FR2492663A1 - T-lymphocyte immunostimulant prepn. - contg. peptide T-activin isolated from thymus gland tissue - Google Patents

Abstract

New preparations regulating the immunological T-system contain (in addition to a pharmaceutical vehicle) as active component peptides ("T-Activin") which have a molecular weight of 1500-6000 Dalton, UV absorption maxima at 208 and 275 nm, and an electrophoretic mobility in polyacrylamide gel relative to bromophenol blue of 0.062 to 0.102, 0.156 to 0.236, 0.354 to 0.374, 0.382 to 0.422, 0.432 to 0.472, 0.485 to 0.545, and 0.850 to 0.930.S The preparation causes re-establishment of immunohomeostasis and exerts a regulatory effect on the immune and haematopoietic system, and indirectly affects the immunological B system. It can thus be used for the treatment of a wide range of primary and secondary immunodeficient states, e.g. disorders of the immune system due to various tumours, inborn immunodeficient disorders (e.g. Lous-Barsyndrome), acute viral infections (e.g. Herpes Zoster), and certain other disorders involving disturbance of immune status (e.g. psoriasis, lupus erythematosus). Details and results of in vitro and in vivo biological tests with T-activin are given in the specification. Also given are results of clinical trials in children with lymphogranulonatosis.

Description

MEDICINAL PRODUCT REGULATOR OF THE T-SYSTEM OF IMMUNITY
AND PROCESS FOR PREPARATION

 The present invention relates to the medical field. It relates more particularly to immunopharmacology, and more specifically to a drug product or regulatory drug of the T-system of immunity and the process for preparing this drug product or drug.

 The drug product according to the invention finds wide application in the treatment of primary and secondary immunodeficiency states.

This drug product promotes the regeneration of the immuno-homoeostasis of the body and has a regulatory action on the immune system as well as on the hematopoietic system. This concerns, in the first place, the T-lymphocytes and the restoration of the balance of various subpopulations of the
T-system of immunity; the product exerts, in fact, an indirect influence on the B-system of the immunity. The medicinal product according to the invention has a clinical application for eliminating disturbances in the function of the T-system of immunity. This relates in particular to the well-known disorders of the immune system resulting from various tumors, to the congenital immunodeficiency pathology. (for example, with manifestation of the syndrome
Louis Bar), acute viral infection (eg Herpes Zoster) and certain other infections with disruption of immune status, for example, psoriasis and lupo-erythemato-visceritis maligna.

 The first attempts to restore the system of immunity in cases of primary and secondary immuno-deficiency states were made by transplantation of the embryonic or neonatal thymus, the thymus en bloc with the sternum (Ju.M.Lopukhin, Ju I. Morozov, RV Petrov: "Current Problems of Organ Transplantation", Moscow, 1974, pp. 286/302).

In patients with ataxia-telangiectasia (Louis-Bar syndrome), a reproductive disorder due to the troubled function of T-cells and the absence of immunoglobulins A and E (IgA, IGE), thymic transplantation causes partial immuno-competence and positive clinical dynamics. However, several biochemical compounds are introduced into the body with the implanted thymus tissue, some of which may be toxic to the container. Inadequate vascularity and immunological conflict are the cause of rapid destruction of the transplant. During the transplantation of the thymus, it is impossible to determine the active principle administered in the body of the patient. Therefore, the therapeutic effect after transplantation of the thymus is short-lived and not always marked.All the aforementioned circumstances stimulate the interest of the development of preparations from the thymus or other organs, which must have a much higher biological activity, a wide spectrum of therapeutic action with respect to the primary and secondary immunodeficiency states caused by various actions of the environment, by malignant neoformations, preparations which do not have immunogenic properties and do not cause complications after administration.

 In the course of studying the drug product isolated from the thymus, it has been established that its biological activity is due to a protein having a molecular weight of 12,600 daltons. The process for preparing the drug product involves homogenizing the thymus tissue in NaCl, removing the precipitate from the homogenate and thermolabile proteins from the solution, precipitating the proteins and peptides in the solution. , the dissolution of the aforementioned proteins and peptides, the release of the peptides, the dissolution of the peptide precipitate, the desalination of the isolated peptides and the freeze-drying of the latter (ALGoldstein, A.Guha, et al., Proc Natl Acad Sci.USA, 69, p.1800, 1972).

Subsequent studies have shown that the protein consists of a series of polypeptides with a molecular weight of less than 1000 daltons (Hopper JA,
McDaniel MC et al., Ann. NY Acad. Sci. 249, p.125, 1975).

 Some polypeptides have been identified by polyacrylamide gel disk electrophoresis and isoelectric focusing into ampholytes.

The fraction of biologically active thymosin (V fraction) has more than 20 molecular weight components of 1000 to 15,000 daltons.

 It should be noted that certain polypeptide fractions of the thymic drug product or combination thereof have biological activity which is found to be different in in vivo and in vitro immunoassays.

Thus, alpha-thymosin with a molecular weight of 3108 and pH = 4.2 has a higher activity in the lymphocyte migration inhibition test (Low TLK, Goldstein ALIn: "The year in Hematology "
R.Silber, J.Lobuc, ASGordon et al., P.281 / 319, Plenum Publishing, New York, 1978). Other peptides obtained from the thymus, for example the ss3 and ss1 peptides, induce the synthesis of the terminal deoxynucleotidyltransferase in the precursors of the T-cells.

A number of thymic polypeptides, for example the ss1 peptide, is entirely identical to the biologically active peptides resulting from other organs, in particular ubiquitin (US Pat. No. 4,002,602, 1977). . Ubiquitin is capable of inducing the expression of in vitro T- and B-markers by activation of the cyclic adenylate ring and influencing ß-adrenergic receptors.

A humoral thymic factor capable of inducing immunohistochemical cells is also known according to the sensitivity of the rosette cells (forming the rosette) to azathioprine. The molecular weight of the humoral factor is about 56,700 daltons; in lower molecular weight fractions, only traces of biological activity were detected (White A, In: "Biochemical Action of Hormons", (G.Lidunk, ed., vol 7,
Academic Press, NY 1979).

Thus, the peptides, polypeptides and proteins of the thymus have a very large heterogeneity as much by the size of the molecules as by their biological properties. It is obvious that the peptide drug product having an effective action must contain molecules of determined molecular mass and relatively high biological activity. In this case, the unique composition of the peptides determines the amplitude and the effectiveness of their therapeutic action. In principle, the biological activity of the peptides can be enhanced by further purification, or by chemical or enzymatic modification thereof. Attempts to clinically apply drugs containing peptides for stimulating the
T-system immunity have already been done on patients with primary and secondary immune deficiency states. In the case of primary immune deficiency status (hypoplasia of thyrnus), the general condition of the patient improves after administration of a drug containing the thymic polypeptides. However, the condition of the patient immediately after the end of the immunotherapy worsens sharply (Goldstein AL, Wara DW et al .: Transplant, Proc V. 7, p.681 / 686, 1975).

The use of the drug containing polypeptides (Ve fraction of the thymus) in the case of secondary immune deficiency states, caused by malignant tumors, gave a positive effect only in 5 patients out of 32 treated patients suffering from leukemia. The general condition of a patient with chorioepithelioma has actually worsened (Shafer LA, Goldstein AL et al .:
Ann. NY Acad. Sci., V.277, p.609 / 620, 1976). It should be noted that the drug containing the polypeptides (Ve fraction) is prescribed at excessively high doses, at 400 mg / m2 / day, usually for 21 days.

Positive dynamics in immunological status have not been noted in patients with severe combined immune deficiency status (Astaldi A.,
Astaldi GCB et al .; Cancer Treat. Rep.62, p.1779, 1978).

Thus, the creation of a drug based on therapeutically more effective polypeptides with respect to primary and secondary immuno-deficits is a current problem in medicine.

 The present invention proposes to develop a medicinal product regulator of the T-system of immunity, having a higher therapeutic activity and having neither immunogeneity nor pyrogeneity.

 The object of the present invention is to seek a drug product based on polypeptides which has a high therapeutic activity and shows neither immunogeneity nor pyrogeneity.

 The problem thus posed has been solved by the development of a medicinal product regulating the T-system of immunity which, according to the invention, is characterized in that it contains as active principle peptides having a molecular weight in the range of 1500 to 6000 daltons, having a maximum in the ultraviolet adsorption spectrum at 208 and 275 nm and an electrophoretic mobility in a gel with respect to bromophenol blue for: 0.062 0.102; 0.156 to 09,236; 0.354 to 0.37; 0.382 to 0.422; 0.432-0.472, 0.485-0.545; 0.850-0.930, and in that it further contains a pharmaceutically acceptable excipient.

 The regulatory drug product of the T-system of immunity is hereafter called "T-activin".

 The excipient for the named drug product is the physiological solution. The physiological solution is a 0.14M solution of NaCl.

The active ingredient content of the claimed drug product is 100-200 mcg / ml injected.

 The drug product according to the invention has an immunostimulatory activity specific for in vitro T-lymphocytes. In the test for restoring the sensitivity of the rosette-forming T-cells to azathioprine, the product exhibits an activity at a dose of 1 mcg / 3 × 10 6 lymphocytes. The drug product has a regulatory action of the T-system of immunity during its disorders in patients with malignant neoformations, psoriasis and some other diseases.

The regulatory action of T-activin is manifested by the normalization of the amount and balance of T-lymphocyte populations in cases of decreased or elevated activity in patients. The product exerts a lytic action on primary neoplastic tissue and metastases.

It potentiates the specific polychemotherapy, allows to perform local radiation therapy of tumors. T-activin causes stable and prolonged regeneration of T-imnnnity in patients with disorders of the immune system due to the primary or secondary immunodeficiency state and exerts a marked therapeutic action in the case of Barr-Barr syndrome , as well as psoriasis. In addition, the drug product abolishes symptoms of toxicity, normalizes body temperature and neurological status for all forms of the conditions mentioned.

 The drug product does not cause complications, pyrogenic effect or appearance of specific antibodies or idiosyncrasy.

The method of preparing the regulatory drug product of the T-system of immunity comprises the following steps:
1 - Homogenization of Thymus Tissue in NaCl
2 - Elimination of the precipitate formed from the product of the homogenization
3 - Elimination of thermolabile proteins from the solution
4 - Precipitation of proteins and peptides in the solution
s - Dissolution of proteins and precipitated peptides
6 - Releasing peptides
7 - Dissolution of the peptide precipitate
8 - desalting peptides and their lyophilization and is characterized, according to the invention, in that:
9 - after step (1), the product of the homogenization is maintained at a
at 2 ° to 60 ° C., for 12 to 16 hours, the peptides are released in three stages:
a first step: in a solution with 20-30% of the saturation in
ammonium sulfate, at a pH of 6.9-751
a second step: in a 40-55% solution of the saturation in
ammonium sulfate, at a pH of 3.9-4.1 and
a third step: in a 45-552 solution of the saturation in
ammonium sulfate, at pH 4.5-6.0; The peptide solution is subjected after step (7) to an ultra
filtration through a membrane having a nominal
retention of 12 000 to 30 000 daltons depending on the proteins globu
and 12 - the ultrafiltrate obtained by isolating the
peptides having a molecular mass of 1500 to 6000 daltons in
buffer solution at a pH of 6.8-8.2 and an ionic strength of
0.1 to 0.4M and after step (8); The peptides are mixed with a pharmaceutically acceptable excipient.

TABLE I
Restoration of rosette cells with in vitro T-activin

<tb><SEP>%<SEP> of <SEP> T-cells <SEP> to <SEP> rosettes <SEP> in
<tb><SEP> The <SEP> blood <SEP> device
<tb><SEP> NO <SEP>
<tb><SEP> of <SEP> Diagnose <SEP> AFTER
<tb> sick <SEP> BEFORE <SEP><SEP> treatment with <SEP> The
<tb><SEP> treatment <SEP> T-attivine
<tb><SEP> (1 <SEP> meg <SEP> on <SEP> 2x105
<tb><SEP> lymphocytes)
<tb><SEP> 1 <SEP> Lymphosarcoma <SEP> 22 <SEP> 46
<tb><SEP> 2 <SEP> lymphosarcoma <SEP> 34 <SEP> 55
<tb><SEP> 3 <SEP> Lymphosarcoma <SEP> 30 <SEP> 56
<tb><SEP> 4 <SEP> Lymphosarcoma <SEP> 16 <SEP> 37
<tb><SEP> 5 <SEP> lymphogranulomatosis <SEP> 34 <SEP> 67
<tb><SEP> 6 <SEP> lymphogranulomatosis <SEP> 54 <SEP> 83
<tb><SEP> 7 <SEP> Lymphogranulomatosis <SEP> 41 <SEP> 80
<tb><SEP> 8 <SEP> Lymphogranulomatosis <SEP> 42 <SEP> 73
<tb><SEP> 9 <SEP> leukemia <SEP> lymphoid <SEP> chronic <SEP> 44 <SEP> 70
<tb><SEP> 10 <SEP> psoriasis <SEP> 42 <SEP> 51
<tb><SEP> 11 <SEP> psoriasis <SEP> 42 <SEP> 59
<tb><SEP> 12 <SEP> psoriasis <SEP> 12 <SEP> 60
<tb><SEP> 13 <SEP> psoriasis <SEP> 23 <SEP> 59
<Tb>
It is desirable to conduct the release in step (10) at a protein and peptide content of 15 to 25 mg / ml of the solution. This results in a more complete yield of biologically active peptides.

 It is advantageous to maintain the product of the homogenization in step (2) such that the ratio of the solid and liquid phases is equal to 1/3, and to carry out gel chromatography of the peptides in step (12). in a buffer solution containing KCl or NaCl.

The drug product according to the invention, consisting of T-activin, has a high immunostimulatory activity with respect to T-lymphocytes in vitro. In the test for inhibition of the spontaneous formation of azathioprine rosettes, its action is seen at the dose of 1 mcg on 3X106 of lymphocytes. The product has the ability to restore the formation of
T-rosettes in case of disorders of the immune system, including the case of oncological conditions and other conditions.

After the treatment of lymphocytes with oncological diseases and other diseases with the aid of the drug product according to the invention, the normalization of the content of rosette-forming T-cells (see Table I) is observed in vitro. above). 1
The ability to regenerate the immunological response in animals under the action of T-activin results from experimental data obtained on hymectomized mice and mice with aplasia of the thymus.

désigne l'introduction des érythrocytes aux animaux thymectomisés normaux; désigne l'introduction des érythrocytes aux animaux thymectomisés; désigne l'introduction des érythrocytes avec administration de La
T-activine aux animaux thymectomisés.The thymectomized mice acquire under the action of the drug product the ability to form the rosette cells in various time after removal of the thymus. Data on tests for the determination of the influence of T-activin at a dose of 40 mcg / m, 5, 35 and 245 days after thymectomy on the immune response of sheep thymectomized mice (rosette cells) are shown on the graph I, on which on the abscissa axis are given the amounts of cells with pink heads in 103 of spleen nuclei cells, and on the ordinate axis, the delays (days) after the thymectomy. ;;

refers to the introduction of erythrocytes to normal thymectomized animals; refers to the introduction of erythrocytes to thymectomized animals; designates the introduction of erythrocytes with administration of La
T-activin to thymectomized animals.

 It can be seen in Chart I that the restoration of T-cell rosette formation to normal values is noted in animals 245 days at the thymectomy, that is to say even during the period of physiological aging of the 'organization.

désigne l'immunisation avec les érythrocytes du mouton, désigne l'immunisation avec Les érythrocytes du mouton avec L'adminis
tration de la T-activine.The indirect influence, through the T-immune system, of T-activin on the B-immune system after immunization of "Nude" mice with sheep erythrocytes, according to the test of the cells forming the antibodies, is shown in graph 2, where on the x-axis is designated the amount of antibody-forming cells on 106 nucleus cells;

refers to immunization with sheep erythrocytes, means immunization with sheep erythrocytes with
T-activin.

 It is found that under the action of T-activin, the mice of the "nude" race acquire a normal immunological response to the T-antigen, particularly dependent on the erythrocytes of the sheep, and that antibody cells are formed. specific. The results obtained show a regulatory action of T-activin on the body's immune system.

 The drug product has a specific action only on the organs of the immune system: the thymus, the lymph nodes and the spleen.

After administration of T-activin9, a stimulation of proliferation of lymphoid cells and deoxynucleic acid synthesis is observed in these organs.

According to the light microscopy data, no morphological changes are noted in other organs: liver, kidneys, heart, intestinal wall, lungs, spinal cord, and encephalon.

The drug product is non-toxic and does not affect the physiological functions of the nervous system, the circulation of the circulation in doses exceeding 100 times the therapeutic dose
T-activin does not cause disorders of hematopoiesis and peripheral blood cell composition in healthy animals, as is evident from the data in Tables II (a, b, c) and T.III ( a and b) below.

 The most important property of T-activin is its positive effect with respect to the normalization of the T-immune system indices and its indirect action on the B-immune system of the body in case of various pathological conditions, as well as that its direct therapeutic action in cases of primary immunodeficiency states: for example, ataxia-telangiectasia (Lois-Barr syndrome) and, in the case of secondary immunodeficiency states, including malignant tumors, in particular lymphogranulomatosis, lymphosarcoma, retino-bl astome, etc.

 In the evaluation of immunological status in patients with primary immunodeficiency states, functional disorders of T- and B-systems have been noted, manifesting in decreased ability of T-lymphocytes to blast-transformation, and by lowering the amount of serum immunoglobulin A (IgA).

The data are shown in Table IV, also below.

ERYTHROCYTE COMPOSITION OF PERIPHERAL BLOOD OF EXPERIMENTAL ANIMALS
TABLE IIa
one week after administration of T-activin

<tb><SEP> Control <SEP> Dose <SEP> Dose <SEP> 20 <SEP> times
<tb><SEP> Indices <SEP> @@@@@@@@ SEP><SEP> therapeutic <SEP> multiplied
<tb><SEP> M <SEP> + <SEP> m <SEP> M <SEP> T <SEP> m <SEP> n <SEP> @p <SEP>: p <SEP> M <SEP> + <SEP> m <SEP> :: p <SEP>
<tb><SEP> Hememglobin <SEP> g /% <SEP> 12.0 # 0.57 <SEP> 11.70 # 0.15 <SEP>> 0.02 <SEP> 11.1 # 0.5 <SEP><SEP>> 0.1
<tb><SEP> Erythrocytes <SEP> mln / l <SEP> 4.2 # 0.21 <SEP><SEP> 5.9 # 0.16 <SEP><0.00<SEP> 6.0 # 0.38 <SEP><SEP><0.001
<tb> Index <SEP> of <SEP> staining <SEP> 0.86 # 0.02 <SEP><SEP> 0.54 # 0.01 <SEP><SEP><0.00<SEP> 0, 54 # 0.01 <SEP><SEP>> 0.001
<Tb>
TABLE IIb .. two weeks after administration of T-activin

<tb><SEP> Control <SEP> Dose <SEP> Dose <SEP> 20 <SEP> times
<tb><SEP> Multiple <SEP> therapeutic index <SEP> multiplied
<tb><SEP> M <SEP>#<SEP> m <SEP><SEP> M <SEP>#<SEP> m <SEP><SEP> p <SEP> M <SEP>#<SEP> m <SEP> p
<tb> Hemoglobin <SEP> g /% <SEP> 12.0 # 0.57 <SEP><SEP> 13.9 # 1.10 <SEP><SEP> 0.01 <SEP> 14.1 # 0 , 40 <SEP><SEP> 0.001
<tb><SEP> Erythrocytes <SEP> mln / l <SEP> 4.0 # 0.21 <SEP><SEP> 6.2 # 0.16 <SEP><SEP> 0.001 <SEP> 6.3 # 0.38 <SEP><SEP> 0.001
<tb><SEP> Index <SEP> of <SEP> staining <SEP> 0.86 # 0.02 <SEP><SEP> 0.7 # 0.02 <SEP><SEP> 0.001 <SEP> 0, 75 # 0.03 <SEP><SEP> 0.001
<Tb>
TABLE one year after administration of T-activin

<tb><SEP> Dose
<tb><SEP> Therapeutic <SEP> Control
<tb><SEP> Indices
<tb><SEP> M <SEP>#<SEP><SEP> m <SEP> M <SEP>#<SEP> m <SEP><SEP> p
<tb> Hemoglobin <SEP> g /% <SEP> 15.2 <SEP>#<SEP><SEP> 0.66 <SEP> 15.5 <SEP>#<SEP> 0.75 <SEP><SEP> 0.2
<tb> Erythrocytes <SEP> mln / l <SEP> 6.2 <SEP>#<SEP> 0.28 <SEP><SEP> 6.7 <SEP>#<SEP><SEP> 0.2 <SEP > 0,1
<tb> Index <SEP> of <SEP> staining <SEP> 0.84 <SEP>#<SEP> 0.04 <SEP><SEP> 0.78 <SEP>#<SEP><SEP> 0.02 <SEP> 0.1
TABLE IIIa Leukocyte Composition of Peripheral Blood of Experimental Animals after Dose Administration
Therapeutic and 20-fold increased dose of T-activin 14 days after introduction of the preparation

<SEP> Control <SEP> Dose <SEP> therapeutic <SEP> Dose <SEP> 20 <SEP> times <SEP> multiplied
<tb><SEP> Indices <SEP> M <SEP>#<SEP> m <SEP> M <SEP>#<SEP> m <SEP> p <SEP> M <SEP>#<SEP> m <SEP> p
<tb><SEP> Leutocytes <SEP> 10 <SEP> in <SEP> l <SEP> 4.7 <SEP>#<SEP> 0.3 <SEQ> 4.95 <SEP>#<SEP> 0, <SEP> 0.2 <SEQ> 4.64 <SEP>#<SEP> 0.12 <SEP> 0.2
<tb><SEP> Eosinophils, <SEP>% <SEP> 3.0 <SEP>#<SEP> 0.8 <SEP> 0.50 <SEP>#<SEP> 0.2 <SEP> 0.01 <SEP> 0.50 <SEP>#<SEP> 0.2 <SEP> 0.01
<tb><SEP> Non-Segmented <SEP> Neutrophils, <SEP>% <SEP> 1.5 <SEP>#<SEP> 0.3 <SEP> 3.50 <SEP>#<SEP> 1.3 <SEP> 0.1 <SEP> 3.50 <SEP> + <SEP> 0.4 <SEP> 0.01
<tb><SEP> Segmented <SEP> Neutrophils, <SEP>% <SEP> 14.0 <SEP>#<SEP> 2.6 <SEP> 18.50 <SEP>#<SEP> 2.7 <SEP > 0.1 <SEP> 13.25 <SEP> + <SEP> 3.6 <SEP> 0.05
<tb><SEP> Lymphocytes, <SEP>% <SEP> 76.5 <SEP>#<SEP> 3.2 <SEP> 74.0 <SEP>#<SEP> 2.5 <SEP> 0.05 <SEP> 81.0 <SEP> + <SEP> 4.2 <SEP> 0.05
<tb><SEP> Monocytes, <SEP>% <SEP> 5.0 <SEP>#<SEP> 1.1 <SEP> 3.5 <SEP>#<SEP> 1.1 <SEP> 0.05 <SEP> 1.75 <SEP>#<SEP> 0.2 <SEP> 0.05
<tb><SEP><SEP><SEP> SEP <SEP><SEP><SEP> Neutrophils <SEP><SEP><SEP><SEP> 0.01 <SEP> 0.19 <SEP>#<SEP> 0.13 <SEP> 0.05 <SEP> 0.26 <SEP>#<SEP> 0.08 <SEP> 0.05
<tb> TABLE IIIb * Experimental animals' peripheral blood leukocyte composition after dose administration
and a 20-fold increase in T-activin one year after introduction of the drug product

<SEP> Control <SEP> Dose <SEP> therapeutic <SEP> Dose <SEP> 20 <SEP> times <SEP> multiplied
####
<tb> Leukocytes, <SEP> 10 <SEP> in <SEP> 1 <SEP> 4.7 <SEP>#<SEP> 0.3 <SEP> 4.10 <SEP>#<SEP> 0.25 <MS> 0.1 <SEP> 3.9 <SEP>#<SEP> 0.3 <SEP> 0.1
<tb> Eosinophils, <SEP> in <SEP>% <SEP> 3.0 <SEP>#<SEP> 0.8 <SEP> 1.50 <SEP>#<SEP> 0.2 <SEP> 0, 02 <SEP> 1.8 <SEP>#<SEP> 0.2 <SEP> 0.02
<tb> Non-segmented <SEP> Neutrophils <SEP><SEP>%<SEP> 1.5 <SEP>#<SEP> 0.3 <SEP> 2.90 <SEP>#<SEP> 0, <SEP> 0.02 <SEP> 3.1 <SEP>#<SEP> 0.5 <SEP> 0.02
<tb> Segmented <SEP> Neutrophils, <SEP>% <SEP> 14.0 <SEP>#<SEP> 2.6 <SEP> 17.10 <SEP>#<SEP> 1.1 <SEP> 0, 02 <SEP> 18.3 <SEP>#<SEP> 2.1 <SEP> 0.03
<tb> Lymphocytes, <SEP>% <SEP> 76.5 <SEP>#<SEP> 3,2 <SEP> 79,10 <SEP>#<SEP> 2,3 <SEP> 0,1 <SEP> 69.1 <SEP>#<SEP> 3.4 <SEP> 0.1
<tb> Monocytes, <SEP>% <SEP> 5.0 <SEP>#<SEP> 1.1 <SEP> 3.40 <SEP>#<SEP> 0.6 <SEP> 0.1 <SEP> 4.5 <SEP>#<SEP> 0.5 <SEP> 0.1
<tb><SEP><SEP><SEP>SEP> SEP <SEP><SEP> Neutrophils <SEP><SEP><SEP><0.01<SEP> 0.15 <SEP>#<SEP> 0.05 <SEP> 0.2 <SEP> 0.13 <SEP>#<SEP> 0.03 <SEP> 0.2
<tb> TABLE IV * Dynamics of change in serum immunoglobulin concentration (in mg /%)
in children with Lois-Barr syndrome

<SEP> BEFORE <SEP> i @@ unotherapy <SEP> AFTER <SEP> immunotherapy <SEP> AFTER <SEP> immunotherapy
<tb><SEP> (7th <SEP> day) <SEP> (14th <SEP> day)
<tb><SEP> N <SEP> i <SEP> m <SEP> m <SEP> u <SEP> n <SEP> o <SEP> g <SEP> l <SEP> o <SEP> b <SEP> u <SEP> l <SEP> i <SEP> n <SEP> e <SEP> s
<tb><SEP> IgA <SEP> IgG <SEP> IgM <SEP> IgA <SEP> IgG <SEP> IgM <SEP> IgA <SEP> IgG <SEP> IgM
####
<tb><SEP> 1 <SEP> 45 <SEP> 350 <SEP> 49 <SEP> 74 <SEP> 330 <SEP> 505 <SEP> 82 <SEP> 560 <SEP> 103
<tb><SEP> 2 <SEP> 110 <SEP> 1680 <SEP> 280 <SEP> 190 <SEP> 2460 <SEP> 500 <SEP> 48 <SEP> 2250 <SEP> 360
<tb><SEP> 3 <SEP> 56 <SEP> 960 <SEP> 70 <SEP> - <SEP> - <SEP> - <SEP> 38 <SEP> 1030 <SEP> 140
<tb><SEP> 4 <SEP> - <SEP> 1260 <SEP> 250 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP>
<SEP> 5 <SEP> 74 <SEP> 1140 <SEP> 90 <SEP> 8 <SEP> 1500 <SEP> 250 <SEP> 16 <SEP> 1350 <SEP> 350
<tb><SEP> 6 <SEP> 58 <SEP> 690 <SEP> 250 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> average <SEP> 69 <SEP>#SEP> 23 <SEP> 964 <SEP>#<SEP> 444 <SEP> 146 <SEP>#<SEP> 93 <SEP> - <SEP> - <SEP> - <SEP> 46 <SEP>#<SEP> 27 <SEP> 1297 <SEP>#<SEP> 198 <SEP> 238 <SEP>#<SEP> 133
<Tb>
As can be seen from Table IV, above, after administration of the drug product for 7 days, there is a tendency to restore the functional activity of T-lymphocytes in the blast-transformation reaction assay. The amount of IgA, G, M serum immunoglobulins increased, indicating indirect stimulation of the B-system of immunity.

 The clinical effect occurs 3 days after administration of the product and is expressed by the positive dynamics of the syndrome of rigid and hyperkinetic acinetic behavior, by the restoration of the speed of propagation of the impulse in the nerve fibers and by the increase of 1.5-2 times the strength of muscle contractions.

 During the evaluation of the immunological status in patients suffering from lymphogranulomatosis with secondary immunodeficiency status, there are quantitative and functional disorders of the T-system of immunity.

Table V below summarizes the data showing the decrease of the absolute content of T-lymphocytes and their functional activity in the blast-transformation test with phyto-hemagglutinin (PhGA). As is apparent from the IgA, IgG, IgM content, functional disorders of the B-system are not observed. [Cf. Table V (a and b) J.

 After administration of T-activin, the recovery of the absolute content of T-lymphocytes is observed. In the group of patients with a diminished T-cell rosette, it is observed that the index is restored to the norm. Functional activity has also recovered to normal levels.

The data obtained induce the regulatory action of T-activin with respect to cellular immunity. The data is represented in the
Table VI (a and b). also below.

TABLE Va * Clinico-immunological Characteristics of Children with Lymphogranulomatosis (LMG) Before Immunotherapy

N <SEP> of <SEP> patient
<tb><SEP> and <SEP> Stage <SEP> Number <SEP>% <SEP> Number
<tb><SEP><SEP> Histological variant <SEP> absent <SEP> of <SEP> from <SEP> E-cells <SEP> absent <SEP> from
<tb><SEP> of <SEP> LGM <SEP> lymphocytes <SEP> to <SEP> rosette <SEP> T-lymphocytes
<tb> its <SEP> age <SEP> (year) <SEP> more <SEP> from <SEP> 1500 <SEP> 65 <SEP> more <SEP> from <SEP> 1000
<tb><SEP> Standards
<tb><SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6
<tb><SEP> 1 <SEP> 7 <SEP> II <SEP> Aa <SEP> variant <SEP><SEP> mixed <SEP> 726 <SEP> 29 <SEP> 211
<tb><SEP> 2 <SEP> 6 <SEP> III <SEP> Ab <SEP>"<SEP>"<SEP>"<SEP> 1300 <SEP> 30 <SEP> 390
<tb><SEP> 3 <SEP> 3 <SEP> II <SEP> Bb <SEP>"<SEP>"<SEP>"<SEP> 1080 <SEP> 36 <SEP> 388
<tb><SEP> 4 <SEP> 3.5 <SEP> III <SEP> Ab <SEP>"<SEP>"<SEP>"<SEP> 2940 <SEP> 32 <SEP> 940
<tb><SEP> 5 <SEP> 7 <SEP> III <SEP> Ab <SEP>"<SEP>"<SEP>"<SEP> 1480 <SEP> 25 <SEP> 370
<tb><SEP> 6 <SEP> 3.5 <SEP> II <SEP> Ab <SEP>"<SEP>"<SEP>"<SEP> 875 <SEP> 46 <SEP> 402
<tb><SEP> 7 <SEP> 13 <SEP> III <SEP> Bb <SEP>"<SEP>"<SEP>"<SEP> 1533 <SEP> 46 <SEP> 720
<tb><SEP> 8 <SEP> 7 <SEP> III <SEP> Ab <SEP>"<SEP>"<SEP>"<SEP> 2394 <SE> 46 <SEP> 1110
<tb><SEP> 9 <SEP> 8 <SEP> II <SEP> Bb <SEP>"<SEP>"<SEP>"<SEP> 1495 <SEP> 52 <SEP> 748
<tb><SEP> 10 <SEP> 7 <SEP> III <SEP> Bb <SEP> predominance <SEP> lymphoid <SEP> 2490 <SEP> 60 <SEP> 1494
<tb><SEP> 11 <SEP> 7 <SEP> II <SEP> Ab <SEP> mixed <SEP> variant <SEP> mixed <SEP> 2223 <SEP> 40 <SEP> 889
<tb><SEP> 12 <SEP> 9 <SEP> II <SEP> Bb <SEP> mixed <SEP> variant <SEP> mixed <SEP> 2070 <SEP> 41 <SEP> 848
<tb><SEP> 13 <SEP> 4 <SEP> III <SEP> Ab <SEP> variant <SEP><SEP> mixed <SEP> 1330 <SEP> 68 <SEP> 904
<tb> TABLE Vb

<SEP> Reaction <SEP> of <SEP> blast-transformation <SEP> Concentration <SEP> in <SEP> serum immunoglobulins <SEP>
<tb><SEP> with <SEP> phHA <SEP> (mg /%)
<tb><SEP> X <SEP> EAC <SEP> * <SEP> A <SEP> M <SEP> G
<tb><SEP> background <SEP> phHA ** <SEP> index
<tb><SEP> plus <SEP> of <SEP> 40 <SEP> 10 <SEP> - <SEP> 30 <SEP> 50 <SEP> - <SEP> 200 <SEP> 70 <SEP> - <SEP> 200 <SEP> 950 <SEP> - <SEP> 1980
<tb><SEP> 7 <SEP> 8 <SEP> 9 <SEP> 10 <SEP> 11 <SEP> 12 <SEP> 13
<tb><SEP> 424 <SEP> 18 <SEP> 869 <SEP> 30.3 <SEP> 40 <SEP> 195 <SEP> 40 <SEP> 950
<tb><SEP> 346 <SEP> 14 <SEP> 355 <SEP> 41.4 <SEP> 20 <SEP> 320 <SEP> 160 <SEP> 2 <SEP> 100
<tb><SEP> 115 <SEP> 3 <SEP> 350 <SEP> 291.0 <SEP> 24
<tb><SEP> 28 <SEP> 208 <SEP> 220 <SEP> 1 <SEP> 400
<tb><SEP> 152 <SEP> 1 <SEP> 357 <SEP> 9.0 <SEP> 20 <SEP> 150 <SEP> 90 <SEP> 1 <SEP> 520
<tb><SEP> 130 <SEP> 2 <SEP> 352 <SEP> 18.0 <SEP> 30 <SEP> 116 <SEP> 60 <SEP> 1 <SEP> 320
<tb><SEP> 22 <SEP> 376 <SEP> 160 <SEP> 2 <SEP> 000
<tb><SEP> 80 <SEP> 2 <SEP> 527 <SEP> 31.3 <SEP> 23 <SEP> 125 <SEP> 135 <SEP> 1 <SEP> 050
<tb><SEP> 439 <SEP> 9 <SEP> 810 <SEP> 22.3 <SEP> 25
<tb><SEP> 5.7 <SEP> 30 <SEP> 210 <SEP> 120 <SEP> 1 <SEP> 680
<tb><SEP> 286 <SEP> 6 <SEP> 830 <SEP> 23 <SEP> 25 <SEP> 62 <SEP> 70 <SEP> 1 <SEP> 500
<tb><SEP> 14
<tb><SEP> 585 <SEP> 16 <SEP> 773 <SEP> 28.6 <SEP> 37 <SEP> 72 <SEP> 130 <SEP> 1 <SEP> 380
<tb> * EAC = B-rosette cells ** phHA = phytohemoglutinine TABLE VIa * Clinoco-immunological characteristics of children with lyophilogranulomatosis

N <SEP> of <SEP> patient
<tb><SEP> and <SEP> Stage <SEP> Number <SEP>% <SEP> Number
<tb><SEP><SEP> Histological variant <SEP> absent <SEP> of <SEP> from <SEP> E-cells <SEP> absent <SEP> from
<tb><SEP> its <SEP> age <SEP> (year) <SEP> of <SEP> LGM <SEP> lymphocytes <SEP> to <SEP> rosette <SEP> T-lymphocytes
<tb><SEP> Standard <SEP> plus <SEP> of <SEP> 1500 <SEP> 65 <SEP> plus <SEP> of <SEP> 1000
<tb><SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 65 <SEP> 6
<tb><SEP> 1 <SEP> 7 <SEP> II <SEP> Aa <SEP> variant <SEP><SEP> mixed <SEP> 1178 <SEP> 66 <SEP> 777
<tb><SEP> 2 <SEP> 6 <SEP> III <SEP> Ab <SEP>"<SEP>"<SEP>"<SEP> 1316 <SEP> 65 <SEP> 855
<tb><SEP> 3 <SEP> 3 <SEP> II <SEP> Bb <SEP>"<SEP>"<SEP>"<SEP> 1566 <SEP> 67 <SEP> 1049
<tb><SEP> 4 <SEP> 3.5 <SEP> III <SEP> Ab <SEP>"<SEP>"<SEP>"<SEP> 2288 <SEP> 68 <SEP> 1556
<tb><SEP> 5 <SEP> 7 <SEP> III <SEP> Ab <SEP>"<SEP>"<SEP>"<SEP> 1184 <SEP> 68 <SEP> 805
<tb><SEP> 6 <SEP> 3.5 <SEP> II <SEP> Ab <SEP>"<SEP>"<SEP>"<SEP> 2544 <SEP> 68 <SEP> 1248
<tb><SEP> 7 <SEP> 13 <SEP> III <SEP> Bb <SEP>"<SEP>"<SEP>"<SEP> 1540 <SEP> 72 <SEP> 1108
<tb><SEP> 8 <SEP> 7 <SEP> III <SEP> Ab <SEP>"<SEP>"<SEP>"<SEP> 1591 <SEP> 68 <SEP> 1081
<tb><SEP> 9 <SEP> 8 <SEP> II <SEP> Bb <SEP>"<SEP>"<SEP>"<SEP> 1664 <SEP> 64 <SEP> 1064
<tb><SEP> 10 <SEP> III <SEP> Bb <SEP> predominance <SEP> Lymphoid <SEP> 3180 <SEP> 55 <SEP> 1749
<tb><SEP> 11 <SEP> 7 <SEP> II <SEP> Ab <SEP> mixed <SEP> variant <SEP> mixed <SEP> 1071 <SEP> 67 <SEP> 717
<tb><SEP> 12 <SEP> 9 <SEP> IV <SEP> Bb <SEP>"<SEP>"<SEP>"<SEP> 1040
<tb><SEP> 13 <SEP> 4 <SEP> III <SEP> Ab <SEP>"<SEP>"<SEP>"<SEP> 1308 <SE> 54 <SEP> 707
<tb> TABLE VIb

<SEP> Reaction <SEP> of <SEP> blast-transformation <SEP> Concentration <SEP> in <SEP> serum immunoglobulins <SEP>
<tb><SEP> with <SEP> phHA <SEP> (mg /%)
<tb><SEP>%<SEP> EAC
<tb><SEP> background <SEP> phHA <SEP> index <SEP> E <SEP> M <SEP> G
<tb><SEP> plus <SEP> of <SEP> 40 <SEP> 10 <SEP> - <SEP> 30 <SEP> 50 <SEP> - <SEP> 200 <SEP> 70 <SEP> - <SEP> 200 <SEP> 950 <SEP> - <SEP> 1980
<tb><SEP> 7 <SEP> 8 <SEP> 9 <SEP> 10 <SEP> 11 <SEP> 12 <SEP> 13
<tb><SEP> 149 <SEP> 23 <SEP> 965 <SEP> 160 <SEP> 26 <SEP> 200 <SEP> 60 <SEP> 860
<tb><SEP> 375 <SEP> 19 <SEP> 962 <SEP> 55 <SEP> 22 <SEP> 400 <SEP> 270 <SEP> 2 <SEP> 500
<tb><SEP> 184 <SEP> 8 <SEP> 720 <SEP> 29 <SEP> 19
<tb><SEP> 107 <SEP> 28 <SEP> 210 <SEP> 144 <SEP> 1 <SEP> 680
<tb><SEP> 714 <SEP> 66 <SEP> 282 <SEP> 92 <SEP> 23 <SEP> 135 <SEP> 90 <SEP> 1 <SEP> 120
<tb><SEP> 248 <SEP> 8 <SEP> 437 <SEP> 34 <SEP> 35 <SEP> 120 <SEP> 120 <SEP> 1 <SEP> 740
<tb><SEP> 330 <SEP> 4 <SEP> 900 <SEP> 14 <SEP> 25 <SEP> 210 <SEP> 310 <SEP> 1 <SEP> 440
<tb><SEP> 308 <SEP> 9 <SEP> 512 <SEP> 30 <SEP> 48 <SEP> 90 <SEP> 85 <SEP> 1 <SEP> 200
<tb><SEP> 200 <SEP> 7 <SEP> 545 <SEP> 38 <SEP> 19 <SEP> 50 <SEP> 30 <SEP> 480
<tb><SEP> 420 <SEP> 17 <SEP> 902 <SEP> 47 <SEP> 20 <SEP> 320 <SEP> 80 <SEP> 1 <SEP> 440
<tb><SEP> 273 <SEP> 17 <SEP> 046 <SEP> 62 <SEP> 20 <SEP> 280 <SEP> 500 <SEP> 200
<tb><SEP> 1513 <SEP> 34 <SEP> 433 <SEP> 22 <SEP> 40 <SEP> 45 <SEP> 70 <SEP> 1 <SEP> 050
<Tb>
A particularly important property of the medicinal product is its therapeutic action with regard to the oncological conditions on the background of immunodeficiency states in patients. The optimal therapeutic action is revealed after administration of the drug product by injections at a daily dose of 20%. at 40 mcg / l m2 of the body surface.

In patients with malignant neoformations with specific intoxication, then, even at the first day, the normalization of temperature, improvement of the general state, and appetite are observed.

On the background of immunotherapy with T-activin in children suffering from lymphogranulomatosis at the stage of generalization of the process (II-IVth stages), the first day after the introduction of the medicinal product, the destruction, the softening and the removal of certain lymph nodes from the conglomerate of affected lymph nodes and subsequently the 60-70% decrease in the affected lymphatic nodes compared to the initial dimensions at day 7-14.

Analysis of cytograms of affected lymph nodes during T-activin treatment, shows increase in lymphocyte lymphocyte content from 70-80% to 95-98% on the background of lymph node effect, the specific Berezovsky-Sternberg cells are destroyed.

In children with a marked manifestation of the biological activity of the process, there is normalization of erythrocyte sedimentation rate (ESV) and cessation of intoxication.

avant administration de la T-activine, après administration de La T-activine.The data are shown in Chart 3a and 3b, where:
a is the influence on the symptoms of specific intoxication
'b is the influence on the biological activity of the process (VSE),

before administration of T-activin after administration of T-activin.

On the background of T-activin, the absolute content of peripheral T-lymphocytes, the content of E-cells with rosettes and the index of stimulation of the lymphocytes are normalized.

After immunotherapy of T-activin of patients with malignant neoformations, it becomes possible to apply multidrug therapy, more effective, or local radiotherapy.

We then note a rapid remission of the malignant disease (Table VII, hereafter) a positive dynamic of the lymphonoduletic effect (Chart 4) where, according to the abscissae, the lymph node surfaces in cm 2 are indicated and, according to the ordinates, the days since the beginning of the treatment.

désigne les périodes d'administration de la T-activine, la ligne désigne la période d'absence de la thérapie médicamenteuse, la ligne désigne la periode de la chimiothérapie. The line

refers to the periods of administration of T-activin, the line refers to the period of absence of drug therapy, the line refers to the period of chemotherapy.

TABLE VII
The effect of T-activin on the deletions of remission of lymphogra
nullomatosis in children

<tb><SEP> Groups <SEP> of <SEP> patients <SEP> Time <SEP> of <SEP><SEP> remission <SEP> (months)
<tb><SEP> No <SEP> of
<tb> Nort <SEP>
<tb> I <SEP> - <SEP> After <SEP> immunothe- <SEP> 1.5 <SEP> - <SEP> 2 <SEP> 6.0 <SEP> 12 <SEP> @ emi @@ ion <SEP> Death <SEP>
<tb><SEP> rapie <SEP> with <SEP> la
<tb><SEP> T-activin, <SEP> Number <SEP> d <SEP> children
<tb><SEP> on <SEP> 13 <SEP> children
<tb><SEP> 12 <SEP> | <SEP> - <SEP> - <SEP>
<tb> 2 <SEP> - <SEP> Children <SEP> no <SEP> rece
<tb><SEP> before <SEP> not <SEP> of <SEP> 2 <SEP> 17 <SEP> l <SEP> I <SEP> | <September>
<tb><SEP> T-activin,
<tb><SEP> on <SEP> 22 <SEP> children
<Tb>
TABLE VIII Effect of T-activin on the frequency of complications in children
with lymphogranulomatosis

<tb><SEP> Groups <SEP> of <SEP> patients <SEP> Forms <SEP> of <SEP> complications <SEP>
<tb><SEP> Insufficiency <SEP> immunolo
<SEP> Cytotoxic <SEP><SEP> induced SEP> Disease
<tb><SEP> (number <SEP> of children) <SEP> (number <SEP> of children)
<tb> With <SEP> Immunotherapy
<tb> to <SEP> the <SEP> T-activin, <SEP> 1
<tb> on <SEP> 13 <SEP> children
<tb> Without <SEP> immunotherapy
<tb> to <SEP> to <SEP> the <SEP> T-activine, <SEP> 11 <SEP> 14
<tb> on <SEP> 22 <SEP> children
<Tb>
T-activin causes a stable and prolonged restitution of immunity.

During the treatment of malignant tumors, the product has a marked direct antitumorigenic action, decreases the delay of the remission approach, potentiates the specific polychemotherapy, allows local radiotherapy.

 Given the regulatory action of the preparation, it is possible to use T-activin for the treatment of hypoplastic secondary states of hematopoiesis. On the background of the immunotherapy, with the drug product, of the hypoplastic acute state in association with the immuno-deficient state, one observes a restitution of the immunity, indices of the peripheral blood and the hematopoese of the bone marrow.

The action of the drug product for the treatment of other conditions, due to disorders of the immune system, such as psoriasis, lupo erythemato-malignant visceritis and others, leads to a good clinical effect already after the first stage of the disease. immunotherapy. The clinical effect of
T-activin results, in this case, by a reduction in the approach time of remission, which is very important for the treatment of patients resistant to specific drug therapy. The prescription of the drug product to patients never gives pyrogenic complications, or appearance of specific antibodies to T-activin. There was no idiosyncrasy.

The claimed process for preparing the drug product is practically carried out as follows:
The thymic gland of the calves is used as the source of the raw material. The tissue of the gland can be preserved before use at the temperature of -20 C for up to 3 months. The thymus is removed from the capsule and cold crushed to pieces of 0.5 to 1 g. To the milled raw material is added any physiological saline solution, usually a 0.14M NaCl solution, in a 1/3 ratio.

The material is then homogenized in a vortex homogenizer for 3 minutes at 8000 rpm. It is possible at this stage to grind the material until a homogeneous mass. This is done cold, at a temperature of +2 to +4 ° C. The ratio indicated between the monger and physiological solution ensures a uniform homogenization product and a more complete yield of biologically active substances. of the raw material in the next step.

The homogenization product is then maintained at a temperature of +2 to 60C for 12 to 16 hours. The conditions under which the maintenance is carried out (autolysis) are determined experimentally, and the modification of the time or temperature parameters leads to a decrease in the yield and the biological activity of the targeted product.

During autolysis, there is an additional destruction of cells, cytoplasmatic and nuclear membranes, and a modification of proteins and peptides by the action of enzymes. This ensures the increase of the yield of biologically active substances and the transformation of these substances with the simultaneous rise of the specific biological activity. Insoluble coarse fragments are then removed by centrifugation at 14,000-20,0009 for one hour at 4 ° C.

The supernatant layer is drained through a marli or capron filter to retain the dispersed fat fragments.

 The supernatant layer is then heated on vigorous stirring at 70 to 90 ° C, generally up to 80 ° C, and maintained at this temperature for 15 minutes.

At this stage, the denaturation of the thermolabile ballast protein components occurs. After cooling to a temperature of 4 ° C., the thermolabile components are removed by centrifugation at 14 000-20 0009 for one hour.The centrifugation can be replaced by any other method which ensures the removal of denatured proteins, in particular filtration on filters having sufficient pore size.

The supernatant layer containing thermostabilally biologically active proteins and peptides is precipitated by acetone.

It is possible to use another organic solvent, for example ethanol.

The operation is carried out at a temperature between -20 and 250C.

A volume of supernatant layer is added dropwise to the 5 volumes of acetone, with simultaneous stirring of the mixture. The mixture is maintained at a temperature of -200C for 2 days. At this stage it is possible to dissolve fats and other ballast substances in an organic solvent. The precipitate contains biologically active proteins and peptides. The liquid part is removed, the precipitate is dried under vacuum at a temperature of 4 ° C. The strict observation of the temperature regime and of the ratio between the supernatant layer and the acetone (from 1/5 to 1/7) is The modification of these parameters causes a decrease in the biological activity of the material and its contamination by the ballast substances. 0.01M buffer, sodium phosphate, pH = 7.0, at a temperature of 18-22 ° C and stirred for one hour. Insoluble products are removed by centrifugation at 10,000-16,000 g for 40 minutes. The supernatant layer is removed and diluted with buffer solution to a concentration of 15-25 mg / ml.

it is recommended to use in particular this concentration of proteins and peptides in solution, since this ensures the minimal losses of biologically active peptides during the subsequent release. Saturated ammonium sulfate solution is added to the resulting solution to a final concentration of 20 to 30%, at a pH of 6.9 to 7.1. The mixture is stirred at a temperature of 2 to 4 C for one hour. An insoluble portion is removed by centrifugation at 16,000 g for 30 to 40 minutes.

To the supernatant layer is then added an acetic acid solution until a pH of 3.9-4.1 is reached. Partial precipitation of the biologically active material is then observed.

A complete precipitation is obtained by introducing into the supernatant layer the pulverulent ammonium sulfate to obtain 45 to 55% saturation, taking into account the final volume of the mixture. The mixture is stirred at a temperature of 2 to 4 C for one hour. The precipitate is obtained by centrifugation at 16 0009 for 30 to 40 minutes. The supernatant layer is removed, the precipitate is dissolved in a 0.01M buffer solution of tris-HCl pH 8.0. The third salting step is carried out in a 45-55% saturation solution of ammonium sulfate at pH 4.5-6.0.

The solution is stirred carefully at 2-4 ° C for one hour and the precipitate is removed by centrifugation at 16,000 g for 30-40 minutes. The realization of the release in three stages makes it possible to carry out an additional purification from the ballast substances, which is necessary to obtain a product having the imposed properties. The precipitate is then dissolved in a 10 μM buffer solution of tris-HCl and at pH 8.0, and the concentration of the protein is raised to 8 to 12 mg / ml using the same buffer solution.

 The solution obtained is subjected to ultrafiltration on membrane filters, at the nominal retention limit according to the globular proteins of 12,000 to 30,000 daltons, in a nitrogen atmosphere, under a pressure of 3.0 to 3.5 atm., and at a temperature of the order of 2 to 4 C.

Pellicon PSED membranes (Millipores) are preferably used, although any other filter with the same characteristics (VM-30, PM-30) can be used. The membrane is then washed with three volumes of 0.01 M Tris-HCl buffer solution and pH 8.0.

This gives a complete passage of the biologically active components in the filtrate. 80 to 85% of the ballast substances are retained on the filter. The filtrate is taken and lyophilized.

The obtained material is dissolved in the minimum volume of distilled water and subjected to desalting by exclusion chromatography in 2.5 x 30 cm columns, loaded with Sefadex G-15 # (medium). Another molecular sieve with similar characteristics can be used. Elution is carried out in distilled water. The material containing protein components is collected and dried again by lyophilization. The dry powder is dissolved in a buffer solution at pH 6.8-8.2 and ionic strength 0.1 to 0.4M, containing NaCl or KCl.

The solution is subjected to gel chromatography in a 2.5 x 100 cm column filled with Sefadex G-509 (medium), previously equilibrated with the same buffer solution. As a vector for gel chromatography, other molecular sieves having the same characteristic can be used.

 The optimal separation of the biologically active peptides in the buffer solution carried out with the indicated characteristics is achieved.

When gel chromatography is complete, the target product with a molecular weight of 1,500 to 6,000 daltons is withdrawn. In this range, there are in particular peptides possessing the regulatory action on the
T-immune system of the body. The peptide solution is again desalted by gel chromatography in columns loaded with Sephadex G-1, freeze-dried, dissolved in saline and put into ampoules of 100 to 200 mcg / ml.

 Other characteristics and advantages of the present invention will appear on reading the following description and examples of preparation of the drug product given for illustrative but not limiting.

EXAMPLE 1
500 g of thymus are freed from freshly frozen calves (at -20 ° C.) and homogenized in a 0.614M solution of NaCl (volume of 1.65 °) at a temperature of +4 ° C. The product is maintained at room temperature. homogenization at this temperature of +4 C for 16 hours. The homogenate is then centrifuged at 20,000 g for 2 hours. The precipitate is removed.

The supernatant layer, having a volume of about 1.5 °, is heated at a temperature of 80 ° C. for 15 minutes in a water bath. The solution is then cooled and the thermolabile denatured components are removed by centrifugation at 20,000 ° C. for 1 hour. The supernatant layer (about 1.35 #) is then treated with the fivefold volume of acetone cooled to -200C for 2 days. The precipitate formed from the liquid portion is separated by decantation and dried under vacuum.

The precipitate is dissolved in 100 ml of a 10 M sodium phosphate buffer solution at pH 7.0, with continuous stirring, at room temperature and for 1 hour. The insoluble portion is removed by centrifugation at 16,0009 for 40 minutes and the supernatant layer is diluted with distilled water to a protein concentration of 25 mg / ml according to the biuret method.

39.6 ml of a saturated solution of ammonium sulfate at 4 ° C. are added to the solution obtained, the pH being 7.0. The mixture is stirred at a temperature of 4 ° C. for 1 hour. The insoluble portion is removed by centrifugation at 16 0009. The supematant layer is acidified with acetic acid to pH 4.0, 24.82 g of ammonium sulphate are added and the mixture is stirred at a temperature of 40 ° C. for 1 hour. . The precipitate formed is removed by centrifugation, dissolved in 80 ml of a UNI Tris-HCl buffer solution, pH 8.0 and precipitated again by the addition of 80 ml of saturated ammonium sulfate solution. at the temperature of 4 and pH 5.0.

We collect it again. precipitated by centrifugation, dissolved in a 10 M buffer solution of tris-HCl and pH 8.0; the protein concentration, determined by the biuret method, is increased to 10 mg / ml using the same buffer solution. The resulting solution is ultrafiltered through the Pellicon PSED membrane (Millipore) at a temperature of 4 C under a nitrogen pressure of 3.5-4 atm. The membrane is washed with three 150 ml portions of 10 μM tris-HCl buffer solution, pH 8.0.

The volume and concentration of the protein for the fraction under the filter and above the filter are respectively 510 ml, 0.7 mg / ml and 0.6 ml, 12 mg / ml. Both fractions were tested after desalting and lyophilization for biological activity in the T-rosette formation system.

Only the fraction under the filter has biological activity; the fraction retained by the filter was found to be inactive. A portion of the biologically active fraction, 30 mg, is dissolved in a 0.2M KCl solution prepared on the tris-HCl buffer solution, pH 8.0, and the gel chromatography is carried out in a column of 1 x 100 cm, filled with Séfadex G-50 #, equilibrated with the same buffer solution. Molecular weight peptides of 1,500 to 6,000 daltons were collected, desalted in the column with Sefadex G-15 and lyophilized. The minimum active dose corresponds to 1-2 mcg per 3,106 lymphocytes in vitro. The overall target product yield is 110 mg / kg of thymus. The activity, determined using the azathioprine inhibition test for spontaneous rosette formation, is at least 3.106 lymphocytes. The peptides obtained are dissolved in a 0.14M NaCl solution.

 To determine the heterogeneity of the drug product of the thymus according to the molecular mass, 10 mg of product are placed in 1 ml of buffer solution (0.14M solution of NaCl - 0.01M solution of tris-HCl), at pH 8.0 in a column of 1 x 100 cm filled with Sefadex G-50 # (fine), balanced by the same buffer solution. The elution rate is 6.0 ml / hour, the fraction harvesting time is 15 minutes. The value of the adsorption at 280 nm is determined in each fraction. The active components of the drug product were distributed in test specimens with a sequence number of 32 to 50. The column was previously calibrated with a mixture of normal substances of known molecular mass. This made it possible to determine the presence of the components in question. molecular weight from 1500 to 6000 daltons in the specimens indicated.

 To determine the heterogeneity of the drug product, analytical disk electrophoresis was also performed in the 15% polyacrylamide gel at pH 8.9 in an apparatus of Reanal (Hungary).

400 to 600 mcg of drug product in 0.02 to 0.03 ml of electrode buffer solution was deposited in each gel tube. Electrophoresis was conducted at a temperature between 5 and 70C and with a current of 2 mA on the tube for 1 to 1.5 hours. The front of the ion movement is marked with bromophenol blue. After the electrophoresis, the tubes are removed from the gel, fixed and stained with a solution of 0.12 amine black in a solution of 7% acetic acid for 40 minutes, and the dye is then washed with the solution of 7% acetic acid. Seven bands are detected, to which 7 components of the drug product whose electrophoretic nobility is estimated according to the formula:
K @ / f = @@ / @ / /
bph in which:
is the length of the gel before staining
# is the length of the gel cleared staining
#p is the distance presented by the protein band, #bph is the distance presented by bromophenol blue.

The results are summarized in Table IX below:
TABLE IX

<tb> No <SEP> of <SEP> band <SEP> of <SEP><SEP> cathode <SEP> 123 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7
<tb><SEP> 0.082 <SEP> 0.169 <SEP> 0.364 <SEP> 0.402 <SEP> 0.452 <SEP> 0.515 <SEP> 0.89
<Tb>
To determine the isoelectric points of thymus drug product components, the thin-layer isoelectrofocusing of the polyacrylamide gel was carried out in a pH range of 3.5 to 10 using the Once the focus is complete, the gel is fixed for 16 hours in trichloroacetic acid in a 25% solution of isopropanol, stained with Cooniassi blue R-250 at 0.05% in a solution of 0.05% strength. 10% acetic acid in the 25% isopropanol solution overnight, then treated with the 10% solution of acetic acid.

The pH gradient is determined according to the position of the known isoelectric point marker proteins. The drug product of the thymus is focused in 13 bands corresponding to the values of the following isoelectric points: 4.0; 4.3; 4.6; 4.7; 4.8; 5.0; 5.2; 5.5; 6.1; 6.3; 6.7; 7.4; 9.0.

 The drug product of the thymus containing polypeptides according to ultraviolet spectral analysis has two adsorption maxima: at 203 and 275 nm.

EXAMPLE 2
The operations are carried out in the same manner as described in Example 1, except that the homogenization product is maintained at a temperature of 2 ° C. for 12 hours, the column filled with Séfadex G-50 # being equilibrated with an O, 14M solution of NaCl in a 0.05M buffer solution of tris-HCl pH 7.2.

 The yield of the targeted product is 118 mg / kg of thymus. The activity determined by means of the azathioprine inhibition test of spontaneous rosette formation is not greater than 1 mcg per 3.106 lymphocytes.

EXAMPLE 3
The procedure is the same as described in Example 1, except that the first release is carried out in a solution at 20% saturation of ammonium sulfate at pH 6.9, the second salting out. being carried out in a solution at 40% saturation of ammonium sulfate, at pH 3.9 and the third salting out, in a solution at 45% saturation of ammonium sulfate, at pH 4.5; the column with Séfadex G-502 is equilibrated with a 0.14M solution of NaCl in a 0.01M solution of tris-HCl, pH 7.2.

 The yield of the targeted product is 115 mg / kg of thymus. The activity determined by the azathioprine inhibition test for spontaneous rosette formation is not greater than 1 mcg per 3 × 10 6 lymphocytes.

Claims (8)

 1.- Regulatory drug product of the T-system of immunity,  characterized in that it contains as active principle pre peptides  molecular weight in the range of 1,500 to 6,000 daltons,  a maximum in the ultraviolet adsorption spectrum for 208 and 275 nm and  having an electrophoretic mobility in a polyacrylamide gel with  with respect to bromophenol blue: 0.062-0.102; 0.156 to 0.236; 0.354-O, 374; 0.382 to 0.422; from 0.432 to 0.472; from 0.485 to 0.545; 0.850-0.930, and additionally contains  a pharmacologically acceptable excipient.  2. Drug product according to claim 1, characterized in that as excipient is used a physiological solution.  3. A medicinal product according to claim 2, characterized in that as a physiological solution is used a 0.14M NaCl solution.  4. The medicinal product according to claim 1, characterized in that the content of active ingredient corresponds to 100-200 mcg / ml injected,  5. A process for preparing the regulatory drug product of the T-system of immunity according to any one of claims 1 to 4,  involving the following steps: homogenization of thymic gland tissue in NaCl, removal of the precipitate from the homogenate, removal of thermolabile proteins from the solution, precipitation of proteins and peptides in solution S - dissolution of proteins and peptides mentioned above, 6-release of peptides, 7 - dissolution of peptide precipitate, 8-desalting of isolated peptides and their lyophilization, and being characterized in that: 9 - after step (1), the homogenization product is maintained at a  at 2 ° to 60 ° C. for 12 to 16 hours, and the peptides are released in three steps:  the first: in a solution with 20-30% of the saturation of the sulphate  ammonium, at a pH of 6.9-7.1,  the second: in a 40-55% solution of sulfate saturation  ammonium, at a pH of 3.9-4.1, and  the third: in a 45-55% solution of the sulfate saturation  of ammonium, at a pH of 4.5-6.0, 11 - after step (7), the solution of peptides is subjected to ultrafiltra  through a membrane with a nominal retention limit  12,000 to 30,000 daltons globular proteins, and the ultrafiltrate obtained with the isolation of  molecular weight of 1500 to 6000 daltons in the buffer solution at one  pH 6.8-8.2 and ionic strength 0.1-0.4M, and 13 - is carried out after step (8) mixing the peptides with the excipient  pharmaceutically acceptable.  6. A process according to claim 5, characterized in that maintains the homogenization product of step (9) at a ratio of solid and liquid parts equal to 1/3.  7. The process as claimed in claim 5, wherein the peptides of step (10) are salted out at a concentration in the solution of 15 to 25 mg / ml.  8. A process according to claim 5, characterized in that the gel chromatography of the peptides in step (12) is carried out in a buffer solution containing KCl or NaCl.