using of the transfer factor according to this invention as a human or veterinary medicine. c) Using of the transfer factor according to this invention as a human or veterinary food supplement. d) Using of the transfer factor according to this invention as a human or veterinary preparation to cure pathological conditions related with cellular immunity disorders, i.e., chronic: infections, septic conditions, atopic eczema, recurring mycoses, psoriasis or immunodeficiencies related with oncologic treatment. b) Using of the transfer factor according to this invention as a human or veterinary prophylactic preparation.
Industrial utilization
The invention is usable upon commercial preparation of any transfer factor, both specific or non-specific, prepared from the peripheral blood leukocytes. The invention is also usable upon manufacture of a human product, as well as in the case of any medicinal products designated for veterinary use. The invention is usable for commercial preparation of any transfer factor, both specific or non-specific, usable as a part of food supplements. The preparation obtained through the preparation process according to this invention is usable to cure pathological conditions related with the cellular immunity disorders {chronic: infections, septic conditions, atopic eczema, recurring mycoses, psoriasis or immunodeficiencies related with oncologic treatment). The preparation is also suitable for prophylactic use. Recommended dosing: a lyophilised tablet is inserted in the mouth where it sticks to the mucosa and speedily melts. The tablet is not swallowed.

Claims

Patent claims
1. Method of preparation of the transfer factor medicine from the peripheral blood leukocytes that is based on a) preparedness of the leukocyte concentrate, b) dialysis and/or filtration characterised by that it comprises the following consecutive steps: c) Removal of salts and concentration with use of nanofiitration; d) Thermal inactivation; e) Tablet formulation; f) Preparation of a lyophilized tablet.
2. Method of preparation of the transfer factor according to claim 1, characterised by that the salt removal from the product through nanofiltration and the product concentration with use of nanofiltration comprises purging of the obtained dialysate of undesired salts and its simultaneous concentration with use of nanofiitration to the required volume, while the temperature during nanofiitration ranges from 14 to 25 °C and should not exceed +30 °C upon processing of the liquid volume not larger 50 litres or smaller than 5 litres, and in an ideal case of approximately of 15 litres and the working pressure at the filter is 20 bar +5 bar, with exclusion of static counter pressure by the permeate and nanofiitration is carried out by maximal separation of the permeate from the filtered dialysate sample by diluting the remaining dialysate sample in the filtration equipment with the demineralised water volume equalling the amount of the collected permeate and conducting ultrafiitration after mixing of such diluted liquid, 'while this cycle repeats 4 times.
3. Method of preparation of the transfer factor according to claim 2, characterised by that for concentration with use of nanofiltration a thin-film polyamide composite is used as a membrane with the porosity allowing for permeability of up to 1,000 Da.
4. Method of preparation according to claims 1, 2 and/or 3, characterised by that the transfer factor active substance is prepared as a lyophilised tablet comprising: Dextranum 40, Iota karagenan, Macrogoium 300, Saccharinum natricum, transfer factor and Aqua purificata.
5. Method of preparation according to claim 4, characterised by that it comprises the following steps: a) preparation of the transfer factor in purified water or water for injection; b) preparation of the matrix in the following steps:
- carrageenan iota is dissolved at increased temperature in a water bath (60 °C) in purified water;
- dextran 40 is added to this solution and dissolved by mixing; to speed up the dissolution of dextran, an ultrasound bath may be used or the mixture may be kept slightly heated in a water bath (40 °C);
- after cooling to the laboratory temperature, we add macrogoi 300 and sodium saccharin supplemented with purified water and we stir until all ingredients are dissolved; c) mixing of the matrix with the prepared transfer factor solution; d) dosing in the blister, i.e., the primary package of the lyophilised tablet.
6. Method of preparation according to any combination of the preceding claims characterised by that the transfer factor active substance is prepared as a lyophilised tablet that is lyophilised directly in the blister pocket; each pocket represents one dose of the preparation.
7. Method of preparation according to claim 6, characterised by that the transfer factor active substance is lyophilised in the blister for 24 hours at the temperatures ranging gradually from -- 55 °C to +25 °C and under the pressure of 0.005 rabar.
8. Method of preparation according to claim 7, characterised by that lyophilisation of the transfer factor active substance is conducted in the following steps:
- preparation of the tablet matrix solution;
- setting of the lyophilisation equipment in the plate freezing mode at the defined temperature of -50 °C, i.e., freezing of the plates to this temperature in 2 hours; the solution dispensing with use of dosing equipment in individual blister pockets;
- setting of blisters with the dispensed material to the frozen plates of the lyophilizer after reaching the required temperature of -50 °C;
- the lyophilisation equipment is switched in the primary drying (sublimation) mode and the values are set to the temperature of - 55 °C and vacuum of 0.3 mbar to freeze the condenser;
- after reaching the set condenser temperature of -55 °C, the lyophilisation process is initiated.
9. Method of preparation according to claim 8 characterised by that lyophilisation of the transfer factor active substance is conducted in an automated or semi-automated regime allowing for modification and switching between the individual stages (freezing, primary drying, secondary drying) through manual control in individual steps as follows: after 2 hours, the lyophilisation process moves to the secondary drying regime (desorption), temperature of the plates is set to -30 °C and vacuum to 0,005 rabar; subsequently, every hour the temperature of the plates is gradually decreased to -20 °C, -103C, 0 °C, +10 *C and +25 °C;
- the entire lyophilisation process is completed in 24 hours from its initiation at the latest;
- the lyophilisation equipment is aerated and blisters with the dried product are removed and sealed with a cover foil using the blistering equipment.
10. The transfer factor medicine prepared according to claims 1 to 9 and made of dialysate or ultrafiltrate of the peripheral blood leukocytes purified of ballast salts and comprising protein substances and nucleic acid substances whose ratio in the medicine, i.e., the absorption index, is not under 1.7, where the molecular weight of none of the medicine substances is greater than 10,000 Daltons.
11. The transfer factor medicine prepared according to claims 1 to 9 and made of dialysate or ultrafiltrate of the peripheral blood leukocytes purified of ballast salts and comprising protein substances and nucleic acid substances whose ratio in the medicine, i.e,, the absorption index, ranges between 1.7 and 2.1, where the molecular weight of none of the medicine substances is greater than 10,000 Daltons.
12. An in vitro method of testing the biological activity of the transfer factor active substance prepared according to any combination of the preceding claims, characterised by that it is conducted through observing the transfer factor impact on the model cellular line viability after it was influenced by an immunosuppressive substance, azathioprine.
13. A method of testing biological activity of the transfer- factor active substance prepared according to any combination of the preceding claims, characterised by that an immunosuppressive substance, azathioprine, is used to monitor the transfer factor medicine.
14. The in vitro method of testing biological activity of the transfer factor medicine prepared according to claims 12 and 13, characterized by that it is conducted in the individual steps as follows:
- the Jurkat cellular line is cultivated in a RPMI media enriched with 10% of foetal bovine serum, 2 raM of glutamine and a mixture of penicillin (100 I/ml) and streptomycin (100 ug/rnl), -where azathioprine with the concentration of 5 pg/ml is used after the model cellular line suppression;
- to determine biological activity, the transfer factor diluted 80 to 160 times is used;
- incubation of the cells takes 72 hours;
- 20 μl of stock MMT solution (5 mg/mi) is added to each hole in the 'plate and the plate is further incubated for 3 h at 37 °C;
- supernatant is removed and the created formazan crystals are dissolved in 150 mΐ of dimethyl sulfoxide;
- the resulting optical density of solutions is measured at 570 nm using a plate spectrophotometer and evaluated as viability of the cells.
15. The in vitro method of testing biological activity of the transfer factor medicine prepared according to claims 12 to 14 is based on using immortalised T-lymphocyte cellular line Jurkat. CCL-81 as a model cellular line.
16. The in vitro method of testing biological activity of the transfer factor medicine prepared according to claims 12 to 16, characterized by that it includes measuring on a flow cytometer with use of the commercial cellular viability MMT test to determine the cellular viability based on tetrazoiiura MTT salt (3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazoiium bromide) reduction to insoluble formazan, while the amount of the created formazan is directly proportional to the number of living cells.
17. The pharmaceutical form tested and prepared by method according to claims 1 to 16 characterized by that it comprises a lyophilised tablet prepared by means of lyophilisation of the liquid matrix in which activity of biologically active substances can be proven; one lyophilised tablet comprises one medicinal dose of the preparation that represents the amount of biologically active substances obtained from approximately 200 million peripheral leukocytes, which are capable of normalising the compromised cellular immune system of the recipient.
18. Use of the transfer factor pharmaceutical form according to claims 1-17 as the pharmaceutical form of a lyophilised tablet administered in the oral cavity where the lyophilised tablet immediately adheres to the mucosa, which prevents its swallowing; it speedily melts on the buccal mucosa and passes directly to the digestive system. 19, Use of the transfer factor pharmaceutical, form according to claims 1-17 as a human veterinary medicine.
20, Use of the transfer factor according to claims 1-17 as a human or veterinary food supplement.
22. Use of the transfer factor pharmaceutical form according to claims 1-17 as a human or veterinary preparation to cure pathological conditions related with cellular immunity disorders, i.e., chronic: infections, septic conditions, atopic eczema, recurring mycoses, psoriasis or immunodeficiencies related with oncologic treatment.
23. Use of the transfer factor pharmaceutical form according to claims 1-17 as a human veterinary prophylactic preparation.
EP21721385.9A 2020-06-12 2021-03-25 Method of preparation of the transfer factor medicine, transfer factor medicine, method of testing its biological activity, pharmaceutical form and its use Pending EP4072569A1 (en)

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