WO1999006441A1 - Proteinic product, process for its preparation, compositions containing it and use in medicaments - Google Patents

Proteinic product, process for its preparation, compositions containing it and use in medicaments Download PDF

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Publication number
WO1999006441A1
WO1999006441A1 PCT/ES1998/000219 ES9800219W WO9906441A1 WO 1999006441 A1 WO1999006441 A1 WO 1999006441A1 ES 9800219 W ES9800219 W ES 9800219W WO 9906441 A1 WO9906441 A1 WO 9906441A1
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WIPO (PCT)
Prior art keywords
strains
cect
fcm
protein
extract
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PCT/ES1998/000219
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Spanish (es)
French (fr)
Inventor
Fernando Chacon Mejias
Original Assignee
Chacon Pabon, Rafael
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Application filed by Chacon Pabon, Rafael filed Critical Chacon Pabon, Rafael
Priority to AU84424/98A priority Critical patent/AU8442498A/en
Publication of WO1999006441A1 publication Critical patent/WO1999006441A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • 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
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention falls within the field of drugs intended for the prevention and treatment of diseases related to immunodeficiency syndromes, autoimmunity, neoplastic processes, degenerative diseases, inflammatory bowel diseases and infectious diseases. More specifically, the invention is encompassed in the field of drugs obtained from protein extracts of microorganisms.
  • SUBSTITUTE SHEET (RULE 26) - immunodeficiency syndromes, including those caused by AIDS and idiopathic CD4 T lymphocytopenia;
  • Neoplastic processes such as those caused by leukemias, myelomas, lymph nodes, brain and spinal cord tumors, skin cancer, thyroid neoplasia, adrenal gland neoplasia, male and female genitourinary tract tumors, heart tumors, gastrointestinal tract tumors , pleura and mediastinal tumors, bone and cartilage neoplasms; - with syndromes related to degenerative diseases, such as osteoarthritis;
  • a protein product comprising proteins, peptides and other molecules, obtained from cell lysis of at least one of the strains of heat-resistant and sporulated bacilli, selected from at least one of the following groups: a first group consisting of strains of B. Licheniformis, B. Circulans 2, B. Pumilus, B. Macerans, B. Amilolicuofaciens, and modified strains thereof; a second group consisting of strains of B. Cereus 1, B. Cereus 2, B. Lentus 1, B. Lentus 2 and modified strains thereof;
  • the product according to the invention comprises - at least a first fraction of proteins, peptides and molecules obtained from cell fractionation, selected from extracts of B. Licheniformis, B. Circulans 2, B. Pumilus, B Macerans, B. Amilolicuofaciens; - at least a second fraction of proteins, peptides and molecules obtained from cell fractionation, selected from extracts of B. Cereus 1, B. Cereus 2, B. Lentus 1, B. Lentus 2;
  • SUBSTITUTE SHEET (RULE 26) product according to the invention comprises
  • the aforementioned strains are selected from natural strains and modified strains.
  • the bacillus strains of the first group may be CECT FCM-1 4913 modified strains;
  • Bacillus strains of the fourth group can be modified CECT FCM-4 4916 strains.
  • the object of the invention is also a process for obtaining a protein product according to the aforementioned characteristics, which basically comprises the following steps:
  • At least one heat-resistant and sporulated strain of at least one of the following groups is selected:
  • the aqueous solution may comprise between 19 and 200g of a culture medium per liter of distilled water.
  • the aqueous solution is adjusted to a pH of 7.0 + 0.2 and sterilized under conventional conditions, for example by autoclaving at 121 ° C + 1 ⁇ C for 15 to 20 minutes.
  • the temperature of the inoculated culture medium is adjusted according to an optimal growth of the strain.
  • the temperature of the inoculated culture medium is adjusted to a temperature between 18 to 40 ° C and forced ventilation by filtered air is applied to the inoculated culture medium during the entire growth phase of the strain, the inoculated culture medium being subjected to an air pressure of 0.5 to 1 1 / min.
  • the strain is grown to a maximum optical density, obtaining a culture containing a bacillary mass and then a protein extract of the bacillary mass is obtained.
  • Said extract is obtained by the steps of separating the bacillary mass from the culture until obtaining bacilli in suspension, for example by cell concentration of the bacillary mass in the culture by centrifugation, cyclone or other conventional procedures, preferably before concentration.
  • Bacillary mass cell separates and analyzes an aliquot part of the culture before concentration to obtain data of "bacilli in suspension” and also to obtain data indicative of the degree of cell concentration in order to control bacillary growth;
  • SUBSTITUTE SHEET (RULE 26) - cross filtration on a filter with a pore diameter of less than 0.22 microns until a concentrated culture with a concentration range of 10 to 20% is obtained. Then, the concentrated culture is subjected to diafiltration with physiological saline exchange. In a preferred embodiment of the process the concentrated culture is subjected to diafiltration with a membrane filter with a pore size equal to or less than 0.22 microns.
  • an extract comprising proteins, peptides and molecules that form the active ingredient through the phases of
  • a cryothermal process comprising a cycle with a first phase at temperatures between 2 and 8 ° C for several hours and a second cold phase at a temperature below -40 ° C for at least 8 hours, preferably at -40 and -50 ° C for 8 to 12 hours, and a third phase in a water bath at temperatures between room temperature and 70 ° C, preferably at a temperature between 60 and 70 ° C, and more preferably at about 65 ° C, and, optionally, a fourth phase in which defrosting is maintained at rest for at least five minutes.
  • Said cycle is repeated at least twice, preferably 4 times, until a concentrate of Cellular Use containing a cellular moiety and the active ingredient is obtained;
  • the first sterilized protein extract is mixed with at least a second protein extract prepared analogously to the first extract.
  • the first sterilized protein extract is obtained from at least one strain of said first group and the second sterilized protein extract is obtained from at least one strain of said second group.
  • the first sterilized protein extract and the second sterilized protein extract are further mixed with a third sterilized protein extract obtained from at least one strain of said third group, said third protein extract sterilized by procedures analogous to those of the preparation procedure of the first extract.
  • the first sterilized protein extract, the second sterilized protein extract, and the third sterilized protein extract are further mixed with a fourth sterilized protein extract obtained from at least one strain of said fourth group, said fourth sterile protein extract having been prepared by analogous procedures
  • the first sterilized protein extract is obtained from CECT FCM-1 4913 strains
  • the second sterilized protein extract is obtained from CECT FCM-2 4914 strains
  • the third sterilized protein extract is obtained at from CECT FCM-3 4915 strains
  • the fourth sterilized protein extract is obtained from CECT FCM-4 4916 strains.
  • the sterilized protein extracts are mixed, in equal proportions, for example 1: 1 when two extracts are mixed, 1: 1: 1 when three extracts are mixed, and 1: 1: 1: 1 when four extracts are mixed, based on their respective protein concentrations.
  • the different extracts may be mixed in unequal proportions, or only one of the extracts may be used.
  • the invention also relates to compositions containing the protein product, consisting of one or more of the aforementioned protein extracts, as active ingredient, with a pharmaceutically acceptable carrier.
  • compositions examples include solid compositions, such as tablets, pills, capsules, granules, or liquids, such as lyophilized solutions, suspensions and emulsions, for oral, topical, parenteral administration or other routes of administration.
  • the doses of these compositions depend on their formulation, the method of administration as well as the place of administration, the individual to be treated. Others
  • SUBSTITUTE SHEET (RULE 26) Factors such as age, body weight, sex, diet, duration of administration, excretion rate, condition of the treated individual, combination with other drugs, sensitivities of the individual and the degree of severity of the disease should also be taken into account. Administration can be carried out continuously or periodically within the maximum tolerated doses.
  • the liquid medium is, for example, distilled water, the solution being formulated as follows:
  • the present invention has special application in the treatment of HIV positive patients.
  • Today it is considered that the immunosuppressive effect of HIV can be attributed to the destruction of CD4 lymphocytes that are considered to play a very important role in the individual's immune response.
  • CD4 lymphocytes that are considered to play a very important role in the individual's immune response.
  • the protein products of the present invention stimulate the proliferation of CD4 lymphocytes.
  • Viable doses for the treatment of patients with AIDS are 1.0 or, preferably, 2.0 units in 2 ml of water for injections, administered every two days intra uscularly.
  • Figure 1 shows the results of a polyacrylamide gel electrophoresis analysis (PAGE) of a product protein according to the invention
  • Figure 2 shows the results of Western Blot analysis to which samples of the protein product according to the invention were subjected
  • Figure 3 summarizes the results of assays related to the in vivo response of antibodies dependent on T cells
  • Figure 4 summarizes the results of the first tests related to in vitro activation of B cells
  • Figure 5 is the control of the first assays related to in vitro activation of B cells in the presence of lipopolysaccharides (LPS)
  • Figure 6 summarizes the results of tests related to in vitro activation of T cells
  • Figures 7 to 11 summarize the results of several assays related to blastogenesis of T cells.
  • Example 1 Materials and equipment
  • thermal water bath containers a conventional PROSTAK cross flow filtration equipment with filtration capacities from 40 to 8001; - a conventional unidirectional filtration device with a capacity to filter volumes up to 100 1;
  • SUBSTITUTE SHEET (RULE 26) - stainless steel containers with a capacity of 50 1, 100 1 and 200 1; CM-P culture medium, marketed by INQUIAROMA; - water for injection (European Pharmacopoeia - FE)
  • the CECT FCM-1 4913 strains were inoculated, in the second solution the CECT FCM-2 4914 strains, in the third culture solution the CECT FCM-3 4915 strain and in the fourth solution the CECT FCM strain -4 4916.
  • the flasks were introduced with the culture solutions inoculated in an incubator and the incubations were carried out at 36 + 1 ° C and constant stirring at 100 rp until a bacillary suspension with an optical density at 620 nm of :
  • the first ferrer contained a culture with 8 1 of the second culture solution and 500 ml of CECT FCM-1 4913 bacillary suspension;
  • the second fermenter contained a culture with 8 1 of the second culture solution and 500 ml of CECT FCM-2 4914 bacillary suspension;
  • the third fermenter contained a culture with
  • the fourth fermenter contained a culture with 8 1 of the second culture solution and 500 ml of CECT FCM-4 4916 bacillary suspension.
  • the cultures were carried out in their respective fermenters at a temperature of 36 + l ° C, a stirring of the culture medium of 500 + 100rpm, with an air flow of 0.8 1 / min, and initial dissolved oxygen equal to 98+ 2% (during cultivation> 0%), until reaching a density
  • a fermenter 800 liters of a third culture solution were prepared by dissolving 35g of CM-P culture medium per liter of water, adding 100 ml of Struktol TM J660, adjusting the pH to 7 + 0.2 with H 3 P0 4 2M, and sterilizing at 121 ° C for 15 minutes.
  • the third solution was inoculated with the second culture solution and 8.5 L of CECT FCM-1 4913 bacillary suspension obtained in the SPS phase, and the fermentation proceeded.
  • batches of third solutions with strains CECT FCM-2 4914, CECT FCM-3 4915 and CECT FCM-4 4916 were prepared, inoculated and similarly fermented, so that they were fermented
  • the four cultures obtained through the MF phase were transferred in batches to various 50 1 containers, each labeling with the initials FP, its preparation date and name of the cultivated strain.
  • Each of the cultures harvested in cross flow was filtered in the PROSTAK filtration equipment with a PVDF membrane of 0.22 microns, until cultures were obtained 20 times more concentrated than the respective crops harvested. Subsequently, each of the concentrated cultures was subjected to diafiltration with exchange of physiological saline in PROSTAK equipment with 0.22 micron membrane filter with 150 1 of physiological saline, 10 1 of each of the concentrated cell suspensions were transferred to containers with a capacity of 20 1, labeling each container with the acronym CCS, its date of preparation and name of the cultivated strain.
  • Cycle strain contained CFU / ml total protein (g / 1) before CECT cycle FCM-1 4913 1x10 8 11.1 1 ⁇ 10 4 11.8 2 2xlO e 10.5 3 2xlO f 10.5 4 2xl0 ( 10.5
  • Cycle strain contained CFU / ml total protein
  • Cycle strain contained CFU / ml total protein
  • phase IA Obtaining protein extracts with the active ingredient free of cell detritus
  • the suspensions of Used cells were subjected, grouped respectively according to the strains of origin, resulting from the cryothermal process to cross filtration in PROSTAK equipment with 0.22 micron membrane filter.
  • Each of the protein extracts resulting from the IAP phase was subjected to diafiltration in PROSTAK equipment with a 0.22 micron membrane filter with a volume of physiological saline solution (0.9% m / v) ten times greater than the volume of the filtered protein extracts, diafiltration being controlled by analysis of the total protein content (Kjeldahl) in the filtrate, until an optimal protein content of:
  • IAP its date of preparation, and the name of the strain of origin.
  • SUBSTITUTE SHEET (RULE 26) It is performing correctly and, therefore, is basically analogous to checking in the IAP phase.
  • the concentrated active ingredients resulting from the IAC phase were introduced in separate containers labeled with the acronym IAC, the date of preparation, and the name of the strain of origin.
  • Each of the concentrated active ingredients resulting from the IAC phase was subjected to filtration in a unidirectional device with a 0.22 micron pore PVDF membrane. From each sterilized principle a sample was taken for sterility testing after filtration. The acceptance criterion is that the retention of microorganisms in the filter is less than 10 / cm. In the present case, the following values were obtained:
  • the protein concentration (Kjeldahl) was also analyzed to keep track of the process and to verify that it was done correctly. For this, a sample of the solution is taken and the protein concentration is measured. This step is analogous to checking in the IAP phase.
  • the resulting active ingredients were placed in a Nalgene package labeled with the acronym IAE, the date of preparation, and the name of the source strain, and stored at 4 ° C in a refrigerator.
  • the bulk product was subjected to sterile filtration using a unidirectional filtration equipment with coupled PVDF membrane of 0.22 microns, obtaining a sterile bulk product.
  • a sample was subjected to sterility and protein concentration tests (Kjeldahl) following the systematic and criteria followed in the sterility tests and protein analysis of the previous stages.
  • the sterile bulk product was packaged in a Nalgene container labeled with the acronym G, the date of preparation, and the names of the origin strains, and stored at 4 ° C in a refrigerated.
  • the sterile bulk product was dispensed in ampoules at a rate of 2 ml per ampoule.
  • Example 2 Three samples of the bulk product obtained in example 1 were analyzed to characterize their
  • the proteins of each of the samples were separated by PAGE techniques, in standard 10% polycylamide trisglycine gels, from BIORAD, USA.
  • the electrode buffer was the Tris-glycine / SDS buffer conventionally used for this type of gel (Laemmli method).
  • the separation parameters were 125 V, 30 mA (starting conditions) for approximately 90 min.
  • this figure shows different bands of different intensities but virtually identical band patterns of the three products tested, which is indicative that the samples had the same protein composition.
  • Example 3 Two other samples of the bulk product of Example 1 were taken and subjected to polyacrylamide gel electrophoresis analysis (PAGE) under the same conditions as described in example 1.
  • PAGE polyacrylamide gel electrophoresis analysis
  • LINUS with LINUS tris-glycine as an electrotransfer buffer, at 200 mA, 40 V and for 2 hours.
  • the membranes on which the PAGE-separated proteins corresponding to each of the samples were transferred were subjected to immunoassay with polyclonal antibodies produced in rabbits that had previously been obtained by treatment with a different batch of the protein product obtained according to the described method. in example 1 and that it was in rabbit serum stored at -180 ° C.
  • SUBSTITUTE SHEET (RULE 26) in the preparation used to immunize rabbits, and, consequently, can serve as a quality control of the new preparations that were generated.
  • Intramuscular administration was performed using disposable syringes with gauge needles.
  • the serum obtained (40ml) was homogenized, aliquoted in 1 and 4.5ml cryotubes, referenced as "Serum-97" and frozen at -180 ° C in liquid nitrogen.
  • each of the second antibodies has a marker (horseradish peroxidase, from PIERCE, Rockford, Illinois, United States of America) that allows visualization of the presence of such antibodies in the membrane when a reagent, 4-chloronaphthol, is added. which produces a violet coloration.
  • the second antibody used in the present assay was an IMMUNOPURE GOAT ANTI-RABBIT IgG (H + L) commercial antibody (product no .: 31460; ote no .: 96060545) from PIERCE of Rockford, Illinois, United States of America.
  • Each of the violet bands in each of the nitrocellulose membranes corresponds to a specific protein that was also in the control preparation of the protein product that had been used to immunize the two rabbits, from which serum the first antibody had been extracted. .
  • Solution 2 solution 1 50 ml
  • Solution 3 solution 1 50 ml
  • First antibody solution (1/100): first antibody 200 ⁇ l solution 3 20 ml
  • Second antibody solution (1/1500) second antibody (PIERCE) 60 ⁇ l Solution 3 30 ml Tris-HCl buffer:
  • Immunostimulation was evaluated based on tests performed with samples of the protein product obtained according to Example 1. The samples are identified as FR-91. 1) T-dependent in vivo response using platelet forming cell assay
  • mice Two mice were immunized with 0.025-0.3 ml of FR-91 for each mouse.
  • mice were sacrificed by cervical dislocation and each spleen was removed.
  • T-dependent antibody producing cells with or without lipopolysaccharides was measured using a platelet forming cell assay.
  • SUBSTITUTE SHEET (RULE 26) cells proliferate, radioactive thymidine is incorporated into the DNA of the descendant cells.
  • the degree of proliferation is determined by harvesting the cells, subjecting them to lysis, and measuring the amount of radioactive thymidine incorporated into the DNA which is directly proportional to the level of proliferation.
  • FR-91 strongly increased the proliferation of T cells.
  • Microwells were treated with lipopolysaccharides, pokeweed mitogen, phytohematoglutinin, concanavalin A as standard mitogen, at a final concentration of 1 ⁇ g / well.
  • Fig. 8 shows that FR-91 produces a synergistic effect with concanavalin A, which is a T-cell mitogen.
  • Fig. 9 evidence that FR-91 has a strong or synergy with phytohematoglutinin, which is a T-cell mitogen.
  • Fig. 10 evidence that FR-91 and lipopolysaccharides, which are mitogens of B cells, have no synergies.
  • Fig. 11 demonstrates, at a concentration of 100: 1, that FR-91 produces some synergistic effect with pokeweed mitogen, which is a common T and B mitogen.
  • FR-91 substantially increases the proliferation and activation of T cells, but has no effect on B cells.

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Abstract

The invention discloses a proteinic product extracted from non-pathogene bacillus and comprising proteins, peptides and other molecules obtained from the cellular lysis over at least one or the two sporulated and thermal resistant bacillus strains, in natural or modified form, and selected among a first group comprised of strains of B. Licheniformis, B. Circulans 2, B. Pumilus, B. Macerans, B. Amilolicuofaciens; a second group comprised of strains of B. Cereus 1, B. Cereus 2, B. Lentus 1, B. Lentus 2; a third group comprised of strains of B. Subtilis; a fourth group comprised of strains of B. Mesentericus. The invention also relates to a process for the preparation of the proteinic product, compositions which contain it and its use in the preparation of medicaments for the treatment of diseases related to syndromes of immunodeficiency, self-immunity, neoplasic processes, degenerative diseases, inflammatory intestinal diseases and infectious diseases.

Description

PRODUCTO PROTEICO, PROCEDIMIENTO PARA SU PREPARACIÓN, COMPOSICIONES QUE LO CONTIENEN, Y SU USO EN MEDICAMENTOS CAMPO DE LA TÉCNICA DE LA INVENCIÓNPROTEIN PRODUCT, PROCEDURE FOR PREPARATION, COMPOSITIONS CONTAINING IT, AND ITS USE IN MEDICATIONS FIELD OF THE INVENTION TECHNIQUE
La presente invención se engloba dentro del campo de los fármacos destinados a la prevención y al tratamiento de enfermedades relacionadas con síndromes de inmunodeficiencia, autoinmunidad, procesos neoplásicos, enfermedades degenerativas, enfermedades inflamatorias intestinales y enfermedades infecciosas. Más concretamente, la invención se engloba en el sector de los fármacos obtenidos a partir de extractos proteicos de microorganismos .The present invention falls within the field of drugs intended for the prevention and treatment of diseases related to immunodeficiency syndromes, autoimmunity, neoplastic processes, degenerative diseases, inflammatory bowel diseases and infectious diseases. More specifically, the invention is encompassed in the field of drugs obtained from protein extracts of microorganisms.
ESTADO DE LA TÉCNICA ANTERIOR A LA INVENCIÓN Los mecanismos de enfermedades relacionadas con síndromes de inmunodeficiencia, autoinmunidad, procesos neoplásicos, enfermedades degenerativas, enfermedades infeccionas e inflamaciones intestinales son complejos y, hasta la fecha, no existen fármacos que permitan la prevención y/o el tratamiento de los síndromes relacionados con los cuadros típicos de estas enfermedades con un grado de eficacia generalizado para los individuos enfermos, y que simultáneamente presenten una toxicidad lo suficientemente baja como para que su administración sea tolerada sin efectos secundarios por el individuo tratado. En los últimos tiempos, se han tratado con éxito esperanzador los síntomas del SIDA mediante combinaciones de medicamentos. Sin embargo, una parte sustancial de los individuos tratados no responde favorablemente a estos "cócteles" de medicamentos que, además son extremadamente caros y precisan un rígido programa de administración con tomas muy frecuentes y producen efectos secundarios adversos en los pacientes.STATE OF THE PRIOR ART OF THE INVENTION The mechanisms of diseases related to immunodeficiency syndromes, autoimmunity, neoplastic processes, degenerative diseases, infectious diseases and intestinal inflammations are complex and, to date, there are no drugs that allow prevention and / or Treatment of the syndromes related to the typical conditions of these diseases with a generalized degree of efficacy for the sick individuals, and that simultaneously present a sufficiently low toxicity so that their administration is tolerated without side effects by the treated individual. In recent times, the symptoms of AIDS have been treated with hopeful success through drug combinations. However, a substantial part of the treated individuals does not respond favorably to these "cocktails" of medications that are also extremely expensive and require a rigid administration schedule with very frequent intakes and produce adverse side effects in patients.
Existe una intensa investigación basada en la creación de moléculas artificiales y el aislamiento de moléculas de productos naturales, para generar fármacos que puedan combatir enfermedades de difícil o imposible curación como muchas de las anteriormente mencionadas . Básicamente, la investigación va dirigida a encontrar moléculas que inhiban o bloqueen determinados mecanismos intracelulares y/o metabólicos que se consideran responsables de las enfermedades, tales como las enfermedades neoplásicas y enfermedades relacionadas con el inmunosistema .There is intense research based on the creation of artificial molecules and the isolation of molecules from natural products, to generate drugs that they can fight diseases of difficult or impossible cure like many of the aforementioned. Basically, the research is aimed at finding molecules that inhibit or block certain intracellular and / or metabolic mechanisms that are considered responsible for diseases, such as neoplastic diseases and diseases related to the immunosystem.
Otros tipos de tratamientos, tales como los tratamientos con interferón, considerados muy esperanzadores en su momento pero criticados en algunos sectores últimamente, son los que combaten las enfermedades mediante el fortalecimiento del sistema inmunológico del paciente . En las patentes españolas ES-A-348986, ES-A-Other types of treatments, such as interferon treatments, considered very hopeful at the time but criticized in some sectors lately, are those that fight disease by strengthening the patient's immune system. In Spanish patents ES-A-348986, ES-A-
410892 y ES-A-414437 describen "vacunas anticancerosas" y su procedimiento de preparación, basadas en extractos obtenidos mediante bacteriolísis de cepas de Bacilos no- termorresistentes y no-esporulados cuyos Usados demostraran una relación positiva en orden a especificidad frente a suero de enfermos afectados por neoplasias. Las "vacunas" descritas en estas patentes españolas, si bien llegaron a emplearse con éxito en una gran cantidad de ensayos clínicos, eran relativamente complicada de preparar en vistas de que se preparaban en función de cada paciente a tratar.410892 and ES-A-414437 describe "anti-cancer vaccines" and their preparation procedure, based on extracts obtained by bacteriolysis of non-thermo-resistant and non-sporulated Bacilli strains whose Used will demonstrate a positive relationship in order to serum specificity of patients affected by neoplasms. The "vaccines" described in these Spanish patents, although they were successfully used in a large number of clinical trials, were relatively complicated to prepare because they were prepared according to each patient to be treated.
OBJETO DE LA INVENCIÓN Es un objeto de la presente invención poner a disposición un fármaco para la prevención y el tratamiento de enfermedades relacionadas con síndromes de inmunodeficiencia, enfermedades autoinmunes, procesos neoplásicos y degenerativos, enfermedades infecciosas y enfermedades infecciosas intestinales, particularmente para la prevención y el tratamiento de individuos afectados por trastornos, como por ejemplo:OBJECT OF THE INVENTION It is an object of the present invention to make available a drug for the prevention and treatment of diseases related to immunodeficiency syndromes, autoimmune diseases, neoplastic and degenerative processes, infectious diseases and intestinal infectious diseases, particularly for prevention and the treatment of individuals affected by disorders, such as:
HOJA DE SUSTITUCIÓN (REGLA 26) - síndromes de inmunodeficiencias, entre los que se encuentran los originados por el SIDA y la linfocitopenia T CD4 idiopática;SUBSTITUTE SHEET (RULE 26) - immunodeficiency syndromes, including those caused by AIDS and idiopathic CD4 T lymphocytopenia;
- síndromes de autoinmunidad, tales como los originados por esclerosis múltiple, Lupus eritematoso sistémico, artritis reumatóide, espondilitis anquilosante, psoriasis y otros de etiopatogenia similar; procesos neoplásicos, tales como los originados por leucemias, mielomas, linfornas, tumores cerebrales y de la médula espinal, cáncer de piel, neoplasia de tiroides, neoplasia de glándulas suprarrenales, tumores del aparato genitourinario masculino y femenino, tumores cardiacos, tumores del tracto gastrointestinal, tumores de pleura y mediastino, neoplasias de huesos y cartílagos; - con síndromes relacionados con enfermedades degenerativas, tales como la artrosis;- autoimmunity syndromes, such as those caused by multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, psoriasis and others of similar pathogenesis; Neoplastic processes, such as those caused by leukemias, myelomas, lymph nodes, brain and spinal cord tumors, skin cancer, thyroid neoplasia, adrenal gland neoplasia, male and female genitourinary tract tumors, heart tumors, gastrointestinal tract tumors , pleura and mediastinal tumors, bone and cartilage neoplasms; - with syndromes related to degenerative diseases, such as osteoarthritis;
- con enfermedades inflamatorias intestinales tales como la enfermedad de Crohn;- with inflammatory bowel diseases such as Crohn's disease;
- con todos los tipos de hepatitis; y - con enfermedades causadas por priones, tales como la encefalopatía espongiforme subaguda (enfermedad de Creutzfeldt-Jakob) , presentando a la vez una alta eficacia y una práctica ausencia de efectos secundarios nocivos para el individuo tratado.- with all types of hepatitis; and - with diseases caused by prions, such as subacute spongiform encephalopathy (Creutzfeldt-Jakob disease), presenting both high efficacy and a practical absence of harmful side effects for the treated individual.
Es otro objeto de la invención poner a disposición un fármaco con las cualidades anteriormente descritas y que además pueda obtenerse de una forma simple y económica. Es un ulterior objeto de la invención, poner a disposición un procedimiento para la obtención de un fármaco de las cualidades anteriormente descritas, cuyo proceso resulte suficientemente simple y estandarizable como para que pueda llevarse a cabo en condiciones económicas y masivas.It is another object of the invention to make available a drug with the qualities described above and which can also be obtained in a simple and economical way. It is a further object of the invention to make available a method for obtaining a drug of the qualities described above, the process of which is sufficiently simple and standardizable so that it can be carried out under economic and massive conditions.
HOJA DE SUSTITUCIÓN (REGLA 26) DESCRIPCIÓN DE LA INVENCIÓNSUBSTITUTE SHEET (RULE 26) DESCRIPTION OF THE INVENTION
Los objetos anteriormente mencionados se consiguen mediante un producto proteico que comprende proteínas, péptidos y otras moléculas, obtenidos a partir de lisis celular de al menos una de las de cepas de bacilos termorresistentes y esporulados, seleccionadas de al menos uno de los siguientes grupos : un primer grupo compuesto por cepas de B. Licheniformis , B. Circulans 2, B. Pumilus, B. Macerans, B. Amilolicuofaciens , y cepas modificadas de las mismas; un segundo grupo compuesto por cepas de B. Cereus 1, B. Cereus 2, B. Lentus 1, B. Lentus 2 y cepas modificadas de las mismas;The aforementioned objects are achieved by a protein product comprising proteins, peptides and other molecules, obtained from cell lysis of at least one of the strains of heat-resistant and sporulated bacilli, selected from at least one of the following groups: a first group consisting of strains of B. Licheniformis, B. Circulans 2, B. Pumilus, B. Macerans, B. Amilolicuofaciens, and modified strains thereof; a second group consisting of strains of B. Cereus 1, B. Cereus 2, B. Lentus 1, B. Lentus 2 and modified strains thereof;
- un tercer grupo compuesto por cepas de B_;_ Subtilis y cepas modificadas de las mismas;- a third group consisting of strains of B _; _ Subtilis and modified strains thereof;
- un cuarto grupo compuesto por cepas de B . Mesentericus y cepas modificadas de las mismas.- a fourth group consisting of strains of B. Mesentericus and modified strains thereof.
En una realización de la invención, el producto según la invención comprende - al menos una primera fracción de proteínas, péptidos y moléculas obtenidas a partir del fraccionamiento celular, seleccionada entre extractos de B. Licheniformis, B. Circulans 2, B. Pumilus, B. Macerans, B. Amilolicuofaciens ; - al menos una segunda fracción de proteínas, péptidos y moléculas obtenidas a partir del fraccionamiento celular, seleccionada entre extractos de B. Cereus 1, B. Cereus 2 , B. Lentus 1, B. Lentus 2;In one embodiment of the invention, the product according to the invention comprises - at least a first fraction of proteins, peptides and molecules obtained from cell fractionation, selected from extracts of B. Licheniformis, B. Circulans 2, B. Pumilus, B Macerans, B. Amilolicuofaciens; - at least a second fraction of proteins, peptides and molecules obtained from cell fractionation, selected from extracts of B. Cereus 1, B. Cereus 2, B. Lentus 1, B. Lentus 2;
- al menos una tercera fracción de proteínas, péptidos y moléculas obtenidas a partir del fraccionamiento celular, seleccionada entre extractos de B. Subtilis;- at least a third fraction of proteins, peptides and molecules obtained from cell fractionation, selected from extracts of B. Subtilis;
- al menos una cuarta fracción de proteínas, péptidos y moléculas obtenidas a partir del fraccionamiento celular, seleccionada entre extractos de B. Mesentericus. En otra realización de la invención, el- at least a fourth fraction of proteins, peptides and molecules obtained from cell fractionation, selected from extracts of B. Mesentericus. In another embodiment of the invention, the
HOJA DE SUSTITUCIÓN (REGLA 26) producto según la invención comprendeSUBSTITUTE SHEET (RULE 26) product according to the invention comprises
- al menos un primer extracto que contiene proteínas, péptidos y moléculas, obtenido a partir del fraccionamiento celular a partir de cepas de bacilos del genero Subtilis;- at least one first extract containing proteins, peptides and molecules, obtained from cell fractionation from bacillus strains of the Subtilis genus;
- al menos un segundo extracto que contiene proteínas, péptidos y moléculas, obtenido a partir del fraccionamiento celular de cepas de bacilos del género Cereus ; - al menos un tercer extracto que contiene proteínas, péptidos y moléculas, obtenido a partir de cepas de bacilos del género Mesentericus;- at least a second extract containing proteins, peptides and molecules, obtained from the cell fractionation of bacillus strains of the genus Cereus; - at least a third extract containing proteins, peptides and molecules, obtained from bacillus strains of the genus Mesentericus;
- al menos un cuarto extracto que contiene proteínas, péptidos y moléculas, obtenido a partir de cepas de bacilos del género Licheniformis ;- at least a fourth extract containing proteins, peptides and molecules, obtained from bacillus strains of the genus Licheniformis;
- al menos un quinto extracto que contiene proteínas, péptidos y moléculas, obtenido a partir de cepas de bacilos del género Lentus ;- at least one fifth extract containing proteins, peptides and molecules, obtained from bacillus strains of the genus Lentus;
- al menos un sexto extracto que contiene proteínas, péptidos y moléculas, obtenido a partir de cepas de bacilos del género Pumilus .- at least one sixth extract containing proteins, peptides and molecules, obtained from bacillus strains of the genus Pumilus.
Las cepas anteriormente mencionadas están seleccionadas entre cepas naturales y cepas modificadas.The aforementioned strains are selected from natural strains and modified strains.
Cuando se emplean cepas modificadas - las cepas de bacilos del primer grupo pueden ser cepas modificadas CECT FCM-1 4913;When modified strains are used - the bacillus strains of the first group may be CECT FCM-1 4913 modified strains;
- las cepas de bacilos del segundo grupo pueden ser cepas modificadas CECT FCM-2 4914;- Bacillus strains of the second group can be modified CECT FCM-2 4914 strains;
- las cepas de bacilos del tercer grupo pueden ser cepas modificadas CECT FCM-3 4915;- Bacillus strains of the third group can be modified CECT FCM-3 4915 strains;
- las cepas de bacilos del cuarto grupo pueden ser cepas modificadas CECT FCM-4 4916.- Bacillus strains of the fourth group can be modified CECT FCM-4 4916 strains.
Las cepas anteriormente mencionadas han sido depositadas de acuerdo con lo dispuesto en el Tratado de Budapest, en la Colección Española de Cultivos Tipo (CECT)The above-mentioned strains have been deposited in accordance with the provisions of the Budapest Treaty, in the Spanish Type Crops Collection (CECT)
HOJA DE SUSTITUCIÓN (REGLA 26) en el Departamento de Microbiología de la Facultad de Ciencias Biológicas de la Universidad de Valencia, con fecha del 25 de junio de 1997.SUBSTITUTE SHEET (RULE 26) in the Department of Microbiology of the Faculty of Biological Sciences of the University of Valencia, dated June 25, 1997.
Así, las cepas modificadas - B. Licheniformis , B. Pumilus B. Macerans,Thus, the modified strains - B. Licheniformis, B. Pumilus B. Macerans,
B .Amilolicuofaciens , han sido catalogadas con el numero de depósito CECT FCM-1 4913;B.Amilolicuofaciens, have been cataloged with the deposit number CECT FCM-1 4913;
- B. Cereus, B. Lentus han sido catalogadas con el número de depósito CECT FCM-2 4914; - B. Subtilis han sido catalogadas con el número de depósito CECT FCM-3 4915;- B. Cereus, B. Lentus have been cataloged with the deposit number CECT FCM-2 4914; - B. Subtilis have been cataloged with the deposit number CECT FCM-3 4915;
- B. Mesentericus ha sido catalogadas con el número de depósito CECT FCM-4 4916.- B. Mesentericus has been cataloged with the deposit number CECT FCM-4 4916.
El producto proteico típicamente tiene las siguientes características físicas:The protein product typically has the following physical characteristics:
Color amarillo pH 7,0 + 0,2 densidad aprox. 1 - 1,01 peso molecular 5.000 - 300.000 daltons La invención también tiene por objeto un procedimiento para la obtención de un producto proteico de acuerdo con las características anteriormente mencionadas, que básicamente comprende la siguientes etapas:Yellow color pH 7.0 + 0.2 density approx. 1 - 1.01 molecular weight 5,000 - 300,000 daltons The object of the invention is also a process for obtaining a protein product according to the aforementioned characteristics, which basically comprises the following steps:
Se selecciona al menos una cepa termorresistente y esporulada de al menos uno de los siguientes grupos :At least one heat-resistant and sporulated strain of at least one of the following groups is selected:
- un primer grupo compuesto por cepas de B . Licheniformis, B. Circulans 2, B. Pumilus, B. Macerans, B. Amilolicuofaciens , y cepas modificadas de las mismas; - un segundo grupo compuesto por cepas de B.- a first group consisting of strains of B. Licheniformis, B. Circulans 2, B. Pumilus, B. Macerans, B. Amilolicuofaciens, and modified strains thereof; - a second group consisting of strains of B.
Cereus 1 , B. Cereus 2 , B. Lentus 1, B. Lentus 2 y cepas modificadas de las mismas;Cereus 1, B. Cereus 2, B. Lentus 1, B. Lentus 2 and modified strains thereof;
- un tercer grupo compuesto por cepas de B . Subtilis y cepas modificadas de las mismas; - un cuarto grupo compuesto por cepas de B_;_- a third group consisting of strains of B. Subtilis and modified strains thereof; - a fourth group consisting of strains of B_ ; _
HOJA DE SUSTITUCIÓN (REGLA 26) Mesentericus y cepas modificadas de las mismas.SUBSTITUTE SHEET (RULE 26) Mesentericus and modified strains thereof.
Seguidamente, se añade una inoculación de dicha cepa a una disolución acuosa de un medio de cultivo, para obtener un medio de cultivo inoculado. En una realización del procedimiento, la disolución acuosa puede comprender entre 19 y 200g de un medio cultivo por cada litro de agua destilada. Preferentemente, la disolución acuosa se ajusta a un pH de 7.0+0,2 y se esteriliza en condiciones en sí convencionales, por ejemplo mediante esterilización en autoclave a 121°C + l^C durante 15 a 20 minutos .Next, an inoculation of said strain is added to an aqueous solution of a culture medium, to obtain an inoculated culture medium. In one embodiment of the process, the aqueous solution may comprise between 19 and 200g of a culture medium per liter of distilled water. Preferably, the aqueous solution is adjusted to a pH of 7.0 + 0.2 and sterilized under conventional conditions, for example by autoclaving at 121 ° C + 1 ^ C for 15 to 20 minutes.
La temperatura del medio de cultivo inoculado se ajusta en función de un crecimiento óptimo de la cepa. Preferentemente, la temperatura del medio de cultivo inoculado se ajusta a una temperatura entre 18 a 40°C y se aplica ventilación forzada por aire filtrado al medio de cultivo inoculado durante toda la fase de crecimiento de la cepa, sometiéndose el medio de cultivo inoculado a una presión de aire de 0,5 a 1 1/min. Se cultiva la cepa hasta alcanzar una densidad óptica máxima, obteniéndose un cultivo que contiene una masa bacilar y, seguidamente se obtiene un extracto proteico de la masa bacilar. Dicho extracto se obtiene mediante las etapas de - separación de la masa bacilar del cultivo hasta obtener bacilos en suspensión por ejemplo mediante concentración celular de la masa bacilar en el cultivo mediante centrifugación, ciclón u otros procedimientos en sí convencionales, preferentemente, antes de la concentración celular de la masa bacilar se separa y analiza una parte alícuota del cultivo antes de la concentración para obtener datos de "bacilos en suspensión" y también para obtener datos indicativos del grado de la concentración celular a efectos de poder controlar el crecimiento bacilar;The temperature of the inoculated culture medium is adjusted according to an optimal growth of the strain. Preferably, the temperature of the inoculated culture medium is adjusted to a temperature between 18 to 40 ° C and forced ventilation by filtered air is applied to the inoculated culture medium during the entire growth phase of the strain, the inoculated culture medium being subjected to an air pressure of 0.5 to 1 1 / min. The strain is grown to a maximum optical density, obtaining a culture containing a bacillary mass and then a protein extract of the bacillary mass is obtained. Said extract is obtained by the steps of separating the bacillary mass from the culture until obtaining bacilli in suspension, for example by cell concentration of the bacillary mass in the culture by centrifugation, cyclone or other conventional procedures, preferably before concentration. Bacillary mass cell separates and analyzes an aliquot part of the culture before concentration to obtain data of "bacilli in suspension" and also to obtain data indicative of the degree of cell concentration in order to control bacillary growth;
HOJA DE SUSTITUCIÓN (REGLA 26) - filtración cruzada sobre un filtro con un diámetro de poro inferior a 0,22 mieras hasta obtener un cultivo concentrado con un rango de concentración de 10 a 20%. Después, se somete el cultivo concentrado a diafiltración con intercambio de solución salina fisiológica. En una realización preferida del procedimiento el cultivo concentrado se somete a la diafiltración con un filtro de membrana con un tamaño de poro igual o inferior a 0,22 mieras.SUBSTITUTE SHEET (RULE 26) - cross filtration on a filter with a pore diameter of less than 0.22 microns until a concentrated culture with a concentration range of 10 to 20% is obtained. Then, the concentrated culture is subjected to diafiltration with physiological saline exchange. In a preferred embodiment of the process the concentrated culture is subjected to diafiltration with a membrane filter with a pore size equal to or less than 0.22 microns.
A continuación, se prepara un extracto que comprende proteínas, péptidos y moléculas que forman el principio activo mediante las fases deNext, an extract is prepared comprising proteins, peptides and molecules that form the active ingredient through the phases of
- someter el cultivo concentrado a lísis celular, mediante un proceso criotérmico que comprende un ciclo con una primera fase a temperaturas entre 2 y 8°C durante varias horas y una segunda fase de frío a una temperatura inferior a -40°C durante al menos 8 horas, preferentemente a - 40 y -50°C durante 8 a 12 horas, y una tercera fase en baño de agua a temperaturas entre temperatura ambiente y 70°C, preferentemente a una temperatura entre 60 y 70°C, y más preferentemente a unos 65°C, y, opcionalmente, una cuarta fase en la que se mantiene en reposo el descongelado durante al menos cinco minutos. Dicho ciclo se repite al menos dos veces, preferentemente 4 veces, hasta que se obtiene un concentrado de Usado celular que contiene un resto celular y el principio activo;- subject the concentrated culture to cell lysis, by a cryothermal process comprising a cycle with a first phase at temperatures between 2 and 8 ° C for several hours and a second cold phase at a temperature below -40 ° C for at least 8 hours, preferably at -40 and -50 ° C for 8 to 12 hours, and a third phase in a water bath at temperatures between room temperature and 70 ° C, preferably at a temperature between 60 and 70 ° C, and more preferably at about 65 ° C, and, optionally, a fourth phase in which defrosting is maintained at rest for at least five minutes. Said cycle is repeated at least twice, preferably 4 times, until a concentrate of Cellular Use containing a cellular moiety and the active ingredient is obtained;
- separar el resto celular del principio activo sometiendo el concentrado de Usado celular a filtración de flujo cruzado con diafiltración en una membrana de tamaño de poro inferior o igual a 0,22 mieras con un rango de dilución de 1:10, para obtener un filtrado de extracto proteico bruto que contiene un principio activo que comprende proteínas, péptidos y moléculas provenientes de- separating the cellular moiety from the active substance by subjecting the cell-use concentrate to cross-flow filtration with diafiltration in a pore size membrane less than or equal to 0.22 microns with a dilution range of 1:10, to obtain a filtrate of crude protein extract containing an active ingredient comprising proteins, peptides and molecules from
HOJA DE SUSTITUCIÓN (REGLA 26) la lísis celular;SUBSTITUTE SHEET (RULE 26) cell lysis;
- concentrar el filtrado utilizándose un filtro de tamaño de poro tal que las proteínas de peso molecular superior a 5000 daltons quedan retenidos por encima del filtro en una solución acuosa;- concentrate the filtrate using a pore size filter such that proteins of molecular weight greater than 5000 daltons are retained above the filter in an aqueous solution;
- filtración estéril del filtrado concentrado por un filtro de tamaño de poro igual o inferior a 0,22 mieras hasta obtener un primer extracto proteico esterilizado que contiene el principio activo. En una realización preferida del procedimiento según la invención, el primer extracto proteico esterilizado se mezcla con al menos un segundo extracto proteico preparado análogamente al primer extracto . Así, en una realización preferente del procedimiento, el primer extracto proteico esterilizado se obtiene a partir de al menos una cepa de dicho primer grupo y el segundo extracto proteico esterilizado se obtiene a partir de al menos un cepa de dicho segundo grupo. Según otra realización preferida del procedimiento, el primer extracto proteico esterilizado y el segundo extracto proteico esterilizado, se mezclan además con un tercer extracto proteico esterilizado obtenido a partir de al menos una cepa de dicho tercer grupo, habiéndose preparado dicho tercer extracto proteico esterilizado por procedimientos análogos a los del procedimiento de preparación del primer extracto.- sterile filtration of the filtrate concentrated by a pore size filter equal to or less than 0.22 microns until a first sterile protein extract containing the active ingredient is obtained. In a preferred embodiment of the process according to the invention, the first sterilized protein extract is mixed with at least a second protein extract prepared analogously to the first extract. Thus, in a preferred embodiment of the process, the first sterilized protein extract is obtained from at least one strain of said first group and the second sterilized protein extract is obtained from at least one strain of said second group. According to another preferred embodiment of the process, the first sterilized protein extract and the second sterilized protein extract are further mixed with a third sterilized protein extract obtained from at least one strain of said third group, said third protein extract sterilized by procedures analogous to those of the preparation procedure of the first extract.
En otra realización aún más preferida del procedimiento según la invención el primer extracto proteico esterilizado, el segundo extracto proteico esterilizado, y el tercer extracto proteico esterilizado, se mezclan además con un cuarto extracto proteico esterilizado obtenido a partir de al menos una cepa de dicho cuarto grupo, habiéndose preparado dicho cuarto extracto proteico esterilizado por procedimientos análogosIn another even more preferred embodiment of the process according to the invention, the first sterilized protein extract, the second sterilized protein extract, and the third sterilized protein extract are further mixed with a fourth sterilized protein extract obtained from at least one strain of said fourth group, said fourth sterile protein extract having been prepared by analogous procedures
HOJA DE SUSTITUCIÓN (REGLA 26) a los del procedimiento de preparación del primer extracto. En las realizaciones preferidas anteriormente descritas, preferentemente el primer extracto proteico esterilizado se obtiene a partir de cepas CECT FCM-1 4913, el segundo extracto proteico esterilizado se obtiene a partir de cepas CECT FCM-2 4914, el tercer extracto proteico esterilizado se obtiene a partir de cepas CECT FCM-3 4915, y el cuarto extracto proteico esterilizado se obtiene a partir de cepas CECT FCM-4 4916. . Para obtener un fármaco "multiuso" es decir, con efectos beneficiosos para prevenir y/o tratar una pluralidad de los síndromes más arriba descritos, los extractos proteicos esterilizados se mezclan, en proporciones iguales, por ejemplo 1:1 cuando se mezclan dos extractos, 1:1:1 cuando se mezclan tres extractos, y 1:1:1:1 cuando se mezclan cuatro extractos, en base a sus respectivas concentraciones de proteínas. Claro está que, cuando se desea potenciar los efectos para sólo una o algunas de las aplicaciones farmacéuticas más arriba descritas, pueden mezclarse los distintos extractos en proporciones desiguales, o puede emplearse sólo uno de los extractos .SUBSTITUTE SHEET (RULE 26) to those of the preparation procedure of the first extract. In the preferred embodiments described above, preferably the first sterilized protein extract is obtained from CECT FCM-1 4913 strains, the second sterilized protein extract is obtained from CECT FCM-2 4914 strains, the third sterilized protein extract is obtained at from CECT FCM-3 4915 strains, and the fourth sterilized protein extract is obtained from CECT FCM-4 4916 strains. In order to obtain a "multipurpose" drug that is, with beneficial effects to prevent and / or treat a plurality of the syndromes described above, the sterilized protein extracts are mixed, in equal proportions, for example 1: 1 when two extracts are mixed, 1: 1: 1 when three extracts are mixed, and 1: 1: 1: 1 when four extracts are mixed, based on their respective protein concentrations. Of course, when it is desired to enhance the effects for only one or some of the pharmaceutical applications described above, the different extracts may be mixed in unequal proportions, or only one of the extracts may be used.
La invención también se refiere a composiciones que contienen el producto proteico, compuesto por uno o más de los extractos proteicos antes citados, como ingrediente activo, con un vehículo farmacéuticamente aceptable .The invention also relates to compositions containing the protein product, consisting of one or more of the aforementioned protein extracts, as active ingredient, with a pharmaceutically acceptable carrier.
Ejemplos de tales composiciones farmacéuticas son composiciones sólidas, tales como tabletas, pildoras, cápsulas, granulos, o líquidos, tales como disoluciones liofilizados, suspensiones y emulsiones, para administración oral, tópica, parenteral u otras vías de administración. Las dosis de estas composiciones dependen de su formulación, el método de administración así como el lugar de la administración, el individuo a tratar. OtrosExamples of such pharmaceutical compositions are solid compositions, such as tablets, pills, capsules, granules, or liquids, such as lyophilized solutions, suspensions and emulsions, for oral, topical, parenteral administration or other routes of administration. The doses of these compositions depend on their formulation, the method of administration as well as the place of administration, the individual to be treated. Others
HOJA DE SUSTITUCIÓN (REGLA 26) factores tales como la edad, el peso corporal, sexo, dieta, duración de la administración, velocidad de excreción, condición del individuo tratado, combinación con otros fármacos, sensibilidades del individuo y el grado de severidad de la enfermedad también deben tenerse en cuenta. La administración puede realizarse continuamente o periódicamente dentro de las dosis máxima toleradas.SUBSTITUTE SHEET (RULE 26) Factors such as age, body weight, sex, diet, duration of administration, excretion rate, condition of the treated individual, combination with other drugs, sensitivities of the individual and the degree of severity of the disease should also be taken into account. Administration can be carried out continuously or periodically within the maximum tolerated doses.
Cuando la composición según la invención es una disolución inyectable, el medio líquido es, por ejemplo, agua destilada, quedando formulada la disolución de la siguiente manera:When the composition according to the invention is an injectable solution, the liquid medium is, for example, distilled water, the solution being formulated as follows:
1 a 99% p/v del producto proteico1 to 99% w / v protein product
0 a 99% v/v de agua destilada0 to 99% v / v distilled water
0,02% v/v de fenol 0-0,12% v/v de formol de grado farmacéutico al 37%.0.02% v / v phenol 0-0.12% v / v formol of 37% pharmaceutical grade.
Realizaciones de tales disoluciones y de su preparación se especifican en los ejemplos.Embodiments of such solutions and their preparation are specified in the examples.
La presente invención tiene especial aplicación en el tratamiento de pacientes HIV positivos. Hoy en día, se considera que el efecto inmunosupresor del HIV puede atribuirse a la destrucción de los linfocitos CD4 que se considera que juegan un papel muy relevante en la inmunorespuesta del individuo. Existen indicios para pensar que los productos proteicos de la presente invención estimulan la proliferación de los linfocitos CD4.The present invention has special application in the treatment of HIV positive patients. Today, it is considered that the immunosuppressive effect of HIV can be attributed to the destruction of CD4 lymphocytes that are considered to play a very important role in the individual's immune response. There is evidence to believe that the protein products of the present invention stimulate the proliferation of CD4 lymphocytes.
Dosis viables para el tratamiento de pacientes con SIDA, son por ejemplo 1,0 o, preferentemente 2,0 unidades en 2 mi de agua para inyecciones, administradas cada dos días intra uscularmente.Viable doses for the treatment of patients with AIDS, for example, are 1.0 or, preferably, 2.0 units in 2 ml of water for injections, administered every two days intra uscularly.
Se ha podido comprobar que en pacientes afectados por el SIDA a los que se les inyectaron intramuscularmente dosis de 2,0 unidades de un producto proteico según la invención, experimentaron incrementos en linfocitos CD4 (y también de linfocitos CD8), sin que seIt has been found that in patients affected by AIDS who were injected intramuscularly with doses of 2.0 units of a protein product according to the invention, experienced increases in CD4 lymphocytes (and also CD8 lymphocytes), without
HOJA DE SUSTITUCIÓN (REGLA 26) produjeran efectos secundarios nocivos de relevancia. BREVE DESCRIPCIÓN DE LAS FIGURASSUBSTITUTE SHEET (RULE 26) produce harmful side effects of relevance. BRIEF DESCRIPTION OF THE FIGURES
Para ilustrar algunos aspectos de la invención, en la descripción detallada de la invención se hará referencia a unas figuras que se anexan, en las que la figura 1 muestra los resultados de un análisis por electroforesis en gel de poliacrilamida (PAGE), de un producto proteico de acuerdo con la invención; la figura 2 muestra los resultados de unos análisis Western Blot al que fueron sometidos muestras del producto proteico según la invención; la figura 3 resume los resultados de unos ensayos relativos a la respuesta in vivo de anticuerpos dependientes de células T; la figura 4 resume los resultados de unos primeros ensayos relativos a la activación in vitro de células B; la figura 5 es el control de los primeros ensayos relativos a la activación in vitro de células B en presencia de lipopolisacáridos (LPS); la figura 6 resume los resultados de unos ensayos relativos a la activación in vitro de células T; las figuras 7 a 11 resumen los resultados de varios ensayos relativos a la blastogénesis de células T.To illustrate some aspects of the invention, in the detailed description of the invention reference will be made to annexed figures, in which Figure 1 shows the results of a polyacrylamide gel electrophoresis analysis (PAGE) of a product protein according to the invention; Figure 2 shows the results of Western Blot analysis to which samples of the protein product according to the invention were subjected; Figure 3 summarizes the results of assays related to the in vivo response of antibodies dependent on T cells; Figure 4 summarizes the results of the first tests related to in vitro activation of B cells; Figure 5 is the control of the first assays related to in vitro activation of B cells in the presence of lipopolysaccharides (LPS); Figure 6 summarizes the results of tests related to in vitro activation of T cells; Figures 7 to 11 summarize the results of several assays related to blastogenesis of T cells.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓNDETAILED DESCRIPTION OF THE INVENTION
A continuación se ilustrará la invención en base a algunos ejemplos de carácter no-limitativo. Ejemplo 1: Materiales y equiposThe invention will now be illustrated based on some examples of a non-limiting nature. Example 1: Materials and equipment
Para obtener un producto proteico según la invención, se seleccionaron las siguientes cepas de bacilos:To obtain a protein product according to the invention, the following bacillus strains were selected:
* B . Licheniformis CECT FCM-1 4913* B. Licheniformis CECT FCM-1 4913
* B . Pumilus CECT FCM-1 4913 * B . Ciculans CECT-FCM-1 4913* B. Pumilus CECT FCM-1 4913 * B. Ciculans CECT-FCM-1 4913
HOJA DE SUSTITUCIÓN (REGLA 26) * B. Amilolicuofaciens CECT FCM-1 4913SUBSTITUTE SHEET (RULE 26) * B. Amilolicuofaciens CECT FCM-1 4913
* B. Lentus CECT FCM-2 4914* B. Lentus CECT FCM-2 4914
* B. Cereus CECT FCM-2 4914* B. Cereus CECT FCM-2 4914
* B. Subtilis CECT FCM-3 4915 * B. Mesentericus CECT FCM-4 4916 y se insertaron en cuatro viales distintos, a saber un primer vial con las DOS cepas CECT FCM-1 4913, un segundo vial con las dos cepas CECT FCM-2-4914, un tercer vial con la cepa CECT FCM-3 4915, y un cuarto vial con las cepas CECT FCM-4 4916.* B. Subtilis CECT FCM-3 4915 * B. Mesentericus CECT FCM-4 4916 and inserted into four different vials, namely a first vial with the two CECT FCM-1 4913 strains, a second vial with the two CECT FCM strains -2-4914, a third vial with CECT FCM-3 4915 strain, and a fourth vial with CECT FCM-4 4916 strains.
Se dispusieron los siguientes equipos y materiales: una incubadora bacteriológica convencional con dispositivo de agitación y regulación de temperatura (36+l°C);The following equipment and materials were arranged: a conventional bacteriological incubator with agitation device and temperature regulation (36 + l ° C);
- recipientes térmicos de baño de agua; un equipo convencional PROSTAK de filtración de flujo cruzado con capacidades de filtración de 40 a 8001; - un equipo convencional de filtración unidireccional con capacidad de filtración de volúmenes hasta 100 1;- thermal water bath containers; a conventional PROSTAK cross flow filtration equipment with filtration capacities from 40 to 8001; - a conventional unidirectional filtration device with a capacity to filter volumes up to 100 1;
- fermentadores convencionales para el cultivo de microorganismos con capacidades de fermentación para 8,5 1 y 800 1; un equipo refrigerador para temperaturas de 4 a 8°C;- conventional fermenters for the cultivation of microorganisms with fermentation capacities for 8.5 1 and 800 1; a refrigerating unit for temperatures from 4 to 8 ° C;
- un equipo congelador para temperaturas de - 70QC; - matraces para la agitación de cultivos con capacidades de 2 a 4 1;- a freezer for temperatures of - 70 Q C; - flasks for the agitation of crops with capacities from 2 to 4 1;
- recipientes de cristal de boca ancha con capacidades de 5 a 20 1;- wide mouth glass containers with capacities from 5 to 20 1;
- membranas de PVDF de 0,22 mieras de tamaño de poro;- PVDF membranes 0.22 microns in pore size;
HOJA DE SUSTITUCIÓN (REGLA 26) - contenedores de acero inoxidable con capacidad de 50 1, 100 1 y 200 1; medio de cultivo CM-P, comercializado por INQUIAROMA; - agua para inyección (Farmacopea Europea- FE)SUBSTITUTE SHEET (RULE 26) - stainless steel containers with a capacity of 50 1, 100 1 and 200 1; CM-P culture medium, marketed by INQUIAROMA; - water for injection (European Pharmacopoeia - FE)
- NaCl de calidad bacteriológica (FE);- Bacteriological quality NaCl (FE);
- fenol ultrapuro (FE);- ultrapure phenol (FE);
- solución acuosa de formaldehido de grado farmacéutico al 37% (FE); - agente antiespumante STRUKTOL™ J 660.- 37% aqueous pharmaceutical grade formaldehyde solution (FE); - STRUKTOL ™ J 660 antifoaming agent.
Propagación primaria de las cepas (=fase PPC)Primary propagation of strains (= PPC phase)
Se prepararon 2 litros de una primera solución de cultivo, disolviéndose 35 g del medio de cultivo CM-P por cada litro de agua, y se llenaron cuatro matraces con 500 mi de solución de cultivo cada uno. Cada una de las disoluciones se esterilizó a 121°C durante 15 min sin necesidad de ajustar el pH.2 liters of a first culture solution were prepared, 35 g of the CM-P culture medium being dissolved per liter of water, and four flasks were filled with 500 ml of culture solution each. Each of the solutions was sterilized at 121 ° C for 15 min without the need to adjust the pH.
En la primera solución de cultivo se inocularon las cepas CECT FCM-1 4913, en la segunda solución las cepas CECT FCM-2 4914, en la tercera solución de cultivo la cepa CECT FCM-3 4915 y en la cuarta solución la cepa CECT FCM-4 4916. Se introdujeron los matraces recipientes con las disoluciones de cultivo inoculadas en una incubadora y se realizaron las incubaciones a 36+l°C y agitación constante a 100 rp hasta que se obtuvo una suspensión bacilar con una densidad óptica a 620 nm de:In the first culture solution the CECT FCM-1 4913 strains were inoculated, in the second solution the CECT FCM-2 4914 strains, in the third culture solution the CECT FCM-3 4915 strain and in the fourth solution the CECT FCM strain -4 4916. The flasks were introduced with the culture solutions inoculated in an incubator and the incubations were carried out at 36 + 1 ° C and constant stirring at 100 rp until a bacillary suspension with an optical density at 620 nm of :
Grupo de Cepas OD620nm tiempo (h)*Strain Group OD 620nm time (h) *
CECT FCM-1 4913 4 10CECT FCM-1 4913 4 10
CECT FCM-2 4914 8 15CECT FCM-2 4914 8 15
CECT FCM-3 4915 7 11CECT FCM-3 4915 7 11
CECT FCM-4 4916 4 12CECT FCM-4 4916 4 12
* = tiempo de finalización cultivo* = crop completion time
HOJA DE SUSTITUCIÓN (REGLA 26) Se extrajeron los matraces y se etiquetó cada uno de los matraces con la sigla PPC seguido de la fecha de preparación y el nombre de la cepa inoculada. Propagación secundaria de las cepas (=fase PSC) Se prepararon 32 litros de una segunda solución de cultivo disolviéndose 35 g de medio de cultivo CM-P por cada litro de agua destilada, ajustándose el pH a 7+0.2 con H3PO4. La segunda solución se vertió en iguales proporciones en cada uno de los 4 fermentadores con capacidad de 8,5 1. Se adicionó 1 mi de STRUKTOL™ J 660 a cada uno de los fermentadores .SUBSTITUTE SHEET (RULE 26) Flasks were removed and each of the flasks was labeled with the acronym PPC followed by the date of preparation and the name of the inoculated strain. Secondary propagation of the strains (= PSC phase) 32 liters of a second culture solution were prepared by dissolving 35 g of CM-P culture medium per liter of distilled water, adjusting the pH to 7 + 0.2 with H 3 PO 4 . The second solution was poured in equal proportions into each of the 4 fermenters with a capacity of 8.5 1. 1 ml of STRUKTOL ™ J 660 was added to each of the fermenters.
La esterilización de la segunda solución de cultivo se realizó durante 15 in a una temperatura de 1212C. Cada una de las suspensiones bacilares obtenidas en la fase PPS se transfirió respectivamente a uno de los cuatro fermentadores con la segunda solución de cultivo esterilizada, de tal manera queSterilization of the second culture solution was carried out for 15 in at a temperature of 1212C. Each of the bacillary suspensions obtained in the PPS phase was transferred respectively to one of the four fermenters with the second sterilized culture solution, such that
- el primer fer entador contenía un cultivo con 8 1 de la segunda solución de cultivo y 500 mi de suspensión bacilar CECT FCM-1 4913;- the first ferrer contained a culture with 8 1 of the second culture solution and 500 ml of CECT FCM-1 4913 bacillary suspension;
- el segundo fermentador contenía un cultivo con 8 1 de la segunda solución de cultivo y 500 mi de suspensión bacilar CECT FCM-2 4914; - el tercer fermentador contenía un cultivo con- the second fermenter contained a culture with 8 1 of the second culture solution and 500 ml of CECT FCM-2 4914 bacillary suspension; - the third fermenter contained a culture with
8 1 de la segunda solución de cultivo y 500 mi de suspensión bacilar CECT FCM-3 4915;8 1 of the second culture solution and 500 ml of CECT FCM-3 4915 bacillary suspension;
- el cuarto fermentador contenía un cultivo con 8 1 de la segunda solución de cultivo y 500 mi de suspensión bacilar CECT FCM-4 4916.- the fourth fermenter contained a culture with 8 1 of the second culture solution and 500 ml of CECT FCM-4 4916 bacillary suspension.
Los cultivos se realizaron en sus respectivos fermentadores a una temperatura de 36+l°C, una agitación del medio de cultivo de 500+100rpm, con un flujo de aire de 0,8 1/min, y oxígeno disuelto inicial igual a 98+2% (durante el cultivo >0%), hasta alcanzar una densidadThe cultures were carried out in their respective fermenters at a temperature of 36 + l ° C, a stirring of the culture medium of 500 + 100rpm, with an air flow of 0.8 1 / min, and initial dissolved oxygen equal to 98+ 2% (during cultivation> 0%), until reaching a density
HOJA DE SUSTITUCIÓN (REGLA 26) óptica a 620 n de:SUBSTITUTE SHEET (RULE 26) 620 n optics from:
Grupo de Cepas OD620nm tiempo (h)*Strain Group OD 620nm time (h) *
CECT FCM-1 4913 4,4 6CECT FCM-1 4913 4.4 6
CECT FCM-2 4914 11 10CECT FCM-2 4914 11 10
CECT FCM-3 4915 8,9 9CECT FCM-3 4915 8.9 9
CECT FCM-4 4916 5,4 8CECT FCM-4 4916 5.4 8
* = tiempo de finalización cultivo* = crop completion time
Fermentación principal (fase MF)Main fermentation (MF phase)
En un fermentador se prepararon 800 litros de una tercera solución de cultivo disolviéndose 35g de medio de cultivo CM-P por cada litro de agua, añadiéndose 100 mi de Struktol™ J660, ajustándose el pH a 7+0,2 con H3P04 2M, y esterilizándose a 121°C durante 15 minutos. Se inoculó la tercera solución con la segunda solución de cultivo y 8,5 L de suspensión bacilar CECT FCM-1 4913 obtenida en la fase SPS, y se procedió a la fermentación. Posterior y sucesivamente, se prepararon, inocularon y fermentaron de forma análoga sendos lotes de terceras soluciones con las cepas CECT FCM-2 4914, CECT FCM-3 4915 y CECT FCM-4 4916, de manera que se fermentaronIn a fermenter 800 liters of a third culture solution were prepared by dissolving 35g of CM-P culture medium per liter of water, adding 100 ml of Struktol ™ J660, adjusting the pH to 7 + 0.2 with H 3 P0 4 2M, and sterilizing at 121 ° C for 15 minutes. The third solution was inoculated with the second culture solution and 8.5 L of CECT FCM-1 4913 bacillary suspension obtained in the SPS phase, and the fermentation proceeded. Subsequently and successively, batches of third solutions with strains CECT FCM-2 4914, CECT FCM-3 4915 and CECT FCM-4 4916 were prepared, inoculated and similarly fermented, so that they were fermented
8,5 1 de cultivo SPS con cepas CECT FCM-18.5 1 of SPS culture with CECT FCM-1 strains
4913 en 800 1 de la tercera solución de cultivo; - 8,5 1 de cultivo SPS con cepas CECT FCM-24913 in 800 1 of the third culture solution; - 8.5 1 of SPS culture with CECT FCM-2 strains
4914 en 800 1 de la tercera solución de cultivo;4914 in 800 1 of the third culture solution;
8,5 1 de cultivo SPS con cepas CECT FCM-38.5 1 SPS culture with CECT FCM-3 strains
4915 en 800 1 de la tercera solución de cultivo;4915 in 800 1 of the third culture solution;
8,5 1 de cultivo SPS con cepas CECT FCM-4 4916 en 800 1 de la tercera solución de cultivo.8.5 1 of SPS culture with CECT FCM-4 4916 strains in 800 1 of the third culture solution.
Cada uno de los cultivos anteriormente mencionados se fermentaron a 36+l°C, con agitación a 300 rp , flujo de aire estéril de 0,8/1 min, pH 7+0,2 y oxígeno disuelto inicial 98+2% (durante el cultivo >0%), hasta que se alcanzó una fase de crecimiento estacionario en cadaEach of the above-mentioned cultures were fermented at 36 + 1 ° C, with agitation at 300 rp, sterile air flow of 0.8 / 1 min, pH 7 + 0.2 and initial dissolved oxygen 98 + 2% (during crop> 0%), until a stationary growth phase was reached in each
HOJA DE SUSTITUCIÓN (REGLA 26) cultivo en una densidad óptica a 620 nm deSUBSTITUTE SHEET (RULE 26) culture at an optical density at 620 nm of
Grupo de Cepas OD620nm tiempo (h)*Strain Group OD 620nm time (h) *
CECT FCM-1 4913 5 10CECT FCM-1 4913 5 10
CECT FCM-2 4914 27 14CECT FCM-2 4914 27 14
CECT FCM-3 4915 27 13CECT FCM-3 4915 27 13
CECT FCM-4 4916 6 12CECT FCM-4 4916 6 12
* = tiempo de finalización cultivo* = crop completion time
CosechaHarvest
Los cuatro cultivos obtenidos mediante la fase MF se transfirieron en lotes a diversos contenedores de 50 1, etiquetándose cada uno de ellos con las siglas FP, su fecha de preparación y nombre de la cepa cultivada.The four cultures obtained through the MF phase were transferred in batches to various 50 1 containers, each labeling with the initials FP, its preparation date and name of the cultivated strain.
Preparación de una suspensión celular concentrada (fase SCOPreparation of a concentrated cell suspension (SCO phase
Se filtró cada uno de los cultivos cosechados en flujo cruzado en el equipo de filtración PROSTAK con una membrana PVDF de 0,22 mieras, hasta que se obtuvieron sendos cultivos 20 veces más concentrados que los respectivos cultivos cosechados. Posteriormente, cada uno de los cultivos concentrados se sometió a diafiltración con intercambio de solución salina fisiológica en equipo PROSTAK con filtro de membrana de 0,22 mieras con 150 1 de solución salina fisiológica, 10 1 de cada una de las suspensiones celulares concentradas se transfirieron a contenedores con capacidad de 20 1, etiquetándose cada contenedor con las siglas CCS, su fecha de preparación y nombre de la cepa cultivada.Each of the cultures harvested in cross flow was filtered in the PROSTAK filtration equipment with a PVDF membrane of 0.22 microns, until cultures were obtained 20 times more concentrated than the respective crops harvested. Subsequently, each of the concentrated cultures was subjected to diafiltration with exchange of physiological saline in PROSTAK equipment with 0.22 micron membrane filter with 150 1 of physiological saline, 10 1 of each of the concentrated cell suspensions were transferred to containers with a capacity of 20 1, labeling each container with the acronym CCS, its date of preparation and name of the cultivated strain.
HOJA DE SUSTITUCIÓN (REGLA 26) Obtención de suspensiones de células usadas por congelación/calentamientoSUBSTITUTE SHEET (RULE 26) Obtaining cell suspensions used by freezing / heating
Cada grupo de contenedores con suspensiones celulares concentradas resultantes de la etapa SCC con la misma cepa, se sometió a un proceso criotérmico repitiéndose cuatro veces el siguiente ciclo: refrigerar a 4°C durante 3 horas en un equipo refrigerador;Each group of containers with concentrated cell suspensions resulting from the SCC stage with the same strain, underwent a cryothermal process repeating the following cycle four times: refrigerate at 4 ° C for 3 hours in a cooling equipment;
- congelar a -40°C durante 18 horas en un equipo congelador;- freeze at -40 ° C for 18 hours in a freezer;
- descongelar poniéndose en un equipo de calentamiento con agua caliente a 65°C hasta la total descongelación .- defrost by placing in a heating equipment with hot water at 65 ° C until the total defrosting.
Al final de cada ciclo se realizó una cuenta de UFC y contenido total de proteínas (Kjeldahl) en cada una de las suspensiones de células lisadas, obteniéndose los siguientes valores:At the end of each cycle, a CFU and total protein content (Kjeldahl) count was performed on each of the lysed cell suspensions, obtaining the following values:
Ciclo cepa contenida CFU/ml proteína total (g/1) antes ciclo CECT FCM-1 4913 1x10 8 11,1 1 <104 11,8 2 2xlOe 10,5 3 2xlOf 10,5 4 2xl0( 10,5Cycle strain contained CFU / ml total protein (g / 1) before CECT cycle FCM-1 4913 1x10 8 11.1 1 <10 4 11.8 2 2xlO e 10.5 3 2xlO f 10.5 4 2xl0 ( 10.5
Ciclo cepa contenida CFU/ml proteína totalCycle strain contained CFU / ml total protein
antes ciclo CECT FCM-2 4914 5xl09 9,4 1 lxlO8 20,5before CECT cycle FCM-2 4914 5xl0 9 9.4 1 lxlO 8 20.5
2 5xl06 18,32 5xl0 6 18.3
3 lxlO4 18,83 lxlO 4 18.8
4 2xl04 20,84 2xl0 4 20.8
HOJA DE SUSTITUCIÓN (REGLA 26) Ciclo cepa contenida CFU/ml proteína totalSUBSTITUTE SHEET (RULE 26) Cycle strain contained CFU / ml total protein
antes ciclo CECT FCM-3 4915 3x10 10 14,7before CECT cycle FCM-3 4915 3x10 10 14.7
1 1x10* 16,81 1x10 * 16.8
2 1x10' 17,02 1x10 '17.0
3 lxlθ 17,03 lxlθ 17.0
4 1x10 17,04 1x10 17.0
Ciclo cepa contenida CFU/ml proteína totalCycle strain contained CFU / ml total protein
antes ciclo CECT FCM-4 4916 6x10' 8,3before CECT cycle FCM-4 4916 6x10 '8.3
1 2xl06 8,61 2xl0 6 8.6
2 2xl06 9,72 2xl0 6 9.7
3 2xl06 9,1 4 lxlO6 9,63 2xl0 6 9.1 4 lxlO 6 9.6
Obtención de extractos proteicos con el ingreidente activo libre de detritus celular (fase IA)Obtaining protein extracts with the active ingredient free of cell detritus (phase IA)
(a) Obtención de extractos proteicos filtrados (fase IAF)(a) Obtaining filtered protein extracts (IAF phase)
Se sometieron las suspensiones de células Usadas, agrupados respectivamente según las cepas de origen, resultantes del proceso criotérmico a filtración cruzada en equipo PROSTAK con filtro de membrana de 0,22 mieras.The suspensions of Used cells were subjected, grouped respectively according to the strains of origin, resulting from the cryothermal process to cross filtration in PROSTAK equipment with 0.22 micron membrane filter.
Para conocer la cantidad de detritus celulares, se tomaron sendas muestras al final de cada proceso de filtración cruzada y se determinaron los respectivos valores D062o antes y después de centrifugar a 4.000 rp durante 15 minutos. La diferencia de absorbancia era en cada muestra menor a 0,1 y, por tanto, los extractos proteicos filtrados tenían calidades aceptables. Cada uno de los extractos resultantes de la fase IAF se etiquetó con las siglas IAF, su fecha de preparación, y el nombre de la cepa de origen.To determine the amount of cell detritus, samples were taken at the end of each cross-filtration process and the respective D0 62 values were determined or before and after centrifugation at 4,000 rp for 15 minutes. The absorbance difference in each sample was less than 0.1 and, therefore, the filtered protein extracts had acceptable qualities. Each of the extracts resulting from the IAF phase was labeled with the acronym IAF, its date of preparation, and the name of the strain of origin.
HOJA DE SUSTITUCIÓN (REGLA 26) (b) Diafiltración con intercambio de solución salina fisiológica (IAP)SUBSTITUTE SHEET (RULE 26) (b) Diafiltration with physiological saline exchange (IAP)
Cada uno de los extractos proteicos resultantes de la fase IAP se sometió a diafiltración en equipo PROSTAK con filtro de membrana de 0,22 mieras con un volumen de solución salina fisiológica (0,9%m/v) diez veces mayor que el volumen de los extractos proteicos filtrados, controlándose la diafiltración por análisis del contenido total de proteínas (Kjeldahl) en el filtrado, hasta alcanzarse un contenido de proteínas óptimo de:Each of the protein extracts resulting from the IAP phase was subjected to diafiltration in PROSTAK equipment with a 0.22 micron membrane filter with a volume of physiological saline solution (0.9% m / v) ten times greater than the volume of the filtered protein extracts, diafiltration being controlled by analysis of the total protein content (Kjeldahl) in the filtrate, until an optimal protein content of:
Cepa contenida Proteína total (g/1)Strain contained Total protein (g / 1)
CECT FCM-1 4913 1,3CECT FCM-1 4913 1.3
CECT FCM-2 4914 5,9 CECT FCM-3 4915 6,9CECT FCM-2 4914 5.9 CECT FCM-3 4915 6.9
CECT FCM-4 4916 2,5CECT FCM-4 4916 2.5
Cada uno de los extractos resultantes de la fase IAP (aproximadamente 40 1 por cada extracto) se introdujo en un contenedor que se etiquetó con las siglasEach of the extracts resulting from the IAP phase (approximately 40 1 per extract) was placed in a container that was labeled with the acronym
IAP, su fecha de preparación, y el nombre de la cepa de origen.IAP, its date of preparation, and the name of the strain of origin.
Obtención del ingrediente activo concentrado (fase IAC) Cada uno de los extractos resultantes de la fase PAI se sometió a concentración mediante un equipo de filtración a flujo cruzado en un equipo PROSTAK con filtro de membrana de 5000 daltons de diámetro nominal de poro (Pellicon, Biomax 5k, Polietersulfon) para obtener una concentración 1:2, es decir un volumen de aproximadamente 20 1 de cada ingrediente activo resultante de la fase IAC .Obtaining the concentrated active ingredient (IAC phase) Each of the extracts resulting from the PAI phase was subjected to concentration by means of a cross-flow filtration equipment in a PROSTAK unit with a membrane filter of 5000 daltons of nominal pore diameter (Pellicon, Biomax 5k, Polyethersulfon) to obtain a 1: 2 concentration, ie a volume of approximately 20 1 of each active ingredient resulting from the IAC phase.
De cada ingrediente activo IAC se tomaron muestras para determinar su contenido en proteínasSamples were taken from each active ingredient IAC to determine their protein content
(Kjeldahl). El análisis de estas muestras se realiza para tener un control para comprobar que el procedimiento se(Kjeldahl). The analysis of these samples is performed to have a check to verify that the procedure is
HOJA DE SUSTITUCIÓN (REGLA 26) está realizando correctamente y, por tanto, es básicamente análogo a la comprobación en la fase IAP.SUBSTITUTE SHEET (RULE 26) It is performing correctly and, therefore, is basically analogous to checking in the IAP phase.
Los ingredientes activos concentrados resultantes de la fase IAC se introdujeron en sendos contenedores etiquetados con las siglas IAC, la fecha de preparación, y el nombre de la cepa de origen.The concentrated active ingredients resulting from the IAC phase were introduced in separate containers labeled with the acronym IAC, the date of preparation, and the name of the strain of origin.
Obtención del principio activo estéril (fase IAE)Obtaining the sterile active substance (IAE phase)
Cada uno de los ingrediente activos concentrados resultantes de la fase IAC se sometió a filtración en un equipo unidireccional con membrana PVDF de 0,22 mieras de poro. De cada principio esterilizado se tomó una muestra para ensayo de esterilidad después de la filtración. El criterio de aceptación es que la retención de microorganismos en el filtro sea menor de 10 /cm . En el presente caso, se obtuvieron los siguientes valores:Each of the concentrated active ingredients resulting from the IAC phase was subjected to filtration in a unidirectional device with a 0.22 micron pore PVDF membrane. From each sterilized principle a sample was taken for sterility testing after filtration. The acceptance criterion is that the retention of microorganisms in the filter is less than 10 / cm. In the present case, the following values were obtained:
Cepa contenida Antes de la Retención Después de la filtración filtro filtración
Figure imgf000023_0001
Strain contained Before Retention After filtration filtration filter
Figure imgf000023_0001
CECT FCM-1 4913 <10 <110 estérilCECT FCM-1 4913 <10 <110 sterile
CECT FCM-2 4914 40 7xl02 estérilCECT FCM-2 4914 40 7xl0 2 sterile
CECT FCM-3 4915 60 4xl02 estérilCECT FCM-3 4915 60 4xl0 2 sterile
CECT FCM-4 4916 <10 <110 estérilCECT FCM-4 4916 <10 <110 sterile
También se analizó la concentración de proteínas (Kjeldahl) para llevar un control del proceso y poder comprobar que éste se ha realizado correctamente. Para ello, se coge una muestra de la solución y se mide la concentración de proteina. Este paso es análogo a la comprobación en la fase IAP.The protein concentration (Kjeldahl) was also analyzed to keep track of the process and to verify that it was done correctly. For this, a sample of the solution is taken and the protein concentration is measured. This step is analogous to checking in the IAP phase.
Los principios activos resultantes se introdujeron en envase Nalgene etiquetados con las siglas IAE, la fecha de preparación, y el nombre de la cepa de origen, y se almacenaron a 4°C en un refrigerador.The resulting active ingredients were placed in a Nalgene package labeled with the acronym IAE, the date of preparation, and the name of the source strain, and stored at 4 ° C in a refrigerator.
HOJA DE SUSTITUCIÓN (REGLA 26) Preparación del producto proteico a granel (fase G)SUBSTITUTE SHEET (RULE 26) Preparation of the bulk protein product (phase G)
En recipiente adecuado, se dispusieron 100 1 de agua para inyección y se añadieron, bajo agitación constante, fenol a una concentración de 0,2% (m/v), con respecto a un volumen de 200 1, solución de formaldehido a una concentración de 0,02% (v/v), también con respecto al volumen de 200 1, y principios activos esterilizados provenientes de la fase IAE, en una relación de IAE CECT FCM-1 4913 : IAE CECT FCM-2 4914 : IAE CECT FCM-3 4915 : IAE CECT FCM-4 4916, de 1:1:1:1, con referencia a sus respectivas concentraciones de proteínas. A la mezcla así obtenida, se añadió agua para inyección hasta completar un volumen de aproximadamente 200 1 y se mantuvo en agitación la mezcla resultante durante 30 minutos, obteniéndose el producto a granel en bruto.In a suitable container, 100 1 of water for injection were placed and phenol at a concentration of 0.2% (m / v) was added, under constant stirring, with respect to a volume of 200 1, formaldehyde solution at a concentration 0.02% (v / v), also with respect to the volume of 200 1, and sterilized active ingredients from the IAE phase, in a ratio of IAE CECT FCM-1 4913: IAE CECT FCM-2 4914: IAE CECT FCM-3 4915: IAE CECT FCM-4 4916, 1: 1: 1: 1, with reference to their respective protein concentrations. To the mixture thus obtained, water for injection was added until a volume of approximately 200 1 was completed and the resulting mixture was kept under stirring for 30 minutes, the crude bulk product being obtained.
El producto a granel, se sometió a filtración estéril empleándose un equipo de filtración unidireccional con membrana acoplada PVDF, de 0,22 mieras, obteniéndose un producto a granel estéril. Se sometió una muestra a pruebas de esterilidad y de concentración de proteínas (Kjeldahl) siguiéndose las sistemáticas y criterios seguidos en las pruebas de esterilidad y análisis de la proteína de las etapas anteriores . El producto a granel estéril se envasó en un contenedor de Nalgene etiquetados con la sigla G, la fecha de preparación, y los nombres de la cepas de origen, y se almacenó a 4°C en un refrigerado.The bulk product was subjected to sterile filtration using a unidirectional filtration equipment with coupled PVDF membrane of 0.22 microns, obtaining a sterile bulk product. A sample was subjected to sterility and protein concentration tests (Kjeldahl) following the systematic and criteria followed in the sterility tests and protein analysis of the previous stages. The sterile bulk product was packaged in a Nalgene container labeled with the acronym G, the date of preparation, and the names of the origin strains, and stored at 4 ° C in a refrigerated.
Preparación del producto dosificado para inveccionesPreparation of the dosed product for inventions
El producto a granel estéril se dispensó en ampollas a razón de 2 mi por ampolla.The sterile bulk product was dispensed in ampoules at a rate of 2 ml per ampoule.
Ejemplo 2: Se analizaron tres muestras del producto a granel obtenido en el ejemplo 1, para caracterizar suExample 2: Three samples of the bulk product obtained in example 1 were analyzed to characterize their
HOJA DE SUSTITUCIÓN (REGLA 26) componente proteico mediante electroforesis en gel de poliacrilamida (PAGE). La muestras analizadas no precisaron preparación previa.SUBSTITUTE SHEET (RULE 26) protein component by polyacrylamide gel electrophoresis (PAGE). The samples analyzed did not require prior preparation.
La proteínas de cada una de las muestras se separaron mediante técnicas PAGE, en geles estándar tris- glicina BIORAD 10% policrilamida, de BIORAD, EE.UU.The proteins of each of the samples were separated by PAGE techniques, in standard 10% polycylamide trisglycine gels, from BIORAD, USA.
Se empleó un tampón desnaturalizador con 10% SDS y 5% 2-mercaptoetanol . El tampón de electrodo era el tampón Tris-glicina/SDS empleado convencionalmente para este tipo de gel (método de Laemmli).A denaturing buffer with 10% SDS and 5% 2-mercaptoethanol was used. The electrode buffer was the Tris-glycine / SDS buffer conventionally used for this type of gel (Laemmli method).
Los parámetros de separación fueron 125 V, 30 mA (condiciones de partida) durante aproximadamente 90 min.The separation parameters were 125 V, 30 mA (starting conditions) for approximately 90 min.
A continuación los geles se colorearon con azul Coomassie y se destiñeron para mostrar las bandas de proteínas, de acuerdo con el siguiente protocolo:The gels were then colored with Coomassie blue and faded to show the protein bands, according to the following protocol:
Solución de tintado de los geles (a 500 mi) Azul Coomassie 500 mg etanol 200 mg agua destilada 250 mi ácido acético glacial 50 miGel tinting solution (at 500 ml) Coomassie Blue 500 mg ethanol 200 mg distilled water 250 ml glacial acetic acid 50 ml
Cada gel coloreó con 100 mi de solución y se dejo permanecer allí durante al menos 30 minutos.Each gel colored with 100 ml of solution and was allowed to remain there for at least 30 minutes.
Solución de destintado de los geles (a 500 mi) etanol 60 mi agua destilada 400 mi ácido acético glacial 40 miSolution for the gels (500 ml) ethanol 60 ml distilled water 400 ml glacial acetic acid 40 ml
Cada gel permaneció en esta solución durante una noche.Each gel remained in this solution overnight.
Los resultados del ensayo PAGE se muestran en la figura 1 en la que los números 1 - 2 tienen los siguientes significados:The results of the PAGE test are shown in Figure 1 in which the numbers 1-2 have the following meanings:
HOJA DE SUSTITUCIÓN (REGLA 26) 1 = Columna de Sigma Marker No. M-4038SUBSTITUTE SHEET (RULE 26) 1 = Sigma Marker column No. M-4038
2 = Columnas de los productos proteicos2 = Columns of protein products
Como puede apreciarse, esta figura muestra diferentes bandas de distintas intensidades pero patrones de bandas prácticamente idénticos de los tres productos ensayados, lo cual es indicativo de que las muestras tenían la misma composición de proteínas.As can be seen, this figure shows different bands of different intensities but virtually identical band patterns of the three products tested, which is indicative that the samples had the same protein composition.
Ejemplo 3: Se tomaron otras dos muestras del producto a granel del ejemplo 1 que se sometieron a análisis de electroforesis en gel de poliacrilamida (PAGE) en las mismas condiciones que las descritas en el ejemplo 1.Example 3: Two other samples of the bulk product of Example 1 were taken and subjected to polyacrylamide gel electrophoresis analysis (PAGE) under the same conditions as described in example 1.
Posteriormente, las proteínas de cada muestra, separadas por PAGE de acuerdo con sus pesos moleculares, se electrotransfirieron a una sola membrana de nitrocelulosa para permitir ulteriores análisis. La metodología correspondiente está descrita por Sambrock, Fritsch y Maniatis, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor (1989). Se utilizó una cámara de electrotransferenciaSubsequently, the proteins in each sample, separated by PAGE according to their molecular weights, were electrotransferred to a single nitrocellulose membrane to allow further analysis. The corresponding methodology is described by Sambrock, Fritsch and Maniatis, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor (1989). An electrotransfer chamber was used
LINUS, con tris-glicina de LINUS como tampón de electrotransferencia, a 200 mA, 40 V y durante 2 horas.LINUS, with LINUS tris-glycine as an electrotransfer buffer, at 200 mA, 40 V and for 2 hours.
Las membranas sobre las que se transfirieron las proteínas separadas por PAGE correspondientes a cada una de las muestras se sometieron a inmunoensayo con anticuerpos policlonales producidos en conejos que se habían obtenido previamente mediante tratamiento con un lote diferente del producto proteico obtenido de acuerdo con el método descrito en el ejemplo 1 y que se encontraba en suero de conejo almacenado a -180°C.The membranes on which the PAGE-separated proteins corresponding to each of the samples were transferred were subjected to immunoassay with polyclonal antibodies produced in rabbits that had previously been obtained by treatment with a different batch of the protein product obtained according to the described method. in example 1 and that it was in rabbit serum stored at -180 ° C.
Dado que la reacción antígeno-anticuerpo es muy específica y, por tanto la unión del producto proteico a las bandas, cada una de las cuales corresponde a una proteína de la preparación, generada por cada una de las muestras es indicativa de la presencia de proteínas comunesSince the antigen-antibody reaction is very specific and, therefore, the binding of the protein product to the bands, each of which corresponds to a protein of the preparation, generated by each of the samples is indicative of the presence of proteins common
HOJA DE SUSTITUCIÓN (REGLA 26) en la preparación empleada para inmunizar los conejos, y, en consecuencia, puede servir de control de calidad de las nuevas preparaciones que se generaron.SUBSTITUTE SHEET (RULE 26) in the preparation used to immunize rabbits, and, consequently, can serve as a quality control of the new preparations that were generated.
Para la obtención del suero con anticuerpos policlonales generados en conejo del producto proteico según la invención se procedió de la siguiente manera.To obtain the serum with rabbit-generated polyclonal antibodies of the protein product according to the invention, proceed as follows.
Se seleccionaron dos conejos albinos New Zealand White, un macho y una hembra, jóvenes y sanos y se mantuvieron aislados y en observación durante 5 días para la aclimatación al entorno de la instalación. Después del periodo de aclimatación y no habiéndose podido observar ninguna anomalía en los animales, se siguió el siguiente programa :Two New Zealand White albino rabbits, one male and one female, young and healthy were selected and kept isolated and under observation for 5 days for acclimatization to the installation environment. After the acclimatization period and not being able to observe any anomaly in the animals, the following program was followed:
Vía de administración: inyección intramuscular Días de administración: 1, 7, 14, 17Route of administration: intramuscular injection Days of administration: 1, 7, 14, 17
La administración intramuscular se realizó mediante jeringuillas desechables con agujas de calibreIntramuscular administration was performed using disposable syringes with gauge needles.
16x0, 5mm y se aplicaron a cada animal en la cara posterior de las pata trasera, alternando los lados derecho e izquierdo.16x0.5mm and were applied to each animal on the back side of the hind legs, alternating the right and left sides.
El día 23 se dio por finalizado el ensayo y se procedió al sacrificio de los animales, previamente anestesiados por inyección intraperitoneal de pentotal sódico en solución acuosa, por exanguinación, extrayéndose la sangre por punción cardiaca. La sangre se recogió sin ningún tipo de aditivo, y el suero se obtuvo por centrifugación a baja velocidad.On day 23, the test was terminated and the animals were slaughtered, previously anesthetized by intraperitoneal injection of sodium pentothal in aqueous solution, by exanguination, the blood being drawn by cardiac puncture. Blood was collected without any additive, and serum was obtained by low speed centrifugation.
El suero obtenido (40ml) se homogeneizó, alicuoteó en criotubos de 1 y 4,5ml, referenciado como "Suero-97" y se congeló a -180°C en nitrógeno líquido.The serum obtained (40ml) was homogenized, aliquoted in 1 and 4.5ml cryotubes, referenced as "Serum-97" and frozen at -180 ° C in liquid nitrogen.
La metodología seguida es en sí convencional y corresponde a los descrito en:The methodology followed is in itself conventional and corresponds to those described in:
Donovan, J. y Brown, P., 1995a, Parenteral injections in current protocols in immunology; 1.6.1- 1.6.10; John Wiley & Sons, New YorkDonovan, J. and Brown, P., 1995a, Parenteral injections in current protocols in immunology; 1.6.1- 1.6.10; John Wiley & Sons, New York
HOJA DE SUSTITUCIÓN (REGLA 26) Current Protocole in Molecular Biology, 1994, Ed. Virginia Benson ChandaSUBSTITUTE SHEET (RULE 26) Current Protocole in Molecular Biology, 1994, Ed. Virginia Benson Chanda
Hurrell J.G.R. ed. 1982, Monoclonal Hybridoma Antibodies: Techniques And Applications, CRC Press, Boca Ratón, Fia.Hurrell J.G.R. ed. 1982, Monoclonal Hybridoma Antibodies: Techniques And Applications, CRC Press, Boca Raton, Fia.
Lagone, J.J. y Van Vunakis, H.H. Eds . 1986, Immunologial Techniques, Part I: Hybridoma Technology And Monoclonal Antibodies, Methods Enzymol., 121:1-947Lagone, J.J. and Van Vunakis, H.H. Eds 1986, Immunologial Techniques, Part I: Hybridoma Technology And Monoclonal Antibodies, Methods Enzymol., 121: 1-947
En vistas de que es posible detectar anticuerpos primarios, que se han unido a las proteínas, empleándose una técnica que implica el uso de un segundo anticuerpo marcado generado en un huésped diferente del que se usó para la generación del primer anticuerpo, se empleó también tal segundo anticuerpo generado en cabras, sensible a la luz y a cadenas pesadas de IgG de conejo, y que en consecuencia era capaz de detectar cualquier anticuerpo de conejo ligado a la proteínas en las membrana de nitrocelulosa. Cada uno de los segundos anticuerpos presenta un marcador (peroxidasa de rábano picante, de PIERCE, Rockford, Illinois, Estados Unidos de América) que permite la visualización de la presencia de tales anticuerpos en la membrana cuando se añade un reactivo, 4- cloronaftol, que produce una coloración violeta.In view of the fact that it is possible to detect primary antibodies, which have bound to the proteins, using a technique that involves the use of a second labeled antibody generated in a different host from that used for the generation of the first antibody, such second antibody generated in goats, sensitive to light and heavy chains of rabbit IgG, and which was consequently capable of detecting any rabbit antibody bound to proteins in nitrocellulose membranes. Each of the second antibodies has a marker (horseradish peroxidase, from PIERCE, Rockford, Illinois, United States of America) that allows visualization of the presence of such antibodies in the membrane when a reagent, 4-chloronaphthol, is added. which produces a violet coloration.
El segundo anticuerpo empleado en el presente ensayo fué un anticuerpo comercial IMMUNOPURE GOAT ANTI- RABBIT IgG (H+L) (n? de producto: 31460; nδ de ote: 96060545) de PIERCE de Rockford, Illinois, Estados Unidos de América.The second antibody used in the present assay was an IMMUNOPURE GOAT ANTI-RABBIT IgG (H + L) commercial antibody (product no .: 31460; ote no .: 96060545) from PIERCE of Rockford, Illinois, United States of America.
Cada una de las bandas violetas en cada una de las membranas de nitrocelulosa corresponde a una proteína específica que también se encontraba en la preparación de control del producto proteico que se había empleado para inmunizar los dos conejos, de cuyo suero se había extraído el primer anticuerpo. Para el tratamiento de cada una de lasEach of the violet bands in each of the nitrocellulose membranes corresponds to a specific protein that was also in the control preparation of the protein product that had been used to immunize the two rabbits, from which serum the first antibody had been extracted. . For the treatment of each of the
HOJA DE SUSTITUCIÓN (REGLA 26) membranas se emplearon las siguientes composicionesSUBSTITUTE SHEET (RULE 26) membranes the following compositions were employed
HOJA DE SUSTITUCIÓN (REGLA 26) Solución 1: PBS (sin Ca ni Mg) 300 miSUBSTITUTE SHEET (RULE 26) Solution 1: PBS (without Ca or Mg) 300 ml
Tween 20 150 μl150 μ l Tween 20
Solución 2 : solución 1 50 miSolution 2: solution 1 50 ml
BSA 500 mgBSA 500 mg
Leche en polvo 2,5g2.5g powdered milk
Solución 3: solución 1 50 miSolution 3: solution 1 50 ml
BSA 250 mgBSA 250 mg
Solución de lavado:Wash Solution:
PBS (sin Ca ni MG) 200 mi leche en polvo 1 gPBS (without Ca or MG) 200 my milk powder 1 g
Tween 20 100 μlTween 20 100 μ l
Primera solución de anticuerpo (1/100): primer anticuerpo 200 μl solución 3 20 miFirst antibody solution (1/100): first antibody 200 μl solution 3 20 ml
Segunda solución de anticuerpo (1/1500): segundo anticuerpo (PIERCE) 60 μl solución 3 30 mi solución Tris-HCl:Second antibody solution (1/1500) second antibody (PIERCE) 60 μ l Solution 3 30 ml Tris-HCl buffer:
Tris HC1 160 mgTris HC1 160 mg
NaCl 820 mg agua destilada 100 mi solución 4-CN:NaCl 820 mg distilled water 100 ml 4-CN solution:
4-cloronaftol (PIERCE) 30 mg etanol 10 mi solución reveladora: solución Tris-HCL 50 mi solución 4-CN 5 mi4-Chloronaphthol (PIERCE) 30 mg ethanol 10 ml developer solution: Tris-HCL solution 50 ml 4-CN solution 5 ml
H202 10%H 2 0 2 10%
Cada membrana de nitrocelulosa con las proteínas transferidas, se sometió al siguiente tratamiento:Each nitrocellulose membrane with the transferred proteins was subjected to the following treatment:
HOJA DE SUSTITUCIÓN (REGLA 26) * Doble lavado en la solución 1 durante 1 min;SUBSTITUTE SHEET (RULE 26) * Double wash in solution 1 for 1 min;
* Incubación en la solución 2 durante 8 horas;* Incubation in solution 2 for 8 hours;
* Doble lavado en la solución 1 durante 30 min;* Double wash in solution 1 for 30 min;
* Incubación en la solución del primer anticuerpo durante 2 horas;* Incubation in the solution of the first antibody for 2 hours;
* Cuatro lavados en la solución de lavado durante 10 min;* Four washes in the wash solution for 10 min;
* Incubación durante 2 horas en la solución del segundo anticuerpo; * Cuatro lavados en la solución de lavado durante 10 min;* Incubation for 2 hours in the solution of the second antibody; * Four washes in the wash solution for 10 min;
* Revelado en la solución de revelado durante 30 min;* Developed in the development solution for 30 min;
* Lavado con agua ultrapura y mantenimiento en oscuridad hasta el momento en el que se realizan las fotografías .* Washing with ultrapure water and maintaining in darkness until the moment in which the photographs are taken.
Después de este tratamiento, las membranas de nitrocelulosa se fotografiaron. Las fotografías se muestran en la fig. 2. De acuerdo con lo que se desprende de la fig.After this treatment, nitrocellulose membranes were photographed. The photographs are shown in fig. 2. According to what follows from fig.
2, las muestras analizadas mostraron patrones de bandas e intensidades de bandas prácticamente idénticas, es decir, fueron homogéneas entre sí en cuanto a sus composiciones en proteínas y frente al suero de conejo. Ensayos de actividad biológica2, the analyzed samples showed band patterns and intensities of virtually identical bands, that is, they were homogeneous with respect to their protein compositions and against rabbit serum. Biological Activity Tests
Se evaluó la inmunoestimulación en base a ensayos realizados con muestras del producto proteico obtenidas según el ejemplo 1. Las muestras se identifican como FR-91. 1) Respuesta in vivo T-dependiente usando el ensayo de células formadoras de plaquetasImmunostimulation was evaluated based on tests performed with samples of the protein product obtained according to Example 1. The samples are identified as FR-91. 1) T-dependent in vivo response using platelet forming cell assay
Se inmunizaron dos ratones con 0,025-0,3 mi de FR-91 por cada ratón.Two mice were immunized with 0.025-0.3 ml of FR-91 for each mouse.
Los ratones se sacrificaron mediante dislocación cervical y se extrajo el bazo de cada uno.Mice were sacrificed by cervical dislocation and each spleen was removed.
HOJA DE SUSTITUCIÓN (REGLA 26) Después de cuatro días, se mezclaron las inmunoglobulinas conjugadas con proteínas SRBC con los linfocitos esplénicos, entre los que se encuentran células productoras de anticuerpos específicos, y se incubaron en un medio semisólido de soporte para permitir que los anticuerpos secretados se unan con la superficie de los eritrocitos. A continuación, se añadió un complemento con especificidad contra anticuerpos conjugados a SRBC a la mezcla, después de lo cual se midieron las zonas libres de lisis (placa) para evaluar la activación de linfocitos.SUBSTITUTE SHEET (RULE 26) After four days, the immunoglobulins conjugated with SRBC proteins were mixed with the splenic lymphocytes, among which specific antibody producing cells are found, and incubated in a semi-solid support medium to allow secreted antibodies to bind with the surface of Erythrocytes Next, a complement with specificity against antibodies conjugated to SRBC was added to the mixture, after which lysis-free zones (plaque) were measured to evaluate lymphocyte activation.
De acuerdo con lo que se desprende de la fig. 3, la muestras con FR-91 presentaron un número aumentado de células productoras de anticuerpos T-dependientes en ratones inmunizados con células sanguíneas rojas de oveja, lo cual es indicativo de que FR-91 es eficaz en el sistema inmune relacionado con muchos tipos de linfocitos .According to what follows from fig. 3, the samples with FR-91 presented an increased number of T-dependent antibody producing cells in mice immunized with red sheep blood cells, which is indicative that FR-91 is effective in the immune system related to many types of lymphocytes
2) Activación in vitro de células B, empleándose ensayo de células formadoras de placas2) In vitro activation of B cells, using plate forming cell assay
Se añadieron muestras de FR-91 a esplenocitos realizándose concentraciones finales desde 100:1 (1%) hastaSamples of FR-91 were added to splenocytes with final concentrations being made from 100: 1 (1%) to
10000:1 (0,01%) y se midió el nivel de células productoras de anticuerpos T-dependientes con o sin lipopolisacáridos empleándose un ensayo de células formadoras de plaquetas.10000: 1 (0.01%) and the level of T-dependent antibody producing cells with or without lipopolysaccharides was measured using a platelet forming cell assay.
De acuerdo con lo que se desprende de las figuras 4 y 5, la adición de FR-91 prácticamente no causó incremento alguno en la proliferación o activación de células B, al tratarse esplenocitos con o sin lipopolisacáridos .According to what is shown in Figures 4 and 5, the addition of FR-91 practically caused no increase in the proliferation or activation of B cells, as splenocytes were treated with or without lipopolysaccharides.
3) Activación in vitro de células T Al cultivarse conjuntamente linfocitos de dos diferentes cepas endogámicas, las células empiezan a proliferar en respuesta a las diferencias antigénicas en los linfocitos alogénicos. La intensidad de esta reacción de linfocitos mixtos (MLR) puede cuantificarse añadiéndose timidina marcada con tritio al medio de cultivo. Dado que3) In vitro activation of T cells When lymphocytes from two different inbred strains are cultured together, the cells begin to proliferate in response to antigenic differences in allogeneic lymphocytes. The intensity of this mixed lymphocyte (MLR) reaction can be quantified by adding tritium-labeled thymidine to the culture medium. Given the
HOJA DE SUSTITUCIÓN (REGLA 26) las células proliferan, la timidina radioactiva se incorpora en el ADN de las células descendientes. El grado de la proliferación se determina cosechándose las células, sometiéndolas a lísis, y midiéndose la cantidad de timidina radioactiva incorporada en el ADN la cual es directamente proporcional al nivel de proliferación.SUBSTITUTE SHEET (RULE 26) cells proliferate, radioactive thymidine is incorporated into the DNA of the descendant cells. The degree of proliferation is determined by harvesting the cells, subjecting them to lysis, and measuring the amount of radioactive thymidine incorporated into the DNA which is directly proportional to the level of proliferation.
En base a la metodología anteriormente definida, se realizó el siguiente ensayo:Based on the methodology defined above, the following test was performed:
* Se mezclaron linfocitos de dos diferentes cepas endogámicas en unos micropocillos .* Lymphocytes from two different inbred strains were mixed in microwells.
* Se añadieron muestras diluidas en serie de FR-91 en concentraciones 100:1, 1000:1 y 10000:1 a los micropocillos .* Serially diluted samples of FR-91 in 100: 1, 1000: 1 and 10,000: 1 concentrations were added to the microwells.
* Después de cultivar 4 días, se añadió timidina tritiada [ H] a las respectivas mezclas (C10 3Hi4N2 * After culturing 4 days, tritiated thymidine [H] was added to the respective mixtures (C 10 3 Hi 4 N 2
05; SIGMA de SIGMA Corp., EE.UU.).0 5 ; SIGMA of SIGMA Corp., USA).
* Después de cultivar durante 20 horas, se cosecharon células de los cultivos en los micropocillos en una tira de papel-filtro, y se midió la timidina [ H] incorporado en el ADN de las respectivas muestras y se compararon con una muestra de control sin FR-91.* After culturing for 20 hours, cells from the cultures were harvested in the microwells in a paper-filter strip, and thymidine [H] incorporated into the DNA of the respective samples was measured and compared with a control sample without FR-91
De acuerdo con lo que se desprende de la figura 6, FR-91 incrementó fuertemente la proliferación de células T. 4) Blastogénesis de células TAccording to what is shown in Figure 6, FR-91 strongly increased the proliferation of T cells. 4) Blastogenesis of T cells
Se añadieron muestras de FR-91 diluidas en serie a esplenocitos, con concentraciones finales de 100:1, 1000:1 y 10.000:1 y se introdujeron en micropocillos.Serially diluted samples of FR-91 were added to splenocytes, with final concentrations of 100: 1, 1000: 1 and 10,000: 1 and introduced into microwells.
Se trataron micropocillos respectivamente identificados, con lipopolisacáridos, mitógeno de pokeweed, fitohematoglutinina, concanavalina A como mitógeno estándar, a una concentración final de lμg/pocillo.Microwells, respectively identified, were treated with lipopolysaccharides, pokeweed mitogen, phytohematoglutinin, concanavalin A as standard mitogen, at a final concentration of 1 µg / well.
Se midió el nivel de proliferación de células T y B mediante ensayos de incorporación de timidina [3H]. Los resultados se reflejan en las figuras 7 - 11.The level of T and B cell proliferation was measured by thymidine incorporation assays [ 3 H]. The results are reflected in figures 7-11.
HOJA DE SUSTITUCIÓN (REGLA 26) De acuerdo con lo que se desprende con la fig. 7, FR-91 incremento intensamente la inmunidad de esplenocitos sin ningún mitógeno.SUBSTITUTE SHEET (RULE 26) According to what follows with fig. 7, FR-91 greatly increased the immunity of splenocytes without any mitogens.
La fig. 8 muestra que FR-91 produce un efecto sinérgico con concanavalina A, la cual es un mitógeno de las células T.Fig. 8 shows that FR-91 produces a synergistic effect with concanavalin A, which is a T-cell mitogen.
La fig. 9 evidencia que FR-91 presenta un sinergis o fuerte con fitohematoglutinina, la cual es un mitógeno de las células T. La fig. 10 evidencia que FR-91 y los lipopolisacáridos, los cuales son mitógenos de las células B, no presentan sinergia alguna.Fig. 9 evidence that FR-91 has a strong or synergy with phytohematoglutinin, which is a T-cell mitogen. Fig. 10 evidence that FR-91 and lipopolysaccharides, which are mitogens of B cells, have no synergies.
La fig. 11 demuestra, a una concentración de 100:1, que FR-91 produce algún efecto sinérgico con mitógeno de pokeweed, el cuál es un mitógeno común de T y B.Fig. 11 demonstrates, at a concentration of 100: 1, that FR-91 produces some synergistic effect with pokeweed mitogen, which is a common T and B mitogen.
De lo anterior se desprende que FR-91 incrementa sustancialmente la proliferación y activación de células T, pero no presenta efectos en cuanto a las células B.It follows that FR-91 substantially increases the proliferation and activation of T cells, but has no effect on B cells.
HOJA DE SUSTITUCIÓN (REGLA 26) SUBSTITUTE SHEET (RULE 26)

Claims

REIVINDICACIONES
1. Producto proteico que comprende fracciones de péptidos, proteínas y otras moléculas, extraídos de bacilos apatógenos, caracterizado porque el producto es al menos un extracto proteico y comprende proteínas, péptidos y otras moléculas, obtenidos a partir de lísis celular de al menos una de las de cepas de bacilos termorresistentes y esporulados, seleccionadas de al menos uno de los siguientes grupos: - un primer grupo compuesto por cepas de B.1. Protein product comprising fractions of peptides, proteins and other molecules, extracted from apatogenic bacilli, characterized in that the product is at least one protein extract and comprises proteins, peptides and other molecules, obtained from cell lysis of at least one of those of strains of heat-resistant and sporulated bacilli, selected from at least one of the following groups: - a first group consisting of strains of B.
Licheniformis , B. Circulans 2, B. Pumilus, B. Macerans, B. Amilolicuofaciens , y cepas modificadas de las mismas;Licheniformis, B. Circulans 2, B. Pumilus, B. Macerans, B. Amilolicuofaciens, and modified strains thereof;
- un segundo grupo compuesto por cepas de B. Cereus 1, B. Cereus 2, B . Lentus 1 , B. Lentus 2 y cepas modificadas de las mismas;- a second group consisting of strains of B. Cereus 1, B. Cereus 2, B. Lentus 1, B. Lentus 2 and modified strains thereof;
- un tercer grupo compuesto por cepas de B. Subtilis y cepas modificadas de las mismas;- a third group consisting of strains of B. Subtilis and modified strains thereof;
- un cuarto grupo compuesto por cepas de B. Mesentericus y cepas modificadas de las mismas.- a fourth group consisting of strains of B. Mesentericus and modified strains thereof.
2. Producto proteico según la reivindicación 1, caracterizado porque comprende2. Protein product according to claim 1, characterized in that it comprises
- al menos una primera fracción de proteínas, péptidos y moléculas obtenidas a partir del fraccionamiento celular, seleccionada entre extractos de B. Licheniformis, B. Circulans 2, B. Pumilus, B. Macerans, B. Amilolicuofaciens ;- at least a first fraction of proteins, peptides and molecules obtained from cell fractionation, selected from extracts of B. Licheniformis, B. Circulans 2, B. Pumilus, B. Macerans, B. Amilolicuofaciens;
- al menos una segunda fracción de proteínas, péptidos y moléculas obtenidas a partir del fraccionamiento celular, seleccionada entre extractos de B. Cereus 1, B. Cereus 2, B. Lentus 1, B . Lentus 2 ;- at least a second fraction of proteins, peptides and molecules obtained from cell fractionation, selected from extracts of B. Cereus 1, B. Cereus 2, B. Lentus 1, B. Lentus 2;
- al menos una tercera fracción de proteínas, péptidos y moléculas obtenidas a partir del fraccionamiento celular, seleccionada entre extractos de B. Subtilis; - al menos una cuarta fracción de proteínas,- at least a third fraction of proteins, peptides and molecules obtained from cell fractionation, selected from extracts of B. Subtilis; - at least a fourth fraction of proteins,
HOJA DE SUSTITUCIÓN (REGLA 26) péptidos y moléculas obtenidas a partir del fraccionamiento celular, seleccionada entre extractos de B. Mesentericus.SUBSTITUTE SHEET (RULE 26) peptides and molecules obtained from cell fractionation, selected from extracts of B. Mesentericus.
3. Producto proteico según la reivindicación 1, caracterizado porque comprende al menos un primer extracto que contiene proteínas, péptidos y moléculas, obtenido a partir del fraccionamiento celular a partir de cepas de bacilos del genero Subtilis; - al menos un segundo extracto que contiene proteínas, péptidos y moléculas, obtenido a partir del fraccionamiento celular de cepas de bacilos del género Cereus; al menos un tercer extracto que contiene proteínas, péptidos y moléculas, obtenido a partir de cepas de bacilos del género Mesentericus;3. Protein product according to claim 1, characterized in that it comprises at least a first extract containing proteins, peptides and molecules, obtained from cell fractionation from bacillus strains of the Subtilis genus; - at least a second extract containing proteins, peptides and molecules, obtained from the cell fractionation of bacillus strains of the genus Cereus; at least a third extract containing proteins, peptides and molecules, obtained from bacillus strains of the genus Mesentericus;
- al menos un cuarto extracto que contiene proteínas, péptidos y moléculas, obtenido a partir de cepas de bacilos del género Licheniformis ; - al menos un quinto extracto que contiene proteínas, péptidos y moléculas, obtenido a partir de cepas de bacilos del género Lentus;- at least a fourth extract containing proteins, peptides and molecules, obtained from bacillus strains of the genus Licheniformis; - at least one fifth extract containing proteins, peptides and molecules, obtained from bacillus strains of the genus Lentus;
- al menos un sexto extracto que contiene proteínas, péptidos y moléculas, obtenido a partir de cepas de bacilos del género Pumilus .- at least one sixth extract containing proteins, peptides and molecules, obtained from bacillus strains of the genus Pumilus.
4. Producto proteico según cualquiera de las reivindicaciones 1 a 3, caracterizado porque las cepas de bacilos están seleccionadas entre cepas naturales y cepas modificadas.4. Protein product according to any of claims 1 to 3, characterized in that the bacillus strains are selected from natural strains and modified strains.
5. Producto proteico según la reivindicación 1, caracterizado porque las cepas de bacilos del primer grupo son cepas modificadas CECT FCM-1 4913.5. Protein product according to claim 1, characterized in that the bacillus strains of the first group are CECT FCM-1 4913 modified strains.
HOJA DE SUSTITUCIÓN (REGLA 26) SUBSTITUTE SHEET (RULE 26)
6. Producto proteico según la reivindicación 1, caracterizado porque las cepas de bacilos del segundo grupo son cepas modificadas CECT FCM-2 4914.6. Protein product according to claim 1, characterized in that the bacillus strains of the second group are CECT FCM-2 4914 modified strains.
7. Producto proteico según la reivindicación 1, caracterizado porque las cepas de bacilos del tercer grupo son cepas modificadas CECT FCM-3 4915.7. Protein product according to claim 1, characterized in that the bacillus strains of the third group are CECT FCM-3 4915 modified strains.
8. Producto proteico según la reivindicación 1, caracterizado porque las cepas de bacilos del cuarto grupo son cepas modificadas CECT FCM-4 4916.8. Protein product according to claim 1, characterized in that the bacillus strains of the fourth group are CECT FCM-4 4916 modified strains.
9. Producto proteico según la reivindicación 2 o 3, caracterizado porque las cepas modificadas de B. Subtilis son cepas CECT FCM-3 4915.9. Protein product according to claim 2 or 3, characterized in that the modified strains of B. Subtilis are CECT FCM-3 4915 strains.
10. Producto proteico según la reivindicación 2 o 3, caracterizado porque las cepas modificadas de B. Cereus son cepas CECT FCM-2 4914.10. Protein product according to claim 2 or 3, characterized in that the modified strains of B. Cereus are CECT FCM-2 4914 strains.
11. Producto proteico según la reivindicación 2 o 3, caracterizado porque las cepas modificadas de B . Mesentericus son cepas CECT FCM-4 4916.11. Protein product according to claim 2 or 3, characterized in that the modified strains of B. Mesentericus are CECT FCM-4 4916 strains.
12. Producto proteico según la reivindicación 2 o12. Protein product according to claim 2 or
3, caracterizado porque las cepas modificadas de B. Licheniformis son cepas CECT FCM-1 4913.3, characterized in that the modified strains of B. Licheniformis are CECT FCM-1 4913 strains.
13. Producto según la reivindicación 2 o 3, caracterizado porque las cepas modificadas de B. Lentus son cepas CECT FCM-2 4914.13. Product according to claim 2 or 3, characterized in that the modified strains of B. Lentus are CECT FCM-2 4914 strains.
14. Producto según la reivindicación 2 o 3, caracterizado porque las cepas modificadas de B. Pumilus son cepas CECT FCM-1 4913.14. Product according to claim 2 or 3, characterized in that the modified strains of B. Pumilus are CECT FCM-1 4913 strains.
HOJA DE SUSTITUCIÓN (REGLA 26) SUBSTITUTE SHEET (RULE 26)
15. Procedimiento para la obtención del extracto proteico, caracterizado porque comprende las etapas de15. Procedure for obtaining the protein extract, characterized in that it comprises the stages of
- seleccionar al menos una cepa termorresistente y esporulada de al menos uno de los siguientes grupos: un primer grupo compuesto por cepas de B. Licheniformis , B. Circulans 2, B. Pumilus, B . Macerans , B. Amilolicuofaciens , y cepas modificadas de las mismas; - un segundo grupo compuesto por cepas de B.- select at least one heat-resistant and sporulated strain from at least one of the following groups: a first group consisting of strains of B. Licheniformis, B. Circulans 2, B. Pumilus, B. Macerans, B. Amilolicuofaciens, and modified strains thereof; - a second group consisting of strains of B.
Cereus 1, B. Cereus 2, B. Lentus 1, B. Lentus 2 y cepas modificadas de las mismas;Cereus 1, B. Cereus 2, B. Lentus 1, B. Lentus 2 and modified strains thereof;
- un tercer grupo compuesto por cepas de B . Subtilis y cepas modificadas de las mismas; - un cuarto grupo compuesto por cepas de B .- a third group consisting of strains of B. Subtilis and modified strains thereof; - a fourth group consisting of strains of B.
Mesentericus y cepas modificadas de las mismas; añadir una inoculación de dicha cepa a una disolución acuosa de un medio de cultivo, para obtener un medio de cultivo inoculado, - ajustar la temperatura del medio de cultivo inoculado en función de un crecimiento óptimo de la cepa; cultivar la cepa hasta alcanzar una densidad óptica máxima hasta obtener un cultivo que contiene una masa bacilar; - obtener un extracto proteico de las masas bacilares mediante las etapas deMesentericus and modified strains thereof; adding an inoculation of said strain to an aqueous solution of a culture medium, to obtain an inoculated culture medium, - adjusting the temperature of the inoculated culture medium according to an optimum growth of the strain; cultivate the strain until it reaches a maximum optical density until a culture containing a bacillary mass is obtained; - obtain a protein extract from the bacillary masses through the stages of
- separación de la masa bacilar del cultivo hasta obtener bacilos en suspensión; filtración cruzada sobre un filtro con un diámetro de poro inferior a 0,22 mieras hasta obtener un cultivo concentrado con un rango de concentración de 10 a 20;- separation of the bacillary mass from the culture until obtaining bacilli in suspension; cross filtration on a filter with a pore diameter of less than 0.22 microns until a concentrated culture with a concentration range of 10 to 20 is obtained;
- someter el cultivo concentrado a diafiltración con intercambio de solución salina fisiológica; - obtener un extracto que comprende proteínas,- subject the concentrated culture to diafiltration with physiological saline exchange; - obtain an extract comprising proteins,
HOJA DE SUSTITUCIÓN (REGLA 26) péptidos y moléculas que forman el principio activo mediante las fases deSUBSTITUTE SHEET (RULE 26) peptides and molecules that form the active substance through the phases of
- someter el cultivo concentrado a lísis celular, mediante un proceso criotérmico que comprende un ciclo con una primera fase a temperaturas entre 2 y 8°C durante varias horas y una segunda fase de frío a una temperatura inferior a -40°C durante al menos 8 horas, y una tercera fase en baño de agua a temperaturas entre temperatura ambiente y 70°C; repetir dicho ciclo al menos dos veces, hasta obtener un concentrado de Usado celular que contiene un resto celular y el principio activo;- subject the concentrated culture to cell lysis, by a cryothermal process comprising a cycle with a first phase at temperatures between 2 and 8 ° C for several hours and a second cold phase at a temperature below -40 ° C for at least 8 hours, and a third phase in a water bath at temperatures between room temperature and 70 ° C; repeat said cycle at least twice, until a concentrate of Cellular Use containing a cellular moiety and the active ingredient is obtained;
- separar el resto celular del principio activo sometiendo el concentrado de usado celular a filtración de flujo cruzado con diafiltración en una membrana de tamaño de poro inferior o igual a 0,22 mieras con un rango de dilución de 1:10, para obtener un filtrado de extracto proteico bruto que contiene un principio activo que comprende proteínas, péptidos y moléculas provenientes de la lísis celular; - concentrar el filtrado utilizándose un filtro de tamaño de poro tal que las proteínas de peso molecular superior a 5000 daltons quedan retenidos por encima del filtro en una solución acuosa;- separating the cellular moiety from the active substance by subjecting the concentrate of used cell to cross flow filtration with diafiltration in a membrane of pore size less than or equal to 0.22 microns with a dilution range of 1:10, to obtain a filtrate of crude protein extract containing an active ingredient comprising proteins, peptides and molecules from cell lysis; - concentrate the filtrate using a pore size filter such that proteins of molecular weight greater than 5000 daltons are retained above the filter in an aqueous solution;
- filtración estéril del filtrado concentrado por un filtro de tamaño de poro igual o inferior a 0,22 mieras hasta obtener un primer extracto proteico esterilizado que contiene el principio activo;- sterile filtration of the filtrate concentrated by a pore size filter equal to or less than 0.22 microns until a first sterile protein extract containing the active ingredient is obtained;
- opcionalmente, mezclar el primer extracto proteico esterilizado, con al menos un segundo extracto proteico preparado análogamente al primer extracto.- optionally, mixing the first sterilized protein extract, with at least a second protein extract prepared analogously to the first extract.
16. Procedimiento según la reivindicación 15, caracterizado porque la disolución acuosa comprende entre 19 y 200g de un medio cultivo por cada litro de agua de calidad icrobiológica.16. Method according to claim 15, characterized in that the aqueous solution comprises between 19 and 200g of a culture medium for each liter of icrobiological quality water.
HOJA DE SUSTITUCIÓN (REGLA 26) SUBSTITUTE SHEET (RULE 26)
17. Procedimiento según la reivindicación 15, caracterizado porque la disolución acuosa se ajusta a un pH de 7.0+0,2.17. Method according to claim 15, characterized in that the aqueous solution is adjusted to a pH of 7.0 + 0.2.
18. Procedimiento según la reivindicación 15, caracterizado porque la disolución acuosa se esteriliza a al menos 121°C durante 15 a 20 minutos.18. Method according to claim 15, characterized in that the aqueous solution is sterilized at at least 121 ° C for 15 to 20 minutes.
19. Procedimiento según la reivindicación 15, caracterizado porque la temperatura del medio de cultivo inoculado en función se ajusta a una temperaturas entre 18 a 40°C.19. Method according to claim 15, characterized in that the temperature of the culture medium inoculated as a function is adjusted to temperatures between 18 to 40 ° C.
20. Procedimiento según la reivindicación 15, caracterizado porque se aplica ventilación forzada por aire filtrado al medio de cultivo inoculado hasta el inicio de la fase de crecimiento de la cepa sometiéndose el medio de cultivo inoculado a una presión de aire de 0,5 a 1 1/min.20. Method according to claim 15, characterized in that forced ventilation by filtered air is applied to the inoculated culture medium until the start of the strain growth phase by subjecting the inoculated culture medium to an air pressure of 0.5 to 1 1 / min
21. Procedimiento según la reivindicación 15, caracterizado porque el cultivo concentrado se obtiene mediante concentración celular de la masa bacilar en el cultivo mediante centrifugación, ciclón u otros procedimientos en si convencionales.21. A method according to claim 15, characterized in that the concentrated culture is obtained by cellular concentration of the bacillary mass in the culture by centrifugation, cyclone or other conventional procedures.
22. Procedimiento según la reivindicación 15, caracterizado porque antes de la concentración celular de la masa bacilar se separa y analiza una parte alícuota del cultivo antes de la concentración para obtener datos de "bacilos en suspensión" para obtener datos indicativos del grado de la concentración.22. Method according to claim 15, characterized in that an aliquot of the culture is separated and analyzed before the concentration of the bacillary mass before the concentration to obtain data of "bacilli in suspension" to obtain data indicative of the degree of concentration .
23. Procedimiento según la reivindicación 15, caracterizado porque el cultivo concentrado somete a la diafiltración con un filtro de membrana con un tamaño de23. Method according to claim 15, characterized in that the concentrated culture is subjected to diafiltration with a membrane filter with a size of
HOJA DE SUSTITUCIÓN (REGLA 26) poro igual o inferior a 0,22 mieras.SUBSTITUTE SHEET (RULE 26) Pore equal to or less than 0.22 microns.
24. Procedimiento según la reivindicación 15, caracterizado porque la segunda fase del ciclo del proceso criotérmico se realiza a - 40 y -50°C durante 8 a 12 horas y porque la tercera fase se realiza a una temperatura entre 60 y 70°C.24. Method according to claim 15, characterized in that the second phase of the cycle of the cryothermal process is performed at -40 and -50 ° C for 8 to 12 hours and that the third phase is performed at a temperature between 60 and 70 ° C.
25. Procedimiento según la reivindicación 15, caracterizado porque el proceso criotérmico se compone de 4 de dichos ciclos.25. Method according to claim 15, characterized in that the cryothermal process is composed of 4 of said cycles.
26. Procedimiento según la reivindicación 15, caracterizado porque el calentamiento para alcanzar la descongelación completa del cultivo concentrado se realiza a una temperatura inferior a 65°C.26. Method according to claim 15, characterized in that the heating to achieve complete defrosting of the concentrated culture is carried out at a temperature below 65 ° C.
27. Procedimiento según la reivindicación 15, caracterizado porque el descongelado se mantiene en reposo durante al menos 5 minutos en una cuarta fase de cada ciclo del proceso criotérmico.27. Method according to claim 15, characterized in that the defrosting is kept at rest for at least 5 minutes in a fourth phase of each cycle of the cryothermal process.
28. Procedimiento según la reivindicación 15, caracterizado porque el primer extracto proteico esterilizado se obtiene a partir de al menos un cepa de dicho primer grupo y porque el segundo extracto proteico esterilizado se obtiene a partir de al menos un cepa de dicho segundo grupo.28. A method according to claim 15, characterized in that the first sterilized protein extract is obtained from at least one strain of said first group and that the second sterilized protein extract is obtained from at least one strain of said second group.
29. Procedimiento según la reivindicación 28, caracterizado porque el primer extracto proteico esterilizado y el segundo extracto proteico esterilizado, se mezclan además con un tercer extracto proteico esterilizado obtenido a partir de al menos una cepa de dicho tercer grupo, habiéndose preparado dicho tercer extracto proteico29. A method according to claim 28, characterized in that the first sterilized protein extract and the second sterilized protein extract are further mixed with a third sterilized protein extract obtained from at least one strain of said third group, said third protein extract having been prepared
HOJA DE SUSTITUCIÓN (REGLA 26) esterilizado por procedimientos análogos a los del procedimiento de preparación del primer extracto .SUBSTITUTE SHEET (RULE 26) sterilized by procedures analogous to those of the preparation procedure of the first extract.
30. Procedimiento según la reivindicación 29, caracterizado porque el primer extracto proteico esterilizado, el segundo extracto proteico esterilizado, y el tercer extracto proteico esterilizado, se mezclan además con un cuarto extracto proteico esterilizado obtenido a partir de al menos una cepa de dicho cuarto grupo, habiéndose preparado dicho cuarto extracto proteico esterilizado por procedimientos análogos a los del procedimiento de preparación del primer extracto.30. The method according to claim 29, characterized in that the first sterilized protein extract, the second sterilized protein extract, and the third sterilized protein extract are further mixed with a fourth sterilized protein extract obtained from at least one strain of said fourth group , said fourth sterilized protein extract having been prepared by procedures analogous to those of the process of preparing the first extract.
31. Procedimiento según la reivindicación 28, caracterizado porque el primer extracto proteico esterilizado se obtiene a partir de cepas CECT FCM-1 4913, el segundo extracto proteico esterilizado se obtiene a partir de cepas CECT FCM-1 4914.Method according to claim 28, characterized in that the first sterilized protein extract is obtained from CECT FCM-1 4913 strains, the second sterilized protein extract is obtained from CECT FCM-1 4914 strains.
32. Procedimiento según la reivindicación 29, caracterizado porque el primer extracto proteico esterilizado se obtiene a partir de cepas CECT FCM-1 4913, el segundo extracto proteico esterilizado se obtiene a partir de cepas CECT FCM-2 4914 y el tercer extracto proteico esterilizado se obtiene a partir de cepas CECT FCM-3 4915.32. Method according to claim 29, characterized in that the first sterilized protein extract is obtained from CECT FCM-1 4913 strains, the second sterilized protein extract is obtained from CECT FCM-2 4914 strains and the third sterilized protein extract is obtained from CECT FCM-3 4915 strains.
33. Procedimiento según la reivindicación 30, caracterizado porque el primer extracto proteico esterilizado se obtiene a partir de cepas CECT FCM-1 4913, el segundo extracto proteico esterilizado se obtiene a partir de cepas CECT FCM-2 4914, el tercer extracto proteico esterilizado se obtiene a partir de cepas CECT FCM-3 4915, y el cuarto extracto proteico esterilizado se obtiene a partir de cepas CECT FCM-4 4916.33. Method according to claim 30, characterized in that the first sterilized protein extract is obtained from CECT FCM-1 4913 strains, the second sterilized protein extract is obtained from CECT FCM-2 4914 strains, the third sterilized protein extract is obtained from CECT FCM-3 4915 strains, and the fourth sterilized protein extract is obtained from CECT FCM-4 4916 strains.
HOJA DE SUSTITUCIÓN (REGLA 26) SUBSTITUTE SHEET (RULE 26)
34. Procedimiento según cualquiera de las reivindicaciones 28 a 33, caracterizado porque los extractos proteicos esterilizados se mezclan en proporciones iguales referidas a sus respectivas concentraciones de proteínas.34. Method according to any of claims 28 to 33, characterized in that the sterilized protein extracts are mixed in equal proportions referred to their respective protein concentrations.
35. Composición farmacéutica caracterizada porque contiene un producto proteico según cualquiera de las reivindicaciones 1 a 14 y un soporte farmacéuticamente aceptable.35. Pharmaceutical composition characterized in that it contains a protein product according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.
36. Composición farmacéutica caracterizada porque contiene un producto proteico según cualquiera de las reivindicaciones 1-14, en disolución en un medio líquido.36. Pharmaceutical composition characterized in that it contains a protein product according to any of claims 1-14, in solution in a liquid medium.
37. Composición según la reivindicación 36, caracterizada porque el medio líquido es una disolución acuosa.37. Composition according to claim 36, characterized in that the liquid medium is an aqueous solution.
38. Composición según la reivindicación 36, caracterizada porque el medio líquido es agua destilada.38. Composition according to claim 36, characterized in that the liquid medium is distilled water.
39. Composición según la reivindicación 38, caracterizada porque contiene39. Composition according to claim 38, characterized in that it contains
1 a 99% p/v del producto proteico1 to 99% w / v protein product
0 a 99% v/v en peso de agua destilada0 to 99% v / v by weight of distilled water
0-0,2% v/v de fenol 0-0,12% v/v de formol de grado farmacéutico al 37%.0-0.2% v / v phenol 0-0.12% v / v formol of 37% pharmaceutical grade.
40. Uso de un producto proteico según cualquiera de las reivindicaciones 1-14, en la fabricación de medicamentos destinados a la prevención y al tratamiento de trastornos en individuos con síndromes de40. Use of a protein product according to any of claims 1-14, in the manufacture of medicaments intended for the prevention and treatment of disorders in individuals with syndromes of
HOJA DE SUSTITUCIÓN (REGLA 26) inmunodeficiencias .SUBSTITUTE SHEET (RULE 26) immunodeficiencies
41. Uso según la reivindicación 40, en la fabricación de medicamentos para la prevención y el tratamiento del SIDA.41. Use according to claim 40, in the manufacture of medicaments for the prevention and treatment of AIDS.
42. Uso según la reivindicación 40, en la fabricación de medicamentos para el la prevención y el tratamiento de linfocitopenia T CD4+ idiopática.42. Use according to claim 40 in the manufacture of medicaments for the prevention and treatment of idiopathic CD 4+ T lymphocytopenia.
43. Uso de un producto proteico según cualquiera de las reivindicaciones 1-14, en la fabricación de medicamentos destinados a la prevención y al tratamiento de trastornos en individuos con síndromes de autoinmunidad.43. Use of a protein product according to any of claims 1-14, in the manufacture of medicaments intended for the prevention and treatment of disorders in individuals with autoimmunity syndromes.
44. Uso según la reivindicación 43, en la fabricación de medicamentos para la prevención y el tratamiento de cualquiera de los síndromes relacionados con esclerosis múltiple, Lupus eritematoso sistémico, artritis reumatoide, espondilitis anquilosante y psoriasis.44. Use according to claim 43, in the manufacture of medicaments for the prevention and treatment of any of the syndromes related to multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis and psoriasis.
45. Uso de un producto proteico según cualquiera de las reivindicaciones 1-14, en la fabricación de medicamentos destinados a la prevención y al tratamiento de trastornos en individuos afectados por procesos neoplásicos .45. Use of a protein product according to any of claims 1-14, in the manufacture of medicaments intended for the prevention and treatment of disorders in individuals affected by neoplastic processes.
46. Uso según la reivindicación 45, en la fabricación de medicamentos para la prevención y el tratamiento de cualquiera de los síndromes relacionados con procesos neoplásicos seleccionados entre leucemias, mielomas, linfornas, tumores cerebrales y de la médula espinal, cáncer de piel, neoplasia de tiroides, neoplasias de glándulas suprarrenales, tumores del aparato genitourinario masculino y femenino, tumores cardiacos,46. Use according to claim 45, in the manufacture of medicaments for the prevention and treatment of any of the syndromes related to selected neoplastic processes among leukemias, myelomas, lymph nodes, brain and spinal cord tumors, skin cancer, neoplasia of thyroid, adrenal gland neoplasms, male and female genitourinary tract tumors, cardiac tumors,
HOJA DE SUSTITUCIÓN (REGLA 26) tumores del tracto gastrointestinal, tumores de pulmón, pleura y mediastino, y neoplasias de huesos y cartílagos.SUBSTITUTE SHEET (RULE 26) Gastrointestinal tract tumors, lung tumors, pleura and mediastinum, and bone and cartilage neoplasms.
47. Uso de un producto proteico según cualquiera de las reivindicaciones 1-14, en la fabricación de medicamentos destinados a la prevención y al tratamiento de trastornos en individuos con síndromes relacionados con enfermedades degenerativas .47. Use of a protein product according to any of claims 1-14, in the manufacture of medicaments intended for the prevention and treatment of disorders in individuals with syndromes related to degenerative diseases.
48. Uso según la reivindicación 47, en la fabricación de medicamentos para la prevención y el tratamiento de la artrosis .48. Use according to claim 47 in the manufacture of medicaments for the prevention and treatment of osteoarthritis.
49. Uso de un producto proteico según cualquiera de las reivindicaciones 1-14, en la fabricación de medicamentos destinados a la prevención y al tratamiento de trastornos en individuos con síndromes relacionados con enfermedades inflamatorias intestinales.49. Use of a protein product according to any of claims 1-14, in the manufacture of medicaments intended for the prevention and treatment of disorders in individuals with syndromes related to inflammatory bowel diseases.
50. Uso según la reivindicación 49, en la fabricación de medicamentos para la prevención y el tratamiento de la enfermedad de Crohn.50. Use according to claim 49, in the manufacture of medicaments for the prevention and treatment of Crohn's disease.
51. Uso según de un producto proteico según cualquiera de las reivindicaciones 1-14, en la fabricación de medicamentos destinados a la prevención y al tratamiento de trastornos en individuos con síndromes relacionados con hepatitis .51. Use according to a protein product according to any of claims 1-14, in the manufacture of medicaments intended for the prevention and treatment of disorders in individuals with hepatitis-related syndromes.
52. Uso según de un producto proteico según cualquiera de las reivindicaciones 1-14, en la fabricación de medicamentos destinados a la prevención y al tratamiento de trastornos en individuos causados por priones .52. Use according to a protein product according to any of claims 1-14, in the manufacture of medicaments intended for the prevention and treatment of disorders in individuals caused by prions.
53. Uso según la reivindicación 52, en la53. Use according to claim 52, in the
HOJA DE SUSTITUCIÓN (REGLA 26) fabricación de medicamentos para la prevención y el tratamiento de la enfermedad de Creutzfeldt-Jakob.SUBSTITUTE SHEET (RULE 26) manufacture of medicines for the prevention and treatment of Creutzfeldt-Jakob disease.
HOJA DE SUSTITUCIÓN (REGLA 26) SUBSTITUTE SHEET (RULE 26)
PCT/ES1998/000219 1997-07-29 1998-07-28 Proteinic product, process for its preparation, compositions containing it and use in medicaments WO1999006441A1 (en)

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AU84424/98A AU8442498A (en) 1997-07-29 1998-07-28 Proteinic product, process for its preparation, compositions containing it and use in medicaments

Applications Claiming Priority (2)

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ESP9701666 1997-07-29
ES009701666A ES2142729B1 (en) 1997-07-29 1997-07-29 PROTEIN PRODUCT, PROCEDURE FOR ITS PREPARATION, COMPOSITIONS THAT CONTAIN IT, AND ITS USE IN MEDICINES.

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Citations (5)

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Publication number Priority date Publication date Assignee Title
ES400418A1 (en) * 1972-03-03 1975-01-01 Chacon Mejias Procedure for the isolation and obtaining of proteins constituting a preventive and curative vaccine of malignant tumor processes. (Machine-translation by Google Translate, not legally binding)
ES410892A1 (en) * 1973-01-23 1975-12-01 Chacon Mejias Process for the production of an injectable preparation
JPS60190720A (en) * 1984-03-09 1985-09-28 Agency Of Ind Science & Technol Antitumor agent having acyl group
WO1994022459A1 (en) * 1993-04-07 1994-10-13 Eisai Co., Ltd. Immunopotentiative and infection-protective agent, containing two or more bacillus, egg white and garlic
WO1995004539A1 (en) * 1993-08-11 1995-02-16 Ahc Inc. Immunopotentiator and method of immunopotentiating animal with the same

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES400418A1 (en) * 1972-03-03 1975-01-01 Chacon Mejias Procedure for the isolation and obtaining of proteins constituting a preventive and curative vaccine of malignant tumor processes. (Machine-translation by Google Translate, not legally binding)
ES410892A1 (en) * 1973-01-23 1975-12-01 Chacon Mejias Process for the production of an injectable preparation
JPS60190720A (en) * 1984-03-09 1985-09-28 Agency Of Ind Science & Technol Antitumor agent having acyl group
WO1994022459A1 (en) * 1993-04-07 1994-10-13 Eisai Co., Ltd. Immunopotentiative and infection-protective agent, containing two or more bacillus, egg white and garlic
WO1995004539A1 (en) * 1993-08-11 1995-02-16 Ahc Inc. Immunopotentiator and method of immunopotentiating animal with the same

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PATENT ABSTRACTS OF JAPAN *

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ES2142729A1 (en) 2000-04-16
AU8442498A (en) 1999-02-22
ES2142729B1 (en) 2000-12-16

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