FR2678949A1 - PROCESS FOR OBTAINING A PROTEIN COMPLEX WITH A STABILIZING ACTION OF DEGENERESCENCE. - Google Patents

Abstract

The process consists in subjecting a culture of living cells to various aggressive treatments. It comprises a first step during which the culture of living cells is washed and suspended in an isotonic solution, a second step during which the culture of living cells in suspension in the isotonic solution is subjected to a succession of aggressive treatments by mechanical, thermal and photonic actions, and a final step during which a physical separation operation is carried out in order to recover the protein complex.

Description

"Process for obtaining a protein action complex
stabilizer of degeneration "
The invention relates to a method for obtaining a protein complex having a stabilizing activity for degeneration and more particularly macular degeneration linked to age and diabetic retinitis.

The invention also relates to the protein complex obtained by the process.

 In patent FR-A-1532489 and BSM 7226M, there is described a process for obtaining a complex of enzymes and nucleic acids, used in particular to combat the manifestations of aging, for example in the treatment of cataract. The method consists in grinding fresh cellular tissues, kneading them in an isotonic solution, subjecting the mixture obtained to a so-called activation treatment comprising one or more cycles each formed by an sensitization phase (for example freezing then reheating) then a phase of irradiation with ultraviolet rays. The sensitization phase has the effect of diffusing the enzymes into the solution and the irradiation phase has the effect of increasing the specific activity of the enzymes released. The complex obtained contains a large amount of active agents.

 It is therefore essentially a method of activating a cell shred.

 When cells are subjected to physicochemical shocks, for example a sudden rise in temperature, a temporary deprivation of oxygen or the presence of toxic substances, a series of so-called protein syntheses is triggered inside the cells. protection, defense or repair, as well as the synthesis of a particular type of protein called shock. These shock proteins have the property of reactivating denatured proteins by restoring their spatial conformation. However, these attacks, especially by thermal shock, strongly damage cells and possible shock proteins.

 The present invention aims to overcome these shortcomings, by proposing a process for obtaining a protein complex by subjecting cellular tissues to aggressive shocks while avoiding damaging the cells.

 To this end, it proposes a method for obtaining a protein complex of the kind resulting from the application to a cellular material of an aggression treatment, characterized in that the tissue material is a culture of living cells.

 The method comprises a first step during which the culture of living cells is washed and suspended in an isotonic solution, a second step during which the culture of living cells suspended in the isotonic solution is subjected to a succession aggression treatments by mechanical, thermal and photonic actions, a last step during which a physical separation operation is carried out to collect the protein complex.

 Such a process makes it possible to obtain a complex comprising on the one hand so-called essential proteins and on the other hand shock proteins.

 The invention will be better understood and the advantages will appear more clearly in the light of the description which follows, given solely by way of example.

 The basic tissue material consists of a culture of living cells, originating from organisms belonging to the animal kingdom, preferably embryonic or placental. The culture can also have a vegetable or bacterial origin.

 Firstly, the culture of living cells is washed and suspended in isotonic liquid. The preparation thus obtained is placed in a container with a flat bottom so as to have a thin layer of solution of the order of a few millimeters in thickness.

 In a second step, the spreading preparation in a thin layer is subjected to the aggression treatments.

 The first phase is a phase of aggression by mechanical action. A non-thermal pulsed laser is used, for example a laser of the so-called Nd-YAG type (aluminum and yttrium garnet doped with neodymium). One proceeds to a monotrace scanning while endeavoring to "describe" by impacts a network of equidistant points on all the volume to be treated, by taking care not to reach a threshold which would destroy the biological activity. The goal is to have a sufficient density of points to obtain a separation of the cells. To preserve the biological activity and proceed to the following phases of aggression, the preparation resulting from this first phase is placed in a sealed bottle. The bottles are then immersed in liquid nitrogen (at -194 C) for 48 hours.

 In a second phase, thermal and photonic aggressions are carried out. The preparation undergoes rapid heating by long microwave pulses until a temperature close to 30 "C. The contents of the bottle are then placed in a container with a flat bottom to be spread out in a thin layer. then using active lasers at different wavelengths (argon or krypton laser) to an aggression by "broad checkerboard" photocoagulation impacts. It is carried out immediately after irradiation with ultraviolet A rays, alternating for 30 minutes maximum action cycles of 5 minutes and minimum action of 5 minutes. The preparation is then immediately transferred to a sealed bottle and frozen in liquid nitrogen. This sequence of operations, namely rapid warming, laser photocoagulations and UV irradiation is renewed every 12 hours For the last irradiation the UVA source is replaced by a source of germicidal UVB so as to sterilize the product.

 Finally, a physical separation is carried out to collect the protein complex. This is then placed in an isotonic solution. An isotonic colloidal solution is obtained in which the components of the complex are in homogeneous and stable suspension. The product is packaged in sealed sterile ampoules.

 Between the different stages of the process, cell culture preparations can be stored in sealed bottles at low temperature.

 The purpose of the aggression treatment is to induce the syntheses on the one hand of a large number of protection and repair proteins and on the other hand the synthesis of shock proteins. This is the effect of general biological processes following the organization of defenses at the cellular and extra-cellular level. By the process of the invention, these hostile conditions are created and even "exaggerated". The response provoked is repetitive and ubiquitous.

 the product can be administered by all the access routes of a liquid.

The product obtained has a neutral pH. It contains a significant amount of essential elements and in particular
- essential proteins
- oxidative anti-radical enzymes, including superoxide dismutases, catalases and glutathionperoxidases;
- DNA repair enzymes including DNA polymerase;
- complex molecules of ambivalent structures which can allow activating and inhibiting actions. This function typically looks like growth factor modulators.

- shock proteins, one of which has been isolated and has a formula close to arginine,
NH2 - C (= NH) - NH (CH2) 3 - CH (NH2) 3 - CH (NH2) - CO2H
Above all, the product has the property of stabilizing age-related macular degeneration (AMD) and diabetic retinitis (DR), the latter in its early stages. The complex acts, on the one hand by rebalancing the regulation of stimulation of angiogenesis by the use of modulators of growth factors present in the complex, on the other hand by using one of the properties of shock proteins which is to reactivate the denatured proteins by giving them a correct spatial structure on which essentially the activity of the protein depends. Stabilization of AMD is obtained in atrophic or "dry" forms and this over a period of ten years. In the case of treatment of DR, a resolution of punctate aneurysmal hemorrhages is observed.

The product has other properties
- the stabilization of the crystalline opacities in the cataract, even resolution of the light opacities at the beginning of affection;
- stabilization of the state of cartilage in degenerative arthritis, even resolution in their very beginning;
- stabilization of the periodontal involution, before the teeth fall, or even resolution of a slight shaking of the teeth;
- stabilization of the condition of the skin, in particular of the dermis and arrest in the epidermis of the decrease in the number of keratinocytes.

 These types of stabilization are carried out by similar mechanisms, knowing that at the level of the components of the extracellular matrix there are specific receptors for growth factors and that the lens, cartilage, periodontium and dermis are constituted by of the extracellular matrix. To stabilize angiogenesis, cells are also provided with specific growth factor receptors.

 The product also makes it possible to increase the speed of elimination and neutralization of free radicals. It also acts on myopigenic degenerations, dermal and ocular localizations of herpes and vasculitis.

 Animal experimentation confirmed for the toxicological and teratological criteria, an absence of short and long term effect.

Claims (7)

 1. Method for obtaining a protein complex of the kind resulting from the application to a tissue material of an aggression treatment, characterized in that the tissue material is a culture of living cells.  2. Method according to claim 1, characterized in that it comprises a first step during which the culture of living cells is washed and suspended in an isotonic solution, a second step during which the culture of living cells in suspension in the isotonic solution to a succession of aggression treatments by mechanical, thermal and photonic actions, a last step during which a physical separation operation is carried out to collect the protein complex.  3. Method according to claim 2, characterized in that the aggression treatment step comprises a first phase of aggression by mechanical action and a second phase of successive aggressions by thermal and photonic actions.  4. Method according to claim 2 or 3, characterized in that the aggression by mechanical action is carried out by pulsed laser impacts.  5. Method according to claim 2 or 3, characterized in that the aggression phase by thermal and photonic actions comprises at least two cycles with the succession of an aggression phase by an abrupt variation in temperature, of large amplitude, then a phase of aggression by impacts of laser photo-coagulation and a phase of aggression by irradiation with ultraviolet rays.  6. Method according to claim 5, characterized in that the irradiations of the last cycle are carried out with germicidal ultraviolet rays (wu).  7. Method according to any one of the preceding claims, characterized in that between the different stages of the method, the cell culture preparations are stored at low temperature.