NL194693C - Method for the preparation of medicinal preparations for treating diabetes. - Google Patents

Description

1 194693

Method for the preparation of medicinal preparations for treating diabetes

The present invention relates to a method for the preparation of medicinal preparations for treating diabetes comprising obtaining a cell suspension containing hemopoietic liver and spleen cells from a human embryo, freezing the obtained cell suspension, and thawing the cell suspension, such that the liver and spleen cells of a human embryo remain viable and can proliferate after administration to a patient.

In recent years, remarkable progress has been made in researching the use of tissues from dead embryos. A completely new field of therapy has been developed, namely that of cell therapy, which makes it possible to compensate for inadequate functioning of damaged and diseased tissues by using medicinal preparations based on suspensions of embryo cells made from fetal tissues.

Transplanting cells appears to be an attractive alternative to transplanting organs and tissues, embryo cells have the advantage that they have not yet built up a pronounced antigenic defense, are easy to transplant and do not cause an immune response of the host. Moreover, those cells are very vital. They multiply, differentiate and are a source of a large number of biologically active substances. Today, suspensions are already being applied from the brain, bone marrow, liver, spleen, thymus, pancreas and gut skin of embryos.

The initially most successful attempts to use suspensions as medicinal preparations concerned liver and spleen from human embryos. It is known that 5 to 16 week old livers and spleen from human embryos perform hemopoiesis with high stem cell yields.

In 1973 a medical preparation was made for the first time based on a suspension of native cells from an embryonic liver after 7 weeks gestation; administration of this preparation led to the recovery of hemopoiesis in a patient suffering from aplastic anemia (E. Kelemen in the second J. Gematol. 10 (No. 4) (1973), pp. 305-308).

In recent years, by varying the methods of preparing suspensions and the procedures for applying them, detective workers have achieved positive results in the treatment of primary and secondary myelodepressions. The preparations for this are mainly described by T. Izzi, O. Polehi et al. In "Fetal liver transplantation" (edited by Alan R. Liss) of 1985, pages 237-249.

Another area of clinical application of the above suspensions involves immunity disorders; Here, most of the experience has been collected by J. Touraine (see "Transplantation Proceedings" 25 (No. 1) (1993) pp. 1012-1013). An excellent article by R. Baechelta et al. In J. Clin. Invest. §1 (March 1993) pp. 1067-1078 shows the final results of treating patients suffering from severe combined immunodeficiency; this not only restored patient immunities, but also demonstrated the availability of split chimeras and the occurrence of tolerance to antigens from both host and donor.

It is known that the development of insulin-responsive diabetes involves autoimmune damage of the islets of Langerhans caused by cells and circulating antibodies. Therefore, the subsequent treatment of insulin-responsive diabetes made use of preparations, substances and tissues aimed at interrupting the autoimmune damage of the β cells of the 45 pancreas, thus correcting the autoimmunity. For this purpose preparations such as isoprinosine, azathioprine, cyclosporine and the like were used, and in particular of thymus preparations.

The prior art therefore comprises the preparation "thymaline" made from the thymuses of calves, and the use thereof to correct the immunity for the treatment of diabetes (TG Koerdanov, BM Kaplbieva and GT Zakiva in Problemi Cytokrinologii 35 (no. 6) (1989) 7-9).

A drawback of the known treatment method, however, is the fact that the daily requirement for insulin only decreases by 15 to 20%. A decrease in the degree of glycemia is only maintained for 5 to 6 months.

It is therefore the main object of the invention to develop a medical preparation with the immunity correcting effect on the basis of a cell suspension, with which an optimum selection of the composition of the cells makes it possible to intensify the mechanism of correction of immunity and achieve positive results in treating diabetes.

According to the invention this object is achieved with a method for the preparation of medicinal preparations for the treatment of diabetes comprising obtaining a cell suspension containing hemopoietic liver and spleen cells from a human embryo, freezing the obtained cell suspension, and defrosting the cell suspension such that the liver and spleen cells of a human embryo remain viable and can proliferate after administration to a patient, characterized in that an embryo aged 5-8 weeks is used and that the obtained cell suspension before freezing is divided by pouring the cell suspension prepared from one human embryo into a separate set of containers to form a sample bank, intended for administration to one patient of at least the contents of one container after thawing, the concentration of nuclear-bearing cells of the cell suspension is 5 to 90-10e / ml, the concentration of colony forming units in the granulomonocytic combination is 20 to 80.103 / ml, the concentration of colony-forming bladder units is 0.5 to 9-103 / ml and the concentration of early precursors of the hemopoietic CCD34) is 1 to 9-10e / ml.

It was empirically determined that the choice of the aforementioned cell composition and the quantitative ratio between its components make it possible to achieve positive results in the treatment of diabetes, with the serious disease indeed declining.

The inventors believe that the anti-diabetic effect of the preparation of the invention can be explained by an effect on the mechanism of the immune correction, and in particular the termination of the aggression of T lymphocytes and immunoglobulins against θ cells.

In applying this invention, it is convenient that the medical preparation additionally contains 3 to 10% dimethyl sulfoxide during freezing. The choice of the lower limit is determined by the best retention of the cells, while the upper limit is determined by the toxic effect on the preparation.

The anti-diabetic effect is reflected in various clinical trials of the inventors of this; some of those tests are described in the examples that follow. Significant improvements in immunity were observed in patients, with recovery after 3 to 11 days (increase in the number of T lymphocytes, change in the ratio between the subpopulations, normalization of the humoral immunity indices). The initial insulin dose was reduced by 40 to 70%. After 3 to 4 weeks 30 there was a decline in the disease.

One aspect of this invention is that it is convenient to administer the medicinal preparation according to the invention in a dose of 0.5 to 5 ml.

Moreover, it is desirable to administer the above-mentioned preparation before and / or after a therapy performed with cytostatic preparations.

It is convenient to select the medicinal preparation from a shaped tissue bank of various samples, taking into account the individual indices of a patient; moreover, at least one portion of the same sample must be used for repeated administration.

The administration of the preparation prepared by the method according to the invention makes it possible for various disorders which determine the course of the diabetes, and in particular the immune resistance, the development of macro and microangiopathies, damage to the liver and kidneys, development of the anemia syndrome and thrombocytopenia, etc., to be corrected by restoring the lost functions of hemopoiesis and immune systems.

A better understanding of the nature of the invention will be obtained from the following detailed description of embodiments thereof, in conjunction with the examples.

The method for preparing medicinal preparations according to the invention can be carried out with the following procedure.

Embryos available from induced abortions of healthy women who were screened for the presence of viruses and blood infections. Embryos with an age of 5-8 weeks are used. From the point of view of maintaining the integrity of an embryo, the vacuum extraction is preferred as the abortion method 50. The embryo is transferred to a sterile vessel containing the solution of Hanks and an antibiotic (from the aminoglycoside group). All the following work is performed in a sterile cupboard. The embryos are transferred to sterile petri dishes containing Hanks and antibiotic solutions; here, after the abdominal cavity has been carefully opened, the liver and spleen are removed and used separately for making suspensions.

55 Liver and spleen are placed in homogenizers, cut into small fragments and ground to a homogeneous mass. With the Hanks solution, cells are flushed from the walls of the device into graduated test tubes, passing first through a filter 3 194693 used for blood transfusion and then through injection needles of decreasing cross-section. The suspensions thus prepared are frozen.

Dimethyl sulfoxide (chemically pure DMSO) is used as protection against frost damage. Before its use, the DMSO passes through a millipore filter (pore diameter 0.22 µm). The same volume of DMSO working solution is added dropwise to the suspension with gentle stirring, preferably to a concentration of 5%.

Suspensions of embryo cells are sunk in polyethylene vessels 0.5 to 2 ml (depending on the later destination).

The containers are introduced into the chamber of a programmable freezer and are cooled to -196 ° C with liquid nitrogen.

Frozen suspensions are stored under liquid nitrogen at -196 ° C in the embryo tissue bank.

After the suspension has been prepared, the following parameters are determined: 1. number of core-bearing cells per ml 2. number of colony-forming units of the granulomonocytic series (CFU-GM) per ml 3. number of colony-forming blastomas (CFU-bl.) Per ml 4. number of early hemopoietic precursors (CD ^) per ml.

The suspensions which are stored in a tissue bank must have the following parameters: 1. 5 to 90-106 / ml 2. 20 to 80-103 / ml 3. 0.5 to 9-103 / ml 4. 1 to 9-106 / ml

The prenatal diagnosis includes tests for syphilis, HIV, HBV and HCV infections, toxoplasmosis and cytomegalovirus. The contents of the vessels are tested for bacterial sterility.

The fetal diagnosis includes tests for HIV, HBV and HCV infections, cytomegalovirus, scarlet fever, herpes and toxoplasmosis.

Depending on the intended clinical treatment, the medicinal preparation is administered with part or all of the material from a frozen sample.

Prior to and in certain cases also after the administration of the medicinal preparation of this invention, treatment with one or more cytostatic preparations is performed which are intended to limit the immunological reactivity of the recipient and to reduce the autoimmune aggression.

The effect of the treatment is evaluated on: - the passage of glycemia, the limitation of the dose of insulin to be administered and the occurrence of the state of hypoglycemia, - changes in the immune status indices (levels of TB lymphocytes and immunoglobulins), - number of erythrocytes containing embryonic hemoglobin,

Detailed description of the application of the present invention in accordance with available clinical practice is now given in Examples 1 to 7.

Example 1

Female patient, D., born in 1946, was admitted to the diabetes department of the Ukrainian research institute for endocrinology and metabolism on August 21, 1993, with complaints of pronounced weakness, dizziness, breathlessness with insignificant physical strain, palpitations, poor appetite , nausea, itching, dizziness, edema of the feet and occasional poor vision.

45 The patient's status revealed that she had shown insulin-sensitive diabetes 13 years ago. She regularly received insulin and was treated occasionally in the hospital. In the last two years the symptoms had become more severe: high blood pressure appeared and the main disease became somewhat unstable; urine was always found to contain protein. In the last year symptoms appeared such as weakness, periodic edema of the legs and poor digestion; in addition, peripheral blood was found to have a too low content of erythrocytes and also hemoglobin. In March 1993, the patient was admitted to the Neurology Department of the Kiev Regional Hospital; then there was a chronic kidney deficiency and a moderate anemia. Four erythrocyte transfusions were administered. After a temporary improvement, the situation in September 1993 was much worse, why it was included.

55 The condition of the patient was serious when admitted to hospital. But her consciousness was clear. Skin and exposed mucous membranes were pale. Peripheral lymph nodes were not thicker. The pulse was 98 beats per minute, in good rhythm and full and clear. The blood pressure was 190/120 mmHg. The heart rate was 194693 4 sufficiently clear and rhythmic, with a systolic noise on top, the accent of the second heart sound of the aorta. Tightening of the blisters had been done in the lungs. The abdomen was soft, moderately bloated and prone to touch in the area of the right hypochondrium. Under the right rib arch, along the right middle clavicle line, the liver protruded 2 cm. The spleen was no larger than 5 normal. Pieces of the large intestine were at times convulsive. Her bowel movements were slimy four times a day. The daily diuresis up to 1 liter. Edema to face and feet.

Diagnosis: Type 1 diabetes, severe, in a state of decompensation. Diabetic general angiopathy (netropathy of the third degree, chronic renal failure of the first degree, vascular form of proliferating kidney damage, micro-macropines in the blood vessels of the legs of the second degree), diabetic 70 polyneuropathy in the legs, severe anemia and symptomatic hypertension .

Upon admission the following indications were found: blood sugar 12.4 mmol / l, in the urine glucose 22 g / l, acetone (++), proteins 3.3 g / l, erythrocytes and cylinders (5 in the field of vision), K + , 5.2 mmol / l, urea 14.42 mmol / l, creatinine 0.220 mmol / l, erythrocytes 2.1-1012 / l blood and hemoglobin 64 g / l.

The following treatment was then prescribed: diet and 22 units of insulin per day; detoxification therapy (intestinal flushing, intestinal assorbents, hemodesis), diuretics, blood pressure lowers, anhyggregants and anhyprotects, electrolysis balance correction, iron preparations and vitamin therapy.

After three weeks the condition of the patient was somewhat better: the carbohydrate metabolism was compensated and the edema had disappeared; the blood pressure was back to 170 / 100-110 mmHg. But a stable and severe anemia syndrome had remained.

On September 15, 1993, a transplantation of native hemopoietic liver cells from a human embryo was performed (sample C038N-2I): the embryo was 5 weeks old and the amount of cells administered was 1.5 ml; number of core-bearing cells 25-106 / ml, KVE-GM 13.9-103 / ml, KVE-b1.

1.3-103 / ml and CD4, 2.4-106 / ml. The method of administration was in the bones (intrasternal).

25 The patient tolerated the transplant well. The syndrome was seen immediately after the transplant after 8 to 10 hours: headache, fatigue and nausea decreased and appetite increased; the patient slept peacefully for the first time in a few months. Hemoglobin, erythrocyte and reticulocyte levels were increased on the third day and were normal on the tenth day (see Table 1). Urine analyzes improved considerably over the course of three weeks (see Table 2). The blood pressure dropped to 160 / 100-90 30 mmHg, which reduced the administration of blood pressure-lowering preparations.

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194693 6

Example 2

The male patient, P., born in 1960, was admitted to the Diabetes Department of the Ukrainian Research Institute for Endocrinology and Metabolism on November 6, 1991, with complaints of general weakness, weight loss, dry mouth, frequent drinking, and polycluria. The patient considered himself sick since September 5, 1991, when those symptoms first appeared. The patient has never been examined before. Drastic worsening of the patient's condition led him to seek medical help in November 1991.

Diagnosis: Type 1 diabetes, first occurring but in severe form, in the state of decompensation and with ketoacidosis.

Upon admission, sugar was found in the blood 15 mmol / l, glucose in the urine 41.6 g / l and acetone in the urine (+++). The following treatment was prescribed: insulin 60 units per day, vitamin therapy and potassium preparations.

Upon reaching the compensation on December 2, 1991, a first transplantation of liver and spleen cells from a human embryo was performed (sample C038N-631L): the embryo was 7 weeks old; 1 ml of cells was administered; number of core-bearing cells 36.2-106 / ml; CFU-GM, 23.6-106 / ml; CFU bl.

2.5-103 / ml and CD34, 1.4-106 / ml. The method of administration was intravenous. The amount of cells relative to the total amount of sample from the bank of frozen tissue was 66%.

The patient tolerated the transplant very well.

Hypoglycaemia occurred three weeks after the transplant, resulting in a gradual decrease in the daily insulin dose; the maximum decrease was observed after 2Vz month (see Table 3).

Normalization of the immune state was already observed on the eleventh day (see Table 4). In the six months following the first transplant, the patient was in a state of compensation, with a stable and relatively low insulin dose.

By the end of May 1992, the patient's condition drastically deteriorated: symptoms of decompensation of the carbohydrate metabolism appeared and the immune condition also worsened (see Table 4). The insulin dose was increased to 32 units per day.

On 3 June 1992 there was another transplant, now from frozen stored hemopoietic cells from the same sample (C038C-63HL), 0.5 ml of cell suspension was administered. The patient tolerated the transfusion very satisfactorily. The positive course of the daily insulin dose and of the immune state can be seen in Tables 3 and 4.

The course of the amount of embryonic, hemoglobin-containing erythrocytes confirms the replacement effect of the transplanted cells.

TABLE 3 35 Analyzes of patient P. before and after transplantation of hemopoietic cells from an embryonic liver (state of compensation) 2 December 1991: 1st transplant June 3, 1992: 2® transplant

Date Indices 01.12.91 23.12.91 17.01.92 23.02.92 07.05.92 03.06.92 06.07.92 17.07.92 21.01.93 40 Before 21® After After After 5 After 6 After 1 After After 7½ the day 1½ 2½ lunar lunar month 1½ month trans-month month den den month plant.

45 Insulin, units per day 48 36 20 20 20 32 24 22 24

Percentage decrease in insulin dose up to.

50 v. The first - 25 40 59 59 34 50 55 50

Erythrocytes containing embryonic hemoglobin,% 0 18 12 14 8 2 12 18 14 55 - 7 194693 TABLE 4

Evolution of patient P.'s immunity data

Date Indices 14.11.91 17.12.91 07.04.92 28.05.92 14.06.92 5 -

Before the 11th day After 4 After 6 11th day after transplant. months months 2nd transplant.

T lymphocytes,% 40 53 50 50 53 10 abs.109 / l 0.7050 0.8904 1.0808 0.7950 0.8480 T help cells,% 37 20 41 37 33 abs.109 / l0, 5328 0.4872 0.7913 0.5883 0.5280 T suppressors,% 12 24 15 13 20 abs.109 / l 0.1728 0.4032 0.2805 0.2067 0.3200 15 T help cells / - 3.1 1.2 2.7 2.85 1.7 T suppressors,% B lymphocytes,% 37 31 18 30 27 abs.109 / l 0.5328 0.5208 0.3474 0.4770 0.4320

IgG, g / l 8.75 11.8 9.70 17.6 10.7 IgA, g / l 7.9 2.1 1.75 1.6 4.25

IgM, g / l 0.43 1.3 0.86 0.86 1.01

Example 3 Female patient R., born in 1961, was admitted on December 1, 1992, to the first therapeutic ward of the central hospital of the Zaliznychni district of Kiev, with powers of pronounced weakness, weight loss, dry mouth despite excessive drinking, pollakiuria, headache and poor appetite.

The patient considered herself sick since October 1991 when she started to feel bad. The patient had never been examined before. A drastic deterioration in the patient's condition made her ask for medical help at the end of November 1992.

Diagnosis: Type 1 diabetes, first observed but in severe form, with condition, and diabetes ketoacidosis. When taken, the blood sugar level was 20.0 mmol / l and in the urine there was 52.4 g / l glucose and a lot of acetone. The following treatment was prescribed: 52 units per day of insulin and therapy with vitamins and potassium preparations; every other day hemodese. Azathioprine 35 was also prescribed with a daily dose of 2 mg per kg of body weight, to be administered in two doses, starting with 25 mg per day with gradually increasing dosage under daily peripheral blood control. Upon reaching the aforementioned therapeutic dose, treatment was performed under daily control of the numbers of leukocytes, thrombocytes and erythrocytes in the peripheral blood.

Upon reaching the subcompensation of the carbohydrate metabolism on the 32 day after the start of treatment, on January 5, 1993, a transplantation of frozen stored hemopoiesis cells from a human embryonic liver was performed (sample C038N-441L); the embryo was 6 weeks old and 1 ml of cells was administered; number of core-bearing cells 17.5-106 / ml, CFU-GM, 24.8-103 / ml, CFU-bl. The method of administration was intravenous. The amount of 45 cells transplanted relative to the total amount of tissue from the sample supplied from the frozen tissue bank was 45%.

The patient tolerated the transplant satisfactorily.

Treatment with azathioprine was continued for 48 days after the transplantation of human liver hemopoietic cells.

Hypoglycaemia occurred two weeks after the transplant, leading to a gradual reduction in the daily insulin dose; the maximum reduction was achieved after 2 months (see Table 5).

By the end of the second week, the patient's immune status was normalized (see Table 6).

The course of the number of erythrocytes with embryonic hemoglobin in the peripheral blood confirms the 55 replacement effect of the transplanted cells.

The recovery of the sick person continues after 9 months. She remains under observation.

194693 8 TABLE 5

Analyzes of female patient P. before and after transplantation of hemopoietic cells from an embryonic liver (state of decompensation) 5 Date Indices 04.01.93 09.01.93 19.01.93 04.02.93 05.03.93

For the 3rd day 14th day After 1 After 2 transplant. month months 10 Insulin, unit. per day 52 44 30 26 16

Percentage decrease of the insulin dose compared to the first - 15.4 42 50 70

Erythrocytes containing embryonic hemoglobin,% 0 14 17 23 26 15 --- TABLE 6

Evolution of immunity in patient R.

Date Indices 04.01.91 19.01.91 07.04.92 20 _

For the 14th day Transplant after 2 months.

T lymphocytes,% 12 56 42 25 abs.109 / l 0.8016 1.0035 0.8064 T help cells,% 10 38 28 abs.109 / l 0.0136 0.3226 0.2352 T suppressors % 2 18 14 abs. 109 / l 0.0136 0.3226 0.2352 30 T help cells / 5.0 2.1 2.0 T suppressors,% B lymphocytes,% 16 24 22 abs. -109 / l 0.1088 0.4301 0.3996

IgG, g / l 8.1 8.75 9.70 IgA, g / l 2.4 1.75 1.6

IgM, g / l 0.38 0.86 0.86

Example 4 40 Female patient L., born in 1991, was admitted to the Endocrinology Department of the specialist children's clinic in Kiev on September 15 and remained there until September 26, 1993.

Diagnosis: Insulin-sensitive diabetes in severe form, labile course, a state of sub-compensation.

The disease was noted in February 1993 when the child was admitted to the precomatosis state for examination 45.

Diagnosis: Type 1 diabetes, insulin therapy was started with 8 units per day. In the following months, the insulin dose was reduced to 6 units per day. The nature of the actual disease was unstable with frequent occurrence of hypoglycemic states and ketoacidosis. The child was placed in the Endocrinology department with the intention of performing a transplantation of embryonic hemopoietic cells 50.

Upon uptake, blood sugar varied from 5.5 to 12.4 mmol / l; in the urine there was 3.2 g / l glucose but no acetone.

On September 12, 1993, a transplantation of frozen stored hemopoietic cells from an embryonic liver was performed (sample CO38N-91H); the embryo was 5 weeks old and 1.5 ml of cells 55 were administered; number of core-bearing cells 25.0-10e / ml, KVE-GM 23.9-103 / ml, KVE-b1. 1.8-103 / ml and CD34 4-106 / ml. The method of administration was intravenous.

The patient passed the transplant satisfactorily. According to her mother the appetite of the 194693 child already improved the first evening after the operation and her sleep became calm and deep. On the fourth day after the transplant, the daily insulin dose was reduced to 5 units. He then remained at that level.

The patient's immune status normalized in one month (see Table 7).

In the time after the transplant, the child's condition improved satisfactorily; the disease stabilized and the blood sugar level remained at a level of 4.5 to 7.3 mmol / l, no episodes of hypoglycaemia or ketoacidosis were observed. Irritability and crying decreased. The child's psychological and physical development matched her age. She remains under observation.

TABLE 7 10 Evolution of female patient L. immunity

Date Indices 09.09.93 08.10.93

For the transplant. After 1 month 15 ------- T lymphocytes,% 37 51 abs.109 / l 0.3907 0.7020 T-help cells,% 29 28 abs.109 / l 0.3062 0.3780 20 T suppressors,% 8 23 abs.109 / l 0.0845 0.3240 T help cells / T suppressors,% 3.6 1.22 B lymphocytes,% 35 27 abs.109 / l 0.3696 0.3645 IgG, g / l 8.4 8.2

IgA, g / l 2.25 2.40

IgM, g / l 0.95 1.30 TABLE 8 30 Evolution of male patient A.

Date Indices 23.07.93 26.08.93

For the transplant. After 1 month 35 T lymphocytes,% 38 54 abs.109 / l 0.4545 1.0206 T help cells,% 29 32 abs.109 / l 0.3468 0.6408 40 T suppressors,% 9 22 abs.109 / l 0.1077 0.4158 T help cells / T suppressors,% 3.2 1.5 B lymphocytes,% 32 29 abs.109 / l 0.3827 0.5481 45 IgG, g / l 8.00 8.60

IgA, g / l 1.5 2.00

IgM, g / l 1.23 1.20 50 Example 5

The male patient A., born in 1969, resided from July 21 to August 12, 1993 in the Therapy Department of the Central Hospital of the Zaliznychni District in Kiev.

Diagnosis: Insulin-sensitive diabetes in the severe form with labile course, state of compensation; general diabetes pain of the second degree, peripheral diabetes polyneuropathy. Chronic 55 cholecystitis, however, declined.

The patient considered himself sick since 1986, when his condition became worse after severe psychological and emotional tensions: fatigue, lots of drinking and a lot of urination and loss of body weight. After 194693 10 2 months, when he asked for medical help, insulin sensitive diabetes was diagnosed. Compensation was achieved with a daily dose of 64 units of insulin.

In the last year the patient had noticed leg pain, generally at night, burning and irritating and sometimes sensitivity and cramps in the gastrocnemi muscles. The actual disease had become unstable 5: a satisfactory condition often varied with occasional ketoacidosis and hypoglycaemia, even with minor changes in diet and physical or emotional stress.

The patient was admitted to the therapeutic ward with the intention of administering a transplantation of hemopoietic cells from an embryonic liver.

At admission the blood sugar content varied from 4.7 to 11.4 mmol / l and there was 22.4 g / l glucose in his urine but no acetone. 58 units of insulin were administered daily.

On July 28, 1993, transplantation of native hemopoietic cells from a human embryonic liver was performed (sample CO38N-101H); the age of the embryo was 6 weeks; number of core-bearing cells 8.5-106 / ml, KVE-GM 23 · 103 / ιύιΙ, amount of cells administered 2.5 ml; CFU bl. 2.4-103 / ml and CD ^ 3, 8-106 / ml. The method of administration was intravenous.

The patient passed the transplant satisfactorily.

In the first two weeks thereafter, the daily insulin dose decreased to 50 units. General clinical research revealed no major changes (within the normal limits before and after the transplant). The patient's immune status normalized in one month (see Table 8).

The number of erythrocytes with embryonic hemoglobin increased to 15%.

The observation was continued for more than 12 months. The patient is feeling well and a state of stable compensation has been reached; no episodes of ketoacidosis were found.

The daily insulin dose was maintained at 50 to 52 units. The level of erythrocytes with embryonic hemoglobin remained between 8 and 10%.

Example 6

The male patient M., born in 1931, was admitted to the Surgery Department of the Central Hospital of the Zaliznychni District of Kiev and remained there from April 12 to May 26, 1993.

Diagnosis: Type 1 diabetes, severe form with subcompensation condition, general diabetes pain: microangiopathy in the vessels of the lower protrusions of the third degree, retinopathy, nephropathy of the second degree, on his right big toe a big ulcer, swollen feet and diabetes polyneuritis.

Upon admission, the patient complained of general fatigue, increased body temperature (up to 38.5 ° C), stiffness in the morning, swelling of the right foot, which feels like it was about to burst, ulceration of the soft tissues on the right big toe, sensation of numbness and irritation in the legs and pain in the legs, both at rest and when stressed.

The patient had suffered from insulin-sensitive diabetes for 22 years. In the past 5 years he had had leg pain when walking, sometimes with stabs in the legs and sometimes numb, generally at night. A month earlier an ulcer in the soft tissue had occurred in the area of the right big toe.

Extra mural care gave no result. After 1 month of footbaths, severe edema of the foot 40 occurred and the pain increased, a marked bleeding of the skin appeared and the body temperature rose to 38 ° C.

Immediately after the patient was admitted to the hospital, the swelling was opened and surgery was performed.

The following treatment was prescribed: therapy with antibiotics and with preparations against 45 inflammations, as well as 38 units per day of insulin. The patient's condition did not improve: he was still suffering from nocturnal temperature rises (up to 38.6 ° C), general fatigue, and a stomach ulcer, where he had been cut, pus emerged. After three days, the right lower region was vaccinated. Blood, urine and faecal samples were examined. Staphylococcus aureus was found in the blood. In the course of three weeks there were 3 treatments with antibiotics (Ampiox, Gentamicin, 50 Rifampicin, Claforam).

Blood counts showed leukocytosis with a differential shift to the left, an acceleration of the S.R. and moderate anemia (see Table 9). The immunogram showed a drastic decrease in the number of T lymphocytes and a slight increase in the number of f3 lymphocytes (see Table 10).

55 For the purpose of improving immune protection, a transplantation with native hemopoiesis cells from a human embryonic liver was performed on 10 May 1993 (sample C388-57H); the embryo was 8 weeks old; number of core-bearing cells 37.5-106 / ml, amount of cells administered 2.5 ml; CFU-GM

11 194693 42-103 / ml, CFU-bl. 1.3 · 103 / πιΙ and CD ^ 4 -106 / ml. The method of administration was intravenous.

Blood tests gradually became normal over the course of 8 to 10 days (see Table 9).

The wound and ulcer began to become free of pus and necrotic tissue. On the seventh day there was good granulation and the growth of the epithelium started. After two weeks, wound and stomach ulcer had become 5 scars.

Recovery of the immune status was observed on the 14th day (see Table 10).

On the fifth day, since hypoglycaemia began to occur, the daily insulin dose was reduced to 34 units.

The patient was discharged from the hospital in a satisfactory condition.

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13 194693 TABLE 10

Evolution of female patient M.

Date Indices 10.05.93 24.05.93 5 --—-

For the transplant. After 2 months T lymphocytes,% 31 53 abs.109 / l 0.5580 1.0196 10 T help cells,% 24 31 abs.109 / l 0.4320 0.5952 T suppressors,% 7 22 abs 109 / l 0.1260 0.4244 T-help cells / T suppressors,% 3.43 1.4 B lymphocytes,% 35 28 abs.-109 / l 0.6300 0.5376

IgG, g / l 8.04 9.12

IgA, g / l 1.7 1.7

IgM, g / l 0.85 1.05 ---

Example 7

Female patient B., born in 1957, was admitted to the Diabetes Department of the Research Institute for Endocrinology and Metabolism in Kiev on September 4, 1992, with complaints of pronounced fatigue, dry mouth despite excessive drinking, polycurgery, weight loss, poor appetite headache.

The patient considered herself sick for three months, when her condition gradually worsened. Drastic deterioration of the situation made her ask for medical help.

Diagnosis: Type 1 diabetes, first expressed, severe form with condition of decompensation, with diabetes ketoacidosis. When taken, the blood sugar content was 20.2 mmol / l and in the urine there was 51.2 g / l glucose and a lot of acetone (++++). The following treatment was prescribed: 62 units per day of insulin and therapy with vitamins and potassium preparations.

Upon reaching the subcompensation, transplantation of native hemopoietic cells from an embryonic liver was performed (sample CO38N-20IL); the embryo was 7 weeks old; number of core-bearing cells 25-106 / ml, amount of cells administered 1 ml; CFU GM 24, 8-103 / ml, CFU bl. 1.7-103 / ml and CD34 35 2-106 / ml. The method of administration was intravenous.

The patient passed the transplant satisfactorily. On the first day the symptom of immediate recovery after transplantation was already seen: reduction of tiredness and headache and better appetite and sleep. From the third day after the transplant, the daily insulin dose was gradually reduced due to the occurrence of hypoglycaemia; the maximum decrease was reached after 2.5 to 3 months (see Table 11).

The positive course of the number of erythrocytes containing embryonic hemoglobin is shown in Table 11.

TABLE 11

Analysis of female patient B before and after transplantation of hemopoietic cells from a human liver 45 (state of compensation)

Date Indices 16.09.92 24.09.92 06.10.92 22.10.92 06.12.92 24.12.92 18.02.92 21.09.92

For the 3rd day 15th After 1 After 2.5 After 3 After 5 After 12 50 trans-day month month months months plant months.

Insulin, one present per day 48 38 32 28 20 18 18 18 55 --- 194693 14 TABLE 11 (continued)

Analysis of female patient B before and after transplantation of hemopoietic cells from a human liver (state of compensation) 5 Date Indices 16.09.92 24.09.92 06.10.92 22.10.92 06.12.92 24.12.92 18.02.92 21.09.92

Before 3rd day 15th After 1 After 2.5 After 3 After 5 After 12 trans-day month month months months plant months.

10 —-—-----—--

Percentage decrease in insulin dose up to. v. the first - 20.8 33.3 41.7 59 62.5 62.5 62.5 Erythrocytes containing embryonic hemoglobin,% 0 12 16 21 25 23 18 14 20 Table 12

Evolution of patient P.'s immunity data

Date Indices 15.09.92 24.09.92 06.10.92 01.12.92 23 For the 3rd day After 15® day After 2.5 transplant. months T lymphocytes,% 44 48 28 37 abs.-109 / l 0.5605 0.9768 1.5958 0.6327 30 T-help cells,% 38 32 21 25 abs.-109 / l 0.4841 0.6512 0.4469 0.4469 T suppressors,% 6 16 7 12 abs.-109 / l 0.0764 0.3256 0.1490 0.2052 T help cells / T suppressors 6.2 2 3 2.1 35 B lymphocytes,% 32 29 26 34 abs.-109 / l 0.4077 0.5902 0.5533 0.5834

IgG, g / l 8.10 11.45 11.45 8.10

IgA, g / l 2.40, 2.55 2.55 2.40

IgM, g / l 0.80 1.40 0.75 1.75 40

The course of the number of erythrocytes with embryonic hemoglobin shows the replacement effect of transplanted cells. The beneficial effect of the bar treatment lasts more than 12 months.

The medicinal preparations prepared by the method according to the invention thus make it possible to: - interrupt the autoimmune aggression against the islets of Langerhans, - reduce the insulin dose by removing the blockage of the remaining beta cells, to eliminate the lability of the disease, - to increase immune resistance, - to restore hemopoiesis, - to limit the complications of diabetes such as pain caused by infections and micro and macro angiopathies and neuropathies.

In addition, the cell suspensions of this invention can be stored in pharmacies and banks of frozen tissues, while it should be noted that histocompatibility does not have to be determined.

15, 194693

Example 8

Male patient Kh, born in 1924. Diagnosis: type 2 diabetes mellitus, state of compensation. Diabetic second-degree retinopathy. maculodystrophy, diabetic micro (stage II) - macro (stage III) angiopathy of vessels in the legs, diabetic foot, non-healing trophic ulcer on left foot, diabetic polyneuropathy. Ischemic heart disease: atherosclerotic cardiosclerosis, blood flow insufficiency-O, cerebral atherosclerosis.

Upon admission, the patient complained of pain in the left foot, a non-healing tropical ulcer on the left foot sole, and trouble seeing. Insulin independent diabetes mellitus was expressed 15 years earlier. For a number of years the patient had been treated by means of a diet, after which he had started taking orally sugar-lowering agents (glibenclamide). At the time of admission, the daily dose of glibenclamide was 0.005 g. No intensive glycemic control had been performed during the entire duration of the disease. Three years before admission, the condition of the patient deteriorated: due to infection of a wound on the big toe of the left foot and gangrene, this toe was amputated. After 18 months a trophic ulcer appeared on the left sole of the foot, which did not heal within 14 months. During the last six months before admission, the patient suffered from a loss of vision. The ophthalmologist's diagnosis was second-degree diabetic retinopathy and maculodystrophy.

During the last year, the patient was admitted three times to the surgery and endocrinology departments of the Donetsk Oblast hospital. The treatment included relief of the affected foot with special footwear and bandage, antibacterial therapy, wound treatment, micro-circulation enhancers, and anticoagulants. The patient received insulin, 18 units twice daily.

The surface of the ulcer became clean although the ulcer did not heal.

When admitted to the Cell Therapy Clinic, the patient was in a state of carbohydrate metabolism compensation: glycemic profile of 5.8-6.6-7.8 mmol / l, aglucosuria.

The treatment was carried out with a medical preparation based on cell suspensions according to the invention (tissue age 7 weeks, number of cells 37.7 106 / ml, KVE-gm, 22 103 / ml, KVE-bl 12 103 / ml, CD34 + 3 , 7 10® / ml, total volume 4.0 ml). The composition was administered dropwise intravenously.

The patient passed the administration satisfactorily. The patient continued to take 0.005 g of glibenclamide daily. No other agents were prescribed. The patient was discharged with the pre-prescribed hypoglycaemic therapy and the recommendation of an intensive glycemic control.

The trophic ulcer healed slowly in 1 month without further medical treatment. After 2 months with regular monitoring of glycemia and glucosuria, the daily dose of glibenclamide was reduced to 0.025 g and after 4 months, glycemia was only controlled with diet.

Example 9

Female patient Kh, born in 1944. Diagnosis: type 2 diabetes mellitus, medium form, condition of subcompensation, climacteric syndrome.

Upon admission, the patient complained of weakness, headache, fear of heights, abnormally dry oral mucosa, presence of hypertension crises accompanied by chills, palpitations, sweating, frequent and excessive urination at the end of each attack.

The above symptoms were observed for two years after the onset of evidence of menstrual cycle disruptions. A menopause was established for one year. Insulin-independent diabetes was diagnosed one year before admission. First the patient was given a diet alone, later she took glycoside (daily dose up to 20 mg); due to poor compensation, 45 glibenclamide was prescribed instead (daily dose 10 mg). During the last month, the patient had taken propranolol (daily dose of 60 mg).

Upon admission, the glycemic profile was 8.4-9.3-11.5-7.4 mmol / l; glucourie 10 g / l. Conventional clinical tests gave results within the standard. ECG: correct synus rhythm. Signs of moderate change in the myocardium.

The patient was treated with the preparation based on cell suspensions according to the invention (tissue age 8 weeks, number of cells 38.7 10® / ml, CFU-gm, 31 103 / ml, CFU-b1 17.3 103 / ml, CD34 + 6.2 10® / ml, total volume 2.0 ml). The composition was administered dropwise intravenously.

The patient withstood the infusion without complications. During the first week, an increase in glycemia with the course of the day was observed. After 2 weeks, a stable state of compensation was obtained using the aforementioned sugar-reducing therapy ((glibenclamide) 10 mg / day). After 1 month the daily dose with an intensive glycemic control was reduced to 5 mg. During the following 7 months of observation, stable compensation of the 194693 carbohydrate metabolism (glycemia during the day 5.4 - 10.3 mmol / l aglucosuria) and normalization of vascular pressure without administration of hypoglycaemic and hypotensive agents were observed. Vegetovascular crises did not actually occur.

Example 10

The results of the treatment of a group of patients with insulin-dependent diabetes mellitus are given here. The diagnosis was made in accordance with WHO classification.

The group consisted of 12 men and 8 women between 1 and 35 years old (average age 17.1 +/- 2.18 years) with clinical diagnosis of first revealed diabetes mellitus.

The group received treatment by administration of human cells. The duration of the disease between diagnosis and treatment was 1 to 6 months (2.6 months on average). The time between the occurrence of classical symptoms and diagnosis was 1 to 5 months (on average 2.2 months). The data regarding this group and the results of the treatment are given in Tables 13 and 14.

A valid reduction in the dose of insulin administered after 4 to 6 weeks was observed in this group, after which the dose remained stable for 1 year after treatment (Table 15). The time of maximum reduction (40.8% on average for the group) was 59.0 +/- 4.38 days. Compared to the control group, the dosage was lower from the third month and throughout the entire observation period (Table 15, Figure 1).

Example 11

The group of 6 patients suffered from severe complications of insulin-dependent diabetes mellitus: general diabetic micro / macro-angiopathy, third-degree diabetic nephropathy, and moderate anemia. The patients were followed for 1 year.

The treatment had a beneficial effect on carbohydrate metabolism: the indices of glycemia reliably decreased and stabilized. The average daily glycaemia reliably decreased on the 10th to 12® days from 9.38 +/- 0.47 mmol / l to 7.04 +/- 0.32 mmol / l (Table 16).

During 2 to 3 months after treatment, the dose of insulin administered was gradually reduced, after which the indices stabilized. For the total group, the average daily dose decreased by 19% during the year, ie from 0.79 +/- 0.09 units / kg to 0.64 +/- 0.09 units / kg.

In all patients, the course of diabetes mellitus before treatment was unstable. The average amplitude of the glycemic variations was 10.27 +/- 1.16 mmol / l. After treatment, this index decreased to 5.11 +/- 0.72 mmol / l and after 2 weeks to 4.75 +/- 0.54 mmol / l (Table 16).

The patients subsequently found that they had no state of hypoglycaemia for several months and that the course of the diabetes was relatively stable.

A haemostimulatory effect was observed in all patients (Table 17).

These indices increased from the 5th to the 7th day and reached maximum values after 1 to 1.5 months (erythrocytes, 3.84 +/- 0.12 1012 / l, hemoglobin 118.8 +/- 4.76 g / l). This effect could be observed for 5 to 11 months. A reliable increase in reticulocytes with a maximum value after 14 to 28 days could be observed on the 3® to 7® day. A positive dynamic of 40 ECR changes was observed, that is, a reliable reduction after 3 to 4 weeks. The changes in leukocyte and platelet counts were not significant.

The immunological indices of patients suffering from diabetic nephropathy, anemia and first-degree chronic renal insufficiency were studied 1 month and 3 months after treatment.

The results are given in Table 18. With the T-cell coupling, the increase in the percentage of 45 CD3 + lymphocytes was statistically reliable, while for the CD4 + and CD8 + lymphocytes, the increase was not statistically reliable. Absolute indices, however, demonstrated a reliable increase in both total T lymphocyte population (CD3 +) and regulatory lymphocyte subpopulations (CD4 + and CD8 +). The initial increase in the percentage of CD22 + lymphocytes was not reliable. During the investigation into the dynamics of the humoral immunity indices, an unreliable reduction in the amount of 50 IgG and IgA was found.

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Change in daily insulin dose (unit / kg / day) in patients suffering from recently expressed insulin-dependent diabetes mellitus (example 10)

5 Time of observation Number Average P

patients shared (standard (unit / - deviation) kg / day) 10 Period 1 Before treatment> 20 0.76 0.06 2 1-7 20 0.74 0.06 0.85 3 7-14 20 0.68 0 05 0.37 4 Days 14-28 20 0.60 0.05 0.06 15 after 28-45 20 0.53 0.05 0.01 * 6 treatment 45-60 20 0.46 0.05 0 00 * 7 60-90 20 0.45 0.06 0.00 * 8 90-180 20 0.47 0.05 0.00 * 9 180-270 19 0.51 0.05 0.00 * 20 10 270-365 15 0.57 0.05 0.03 * 11 Years 2 9 0.58 0.03 0.06 * p <0.05 (statistically significant) 25 ----

Change in daily insulin dose in patients suffering from recently expressed insulin-dependent diabetes mellitus who do (marked)

30 or not treated (not marked). I

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Claims (2)

194693 24 · | A method for preparing medicinal preparations for treating diabetes comprising obtaining a cell suspension containing hemopoietic liver and spleen cells from a human embryo, freezing the obtained cell suspension, and thawing the cell suspension such that the cell suspension liver and spleen cells of a human embryo remain viable and can proliferate after administration to a patient, characterized in that an embryo aged 5-8 weeks is used and that the cell suspension obtained is distributed before freezing by pouring one human embryo prepared cell suspension into a separate set of containers to form a sample bank, intended for administration to at least one patient's contents of one container after thawing, wherein the concentration of nucleus-bearing cells of the cell suspension is 5 to 90- Is 106 / ml, the concentration of colony forming units in the granulomonocytic combination is 20 to t is 80-103 / ml, the concentration of colony forming bladder units is 0.5 to 9-103 / ml and the concentration of early precursors of the hemopoietic CCD34) is 1 to 9.106 / ml. The method of claim 1, wherein the content of each container is 0.5 to 5 ml.