RU2126260C1 - Cell suspension-based immunocorrecting drug and using this drug in a method of treating diabetes mellitus - Google Patents

Abstract

FIELD: diabetes. SUBSTANCE: drug is characterized by a definite composition and quantitative characteristics of cell suspension prepared from native or cryopreserved hemopoietic cells of human fetal liver and/or spleen. Diabetes mellitus is treated by injecting a drug selected from preliminarily formed tissue bank of different samples taking into account individual features of patient. For reuse, preparation of the same sample is selected. EFFECT: strengthened immunocorrection mechanism in diabetes treatment. 6 cl, 12 tbl, 7 ex

Description

 The invention relates to medicine, in particular to cell therapy and may find application for the treatment of diseases caused by impaired immunity, for example, diabetes mellitus.

 In recent years, dramatic progress has been made in research into the use of cadaveric human embryonic tissues. A whole new branch of therapy is being developed - cell therapy, which allows using drugs based on cell suspensions prepared from embryonic tissues to make up for the insufficient functional activity of damaged, diseased tissues.

 Cell transplantation is an attractive alternative for organ and tissue transplantation, since the advantage of embryonic cells is that they did not form hard individual signs of antigenic histocompatibility, they easily take root and do not cause a graft versus host reaction. In addition, cells have powerful life potential. They proliferate, differentiate, are a source of a huge amount of biologically active substances. Currently, cell suspensions prepared from the embryonic brain, bone marrow, liver, spleen, thymus, pancreas, and skin-muscle flap are used.

 The first most successful attempts to use cell suspensions as medications were associated with the human embryonic liver and spleen. It is known that hematopoiesis with a high content of stem cells is carried out in the liver or spleen of a human embryo aged 5 to 16 weeks. In these developmental periods, cells of the embryonic liver and spleen only to a small extent express antigens of the first and second class of tissue histocompatibility.

 In 1973, a drug was first created on the basis of a cell suspension of native liver cells of an embryo of a 7-week gestation and, due to its administration, the hematopoiesis of a patient with aplastic anemia was restored (Kelemen E. Second J. Gematol., 1973, v.10, N 4, p. 305-308).

 In recent years, by varying various methods of preparing cell suspensions, as well as various methods of their application, researchers have been able to achieve positive results in the treatment of primary and secondary myelosuppressive conditions. These preparations, in particular, are described in Izzi T., Polehi O., et al, Fetal liver transplantation, Alan R. Liss, 1985, pp 237-249).

 The second direction of the clinical use of the indicated cell suspensions was immunity disorders, here the most significant experience belongs to Touraine J.I (Transplantation Proceedings, 1993, v. 25, No. 1, pp 1012-1013). In the excellent work of Baechelta R, et al in I. Clin. Invest, 1993, v.91, March, 1067-1078) showed the long-term results of treatment of patients with severe combined immune deficiency, when not only the immunity of these patients was restored, but the presence of split chimerism and the appearance of tolerance to antigens of both the host and the donor.

 The authors also have their own experience in treating immune disorders in blood diseases using cell suspensions.

 However, the authors are not aware of the use of these drugs for the treatment of, in particular, diabetes mellitus.

 As is known, the mechanism of development of insulin-dependent diabetes mellitus includes autoimmune damage to the islets of Langerhans by cellular and circulating antibodies. Therefore, in the subsequent treatment of insulin-dependent diabetes mellitus, drugs, substances, tissues are used to interrupt the autoimmune lesion of β-cells of the pancreas by the type of immunocorrection. For this purpose, isoprinosine, azathioprine, cyclosporine, as well as preparations based on biological materials, in particular thymus preparations, were used.

 So, the drug obtained from the thymus gland, thymalin isolated from the thymus of calves, and its use as an immunocorrector in the treatment of diabetes mellitus (Kurbanov T.G., Kalbieva B.M., Zakieva G.T., Problems of endocrinology, 1989, t. 35, N 6, p. 7-9).

 However, a drawback of the known treatment method is that the daily requirement of insulin is reduced by only 15-20%. The decrease in glycemia is maintained only for 5-6 months. In addition, thymalin often does not completely restore immunological parameters, but increases them selectively by a certain percentage without reaching a normal level.

 The basis of the invention is the task of creating such an immunocorrective drug on the basis of a cell suspension in which, thanks to the optimal selection of cell composition indicators, it would be possible to strengthen the immunocorrection mechanism and achieve positive results in the treatment of diabetes mellitus.

The problem is solved in that in a medicament of immunocorrective action based on a cell suspension prepared from native or cryopreserved hematopoietic cells of the liver and / or spleen of a human embryo, according to the invention, the number of nucleated cells in this suspension is from 5 to 90 • 10 ⁶ / ml , the number of colony forming units granulemonotsitarnogo series - from 20 to 80 • ¹⁰ March / ml, the number of colony forming units blasts - from 0.5 to 9 • ¹⁰ March / ml and the number of earlier progenitor gemopo for - from 1 to 9 • June ¹⁰ ml.

 Empirically, it was found that the choice of just such indicators of the cellular composition and their quantitative ratios make it possible to achieve positive treatment results for diabetes mellitus, namely, the transition of the underlying disease to the remission stage.

 The authors believe that it is precisely this composition of the claimed drug that can most effectively influence the mechanism of immunocorrection, in particular, stop the aggression of T-lymphocytes and immunoglobulins against β-cells. In addition, the drug of the claimed composition, being a source of biologically active substances, causes a powerful trophic effect, manifested in an increase in the number of functioning capillaries, normalization of permeability, cell migration, their differentiation, which, ultimately, determines the completeness of the inflammatory process and a decrease in the phenomenon of dystrophy.

 According to the invention, it is advisable that when cryopreserved, the drug additionally contains dimethyl sulfoxide in an amount of from 3 to 10%. The choice of the lower limit is due to the highest preservation of cells, and the upper one is based on the toxic effect of the drug.

 The problem is also solved by the fact that in the method of treating diabetes mellitus, which consists in introducing biological material of immunocorrective action, according to the invention, a patented drug is used as biological material of immunocorrective action.

 The authors believe that cell suspension-based drugs have never been used to treat diabetes. However, long-term research work on the mechanisms of action of drugs based on cell suspensions on the treatment processes of various diseases suggests that cell suspensions of the claimed composition can be very successfully used to treat diabetes. This is evidenced by all kinds of clinical trials conducted by the authors, some of which are described in detail in the Examples below. Patients observed a significant improvement in their immune status, up to a recovery of 3-11 days (an increase in the number of T-lymphocytes, a change in the ratio of subpopulations, normalization of indicators of humoral immunity). The initial dose of insulin was reduced by 40-70%. Within 3-4 weeks, a remission of the disease was achieved.

 In accordance with one aspect of the invention, it is advisable to administer a patented drug in a dose of 0.5 ml to 5 ml.

 In addition, it is desirable that this drug be administered before and / or after therapy with cytotoxic drugs.

 It is advisable to choose a drug from the formed tissue bank of various samples, taking into account the individual indicators of the patient, and with repeated administration, use the drug of the same sample.

 The proposed method for the treatment of diabetes mellitus allows the introduction of a patentable cell suspension to correct various disorders that determine the course of diabetes mellitus, in particular, insulin resistance, the development of macro- and microangiopathies, damage to the kidneys, liver, development of anemic syndrome, thrombocytopenia, etc., restoring lost blood formation functions and the immune system.

 Further, the essence of the invention will become more clear from the following detailed description of its implementation set forth in specific Examples of its implementation.

A drug of the claimed composition can be made using the following methodology:
Embryos are obtained by abortion in healthy women previously examined for the presence of viral and hemic infections. Using embryos for a period of 5 to 8 weeks of gestation. In order to maintain the integrity of the embryo, abortion is performed by vacuum extraction. The embryo is transferred into a sterile vessel with Hanks and antibiotic solution / aminoglycoside group /. Further work is carried out in sterile boxing conditions. Embryos are transferred to sterile Petri dishes filled with Hanks solution with antibiotic, where, carefully opening the abdominal cavity, the liver and spleen are removed, from which cell suspensions are separately prepared.

 Hematopoietic organs are placed in homogenizers, cut into small fragments and crushed to obtain a homogeneous mass. Cells are washed off the walls and pestle of the homogenizer with Hanks solution in volumetric tubes, passing through a filter for transfusion of blood products, and then through needles of decreasing diameter. Part of the prepared suspension is transferred to a plastic container and sealed. It will be used for transplantation of native cell suspension. The other part is subject to cryopreservation.

 Dimethyl sulfoxide (DMSO, chemically pure) is used as a cryoprotectant. Before use, DMSO is passed through a millipore filter (with a pore diameter of 0.22 μm.). With gentle stirring of the cell suspension, an equal volume of the DMSO working solution is added dropwise, reaching a concentration of preferably 5%.

 Cell suspensions are poured into plastic containers, depending on further purposes, from 0.5 to 2 ml.

The containers are placed in a chamber of a liquid nitrogen program freezer and frozen to -196 ^o C.

Cryopreserved suspensions are stored in a bank of embryonic tissues in liquid nitrogen at a temperature of -196 ^o C.

After preparation of the cell suspension, the following indicators are determined: 1st — the number of nucleated cells in 1 ml, 2nd — the number of colony forming units of the granulomonocytic row (CFU GM) in 1 ml, 3rd — the number of colony forming blast units (CFU bl.) In 1 ml, 4th - the number of early hematopoiesis precursors (CD ₃₄ ) in 1 ml.

And then cell suspensions of the following parameters are used and stored in a tissue jar:
1st - from 5 to 90 • 10 ⁶ ml ^-1
2nd - from 20 to 80 • 10 ³ ml ^-1
3rd - from 0.5 to 9 • 10 ³ ml ^-1
4th - from 1 to 9 • 10 ⁶ ml ^-1
Prenatal diagnosis includes studies for syphilis, HIV infection, viral hepatitis B and C, toxoplasmosis, cytomegalovirus infection. Studies of the contents of containers on bacterial sterility are being conducted.

 Fetal diagnostics include studies for the presence of HIV infection, viral hepatitis B and C, cytomegalovirus, rubella virus, herpes and toxoplasmosis.

Depending on the clinical treatment option, the drug is administered using one of the following methods:
- the introduction (transplantation) of a portion of native hematopoietic cells, subsequently the introduction of cryopreserved cells stored in the bank of this sample;
- the introduction of the entire native sample;
- the introduction of part or all of the cryopreserved material of this sample;
Before and in some cases, after the administration of a patented drug, one or a combination of cytostatic drugs is treated in order to reduce the immunological reactivity of the recipient, and to reduce autoimmune aggression.

 The treatment effect is assessed by the level of glycemia reduction, a decrease in the dose of insulin administered, and the occurrence of hypoglycemic conditions; a change in indicators of the immune status / blood content of T-B lymphocytes, immunoglobulins /; the number of peripheral blood red blood cells containing fetal hemoglobin; the presence of cells containing the Y chromosome, determined by the method of chain reaction of polymerization using oligonucleotide primers.

 A detailed description of the use of the patented invention in the clinical practice of the authors is presented, also in Examples 1-7.

 Example 1. Patient D., born in 1946, was admitted to the diabetology department of the Ukrainian Research Institute of Endocrinology and Metabolism on 08.21.93. with complaints of severe weakness, dizziness, shortness of breath with slight physical exertion, palpitations, poor appetite, nausea, skin itching, headache, swelling in the legs, decreased vision.

 From the anamnesis it is known that 13 years ago, insulin-dependent diabetes mellitus was detected. She constantly received insulin therapy, was observed by an endocrinologist, and was periodically treated in a hospital. Over the past two years, a worsening condition has been noted: arterial hypertension has appeared, the course of the underlying disease has become labile, in urine tests it has constantly become protenuria. Over the past year, there has been weakness, periodically swelling of the lower extremities, dyspeptic phenomena, a decrease in the level of red blood cells and hemoglobin of peripheral blood. In March 1993, she was treated in the neurological department of the regional hospital, the presence of chronic renal failure, moderate anemia was established. Made 4 transfusions of red blood cell mass. After a temporary improvement, the condition worsened sharply in September 1993, which was the reason for hospitalization.

 Upon admission, the patient is in serious condition. Consciousness is clear. The skin and visible mucous membranes are pale. Peripheral lymph nodes are not enlarged. Pulse - 98 beats / min, rhythmic, satisfactory filling and tension. Blood pressure 190/120 mm RT. Art. Heart sounds of sufficient sonority, rhythmic, systolic murmur at the apex, emphasis II tone on the aorta. In the lungs, vesicular breathing is heard. The abdomen is soft, moderately swollen, sensitive to palpation in the right hypochondrium. The liver protrudes from under the edge of the right costal arch along the right midclavicular line by 2 cm. The spleen is not enlarged. Segments of the colon are spasmodic. Stool - 4 times a day, mushy. Daily diuresis up to 1 liter. The face, feet, legs are swollen.

 Diagnosed with diabetes mellitus, type 1, severe, in a state of decompensation. Diabetic universal angiopathy (nephropathy of the III degree, chronic renal failure of the 1st degree; vascular form of proliferating retinopathy; micro-macroangiopathy of the vessels of the lower extremities of the 2nd degree); Diabetic polyneuropathy of the lower extremities. Severe anemia. Symptomatic hypertension.

Upon receipt of glycemia - 12.4 mmol / l, glucosuria - 22 g / l, acetonuria - (+ +), proteinuria - 3.3 g / l, erythro- and cylindruria (5 in the field of view), K ⁺ - 5 , 2 mmol / L, urea - 14.42 mmol / L, creatinine - 0.220 mmol / L, red blood cells - 2.1 • 10 ¹² / L, hemoglobin - 64 g / L.

 The treatment was prescribed: diet therapy, insulin therapy - 22 units / day, detoxification therapy (enterosorbents, hemodesis, bowel lavage), diuretics, antihypertensives, angiaggregants, angioprotectors, electrolyte balance correction, iron preparations, vitamin therapy.

 Three weeks later, the patient's condition improved slightly: carbohydrate metabolism was compensated, swelling subsided, blood pressure decreased to 170/100 - 110 mm Hg. Art. However, persistent severe anemic syndrome persisted.

09/15/93 transplantation of native hematopoietic cells of the human embryonic liver (sample Z038C-82H) was performed: the embryo was 5 weeks old, the number of nucleated cells was 25 • 10 ⁶ / ml, the volume of tissue introduced was 1.5 ml, CFU GM-13.9 • 10 ³ , CFU bl. -1.3 • 10 ³ / ml, CD ₃₄ - 2.4 • 10 ⁶ / ml. The route of administration is intraosseous (intrathoracic).

 The patient underwent transplantation satisfactorily. After 8-10 hours, the syndrome of early post-transplant improvement was observed: headache, weakness, nausea decreased, appetite improved, the patient slept quietly for the first time in the last few months. On the 3rd day, the level of hemoglobin, erythrocytes and reticulocytes of peripheral blood increased with normalization by the 10th day (Table 1), urine tests were significantly improved over the course of three weeks (Table 2). Blood pressure decreased to 160 / 100-90 mm RT. Art., which allowed to reduce the dose of antihypertensive drugs.

 Example 2. Patient P., born in 1960, was admitted to the diabetology department of the Kiev Research Institute of Endocrinology and Metabolism on 6.11.91 with complaints of general weakness, weight loss, dry mouth, thirst, and excessive urination. Considers herself ill since September 1991, when such complaints first appeared. Not surveyed. A sharp deterioration in November 1991 forced to seek medical help.

 Diagnosed with diabetes mellitus, type 1, first detected, severe, in a state of decompensation, diabetic ketoacidosis.

 On admission, the level of glycemia was 15 mmol / l, glucosuria - 41.6 g / l, acetonuria - / +++ /. The treatment was prescribed: insulin therapy - 60 units per day, vitamin therapy, potassium preparations.

After achieving the compensation of the state on 2.12.1991. the first transplantation of native hematopoietic cells of the human embryonic liver and spleen was performed (sample Z038C - 63HL): the age of the embryo was 7 weeks, the volume of introduced cells was 1 ml, the number of nucleated cells was 36.2 • 10 ⁶ / ml, CFU GM - 23.6 • 10 ³ / ml, CFU bl. - 2.5 • 10 ³ / ml, CD ₃₄ - 1.4 • 10 ⁶ / ml. The route of administration is intravenous. The volume of transplanted cells of the total tissue mass of this sample located in the cryopreserved tissue bank was 66%.

 The patient had a satisfactory transplantation. On the first day, we observed a syndrome of early post-transplant improvement in general condition: a decrease in weakness, an improvement in appetite, the patient became more alert, more active.

 Three weeks after transplantation, hypoglycemic conditions appeared, which caused a gradual decrease in the daily dose of insulin administered, with a maximum decrease after 2.5 months (Table 3).

 We observed the normalization of indicators of immune status on the 11th day (Table 4). Within six months after the first transplantation, the patient was in a compensated state with a stable relatively low daily dose of insulin.

 At the end of May 1992, the patient's condition worsened: signs of decompensation of carbohydrate metabolism appeared, indicators of immune status worsened (Table 4). The daily dose of insulin was increased to 32 units.

 06/03/92 repeated transplantation of cryopreserved hematopoietic cells of the human embryonic liver of the same sample was performed: N З038С - 63HL - 0.5 ml of a cell suspension was introduced. The patient suffered a transfusion satisfactorily. The positive dynamics of the daily dose of insulin administered, indicators of immune status are shown in Table. 3, 4.

 The dynamics of changes in the number of red blood cells containing fetal hemoglobin indicates the substitution effect of transplanted cells.

 Example 3. Patient R., born in 1961, was admitted to the 1st therapeutic department of the CRH in the Zaliznychny district of Kiev on December 1, 1992 with complaints of severe weakness, weight loss, dry mouth, thirst, excessive urination, headache, and bad appetite.

 Considers herself a patient since October 1992, when she felt deterioration. Not surveyed. A sharp deterioration in condition at the end of November 1992 was the reason for seeking medical help.

 Diagnosed with diabetes mellitus, type 1, first detected, severe, compensated, diabetic ketoacidosis. Upon admission, the level of glycemia was 20.0 mmol / L, glucosuria - 52.4 g / L, acetonuria - / +++ /. The treatment was prescribed: insulin therapy - 64 units per day, vitamin therapy, potassium preparations, hemodesis 200.0 every other day N 6. The patient was prescribed azathiopyrine in a daily dose of 2 mg per 1 kg of body weight in two divided doses, starting from 25 mg per day, gradually increasing dose under daily control of peripheral blood. Upon reaching the indicated therapeutic dose, the treatment was carried out with weekly monitoring of the number of leukocytes, platelets, red blood cells in the peripheral blood.

32 days after the initiation of treatment with azathioprine, upon reaching subcompensation of carbohydrate metabolism disorders on 5.1.93, cryopreserved hematopoietic cells of the human embryonic liver were transplanted / sample З038С - 44H /: embryo age 6 weeks, injected cell volume - 1 ml, number of nucleated cells - 17 , 5 • 10 ⁶ / ml, CFU GM - 24.8 • 10 ³ / ml, CFU bl. - 1.7 • 10 ³ / ml, CD ₃₄ -2 • 10 ⁶ / ml, the route of administration is intravenous. The volume of transplanted cells from the total tissue mass of this sample located in the cryopreserved tissue bank was 45%.

 The patient underwent transplantation satisfactorily. On the first day, an syndrome of early post-transplant improvement of the general condition was observed: a decrease in weakness, headache, and an improvement in appetite.

 Treatment with azathioprine continued for 48 days after transplantation of hematopoietic cells of the human embryonic liver.

 2 weeks after transplantation, hypoglycemic conditions appeared, which caused a gradual decrease in the daily dose of insulin administered, with a maximum decrease after 2 months, / Table. 5/.

 By the end of the second week, indicators of the patient’s immune status returned to normal / Table. 6 /.

 The dynamics of changes in the number of peripheral blood erythrocytes containing fetal hemoglobin indicates the substitution effect of transplanted cells.

 Remission of the disease lasts more than 9 months.

 Observation continues.

 Example 4. Patient L., born in 1991, was in the endocrinology department of the Children's Specialized Clinical Hospital N 14 in Kiev from 09/15/93 to 09/26/93.

 Diagnosis: Insulin-dependent diabetes mellitus, severe form, labile course, subcompensation state.

 The disease was detected in February 1993, when in a precomatous state the child was taken to the intensive care unit.

 The diagnosis was established: Type 1 diabetes mellitus. Insulin therapy was started - 8 units / day. In the following months, the daily dose of insulin was reduced to 6 units. The nature of the course of the main process was labile: frequent hypoglycemic conditions in alternation with ketoacidosis. The girl was hospitalized in the endocrinology department for transplantation of hematopoietic embryonic cells.

 Upon receipt of glycemia during the day from 5.5 to 12.4 mmol / l, glucosuria - 3.2 g / l, acenoturia - is absent.

09/12/93. transplantation of cryopreserved hematopoietic cells of the human embryonic liver (sample З038С - 91H) was performed: the embryo was 5 weeks old, the number of nucleated cells was 25.0 • 10 ⁶ / ml, the volume of introduced tissue was 1.5 ml, CFU GM - 23.9 • 10 ³ / ml, CFU bl. - 1.8 • 10 ³ / ml, CD ₃₄ - 4 • 10 ⁶ / ml. The route of administration is intravenous.

 The patient underwent transplantation satisfactorily. According to the mother, the child’s appetite improved in the evening after transplantation, the sleep became calm, deep. On the fourth day after transplantation, the daily dose of insulin administered was reduced to 5 units. Subsequently, the daily dose of insulin administered remained the same.

 Within a month, the patient's immunological parameters returned to normal (Table 7).

 Throughout the time after transplantation, the condition of the child remains satisfactory: the course of the disease stabilized - glycemia during the day at the level of 4.5 - 7.3 mmol / l, hypoglycemia and episodes of ketoacidosis were not observed. Increased irritability, tearfulness decreased. Psychophysically, the girl develops according to age. Observation continues.

 Example 5. Patient A., born in 1969, was in the therapeutic department of the Central Clinical Hospital of the Zaliznychny District of Kiev from 07/21/93 to 08/12/93.

 Diagnosis: Insulin-dependent diabetes mellitus, severe form, labile course, compensation state, diabetic universal angiopathy of the II stage, diabetic peripheral polyneuropathy. Chronic cholecestitis, remission phase.

 Considers herself sick since 1986, when, after a strong psycho-emotional overstrain, health began to deteriorate: weakness, polydipsia, polyuria, loss in body weight. After 2 months, when he sought medical help, he was diagnosed with insulin-dependent diabetes mellitus. Compensation of the state was achieved with a daily dose of insulin administered in 64 units.

 Over the past year, the appearance of pain in the legs, mainly at night, a burning sensation, tingling, sometimes numbness, cramps in the calf muscles. The main disease has acquired a labile nature: frequent changes in a satisfactory state of ketoacidosis or hypoglycemia with minor changes in food intake, physical and emotional stress.

 The patient was hospitalized in the therapeutic department for transplantation of hematopoietic cells of the human embryonic liver.

 Upon receipt of glycemia during the day from 4.7 to 11.4 mmol / l, glucosuria - 22.4 g / l, acetonuria is absent. The daily dose of insulin is 58 units.

On July 28, 1993, native hematopoietic cells of the human embryonic liver were transplanted (sample З038 - 101H): the age of the embryo was 6 weeks, the number of nucleated cells was 8.5 • 10 ⁶ / ml, the volume of introduced tissue was 2.5 ml, CFU GM - 23 • 10 ³ / ml, CFU bl. - 2.4 • 10 ³ / ml, CD ₃₄ - 3.8 • 10 ⁶ . The route of administration is intravenous.

 The patient transplanted satisfactorily. After 8-10 hours, the appearance of the syndrome of primary post-transplant improvement of the general condition was observed: a decrease in weakness, an increase in working capacity, and an improvement in sleep.

 Within two weeks, the dose of insulin administered was reduced to 50 units per day. In general clinical studies, no significant changes occurred (within normal limits before and after transplantation). The indicators of immunological status were restored after 1 month (Table 8).

 The level of erythrocytes containing fetal hemoglobin increased to 15%.

 Observation continues for more than 12 months. The patient feels satisfactorily: stable compensation of the condition has been achieved, there have been no episodes of ketoacidosis.

 The daily dose of insulin is maintained at the level of 50-52 units. Red blood cells containing fetal hemoglobin are determined in the range of 8-10%.

 Example 6. Patient M., born in 1931, was in the surgical department of the Central Clinical Hospital of the Zaliznychny District of Kiev from 04/12/93 to 05/26/93. Diagnosis: Diabetes mellitus, type 1, severe form of subcompensation, universal diabetic angiopathy: micro-macroangiopathy of the vessels of the lower extremities of the III st., retinopathy, nephropathy of the II st. ; trophic ulcer of 1 finger of the right foot, foot phlegmon. Diabetic polyneuritis.

Upon admission, the patient complained of general weakness, fever up to 38.5 ^o C, chills in the morning, swelling, feeling of fullness in the right foot, ulceration of soft tissues of 1 toe of the right foot, numbness, tingling in the legs, leg pain, both at rest and during physical exertion.

 Suffers from insulin-dependent diabetes mellitus for 22 years. Over the past 5 years there have been complaints of pain in the legs when walking, calming down at rest, as well as a feeling of numbness, tingling in the legs, mainly at night. A month ago, in the area of 1 toe of the right foot, an ulcer of soft tissues opened.

The patient was treated on an outpatient basis without result. After the next bath, the foot sharply swelled, the lower leg, the pain intensified, severe hyperemia of the skin appeared, the body temperature increased to 38 ^o C. Immediately after receipt, the phlegmon was opened, surgical treatment was performed.

Prescribed antibiotic therapy, anti-inflammatory drugs, insulin therapy - 38 units per day. The patient's condition did not change: body temperature rises in the evenings to 38.6 ^o C continued, general weakness, purulent discharge from the incision and trophic ulcer continued. Three days later, the patient listened to the right-sided lower-lobe pneumonia. Bacteriological cultures of blood, urine, feces were made. Staphylococcus aureus is seeded from the blood. The patient received 3 courses of antibiotic therapy for three weeks / ampiox, gentamicin, rifampicin, claforam /. In the analysis of blood leukocytosis with a shift of the leukocyte formula to the left, accelerated ESR, moderate anemia / Table 9 /. In the immunogram, the number of T-lymphocytes is sharply reduced, the number of β-lymphocytes is slightly increased / Table 10 /.

In order to strengthen the immune defense, on 10.05.93, native hematopoietic cells of the human embryonic liver were transplanted / sample З038С - 57H /: the embryo was 8 weeks old, the number of nucleated cells was 37.5 • 10 ⁶ / ml, the volume of tissue introduced was 2, 5 ml, CFU GM - 42 • 10 ³ / ml, CFU bl. - 1.3 • 10 ³ / ml, CD ₃₄ - 1.4 • 10 ⁶ / ml, the route of administration is intravenous.

8-10 hours after transplantation, the manifestations of the syndrome of the primary post-transplant improvement of the general condition were observed: in the evening the body temperature decreased to 37 ^o C, the weakness decreased, the appetite appeared, and slept calmly at night. Gradually, blood counts returned to normal within 7-10 days / Tab. nine/.

 The cleansing of the wound and ulcer from purulent necrotic masses began. On the seventh day, healthy granulations appeared and epithelization began. Two weeks later, the ulcer and wound healed.

 Observed the restoration of indicators of the immune status on the 14th day / Table. ten/.

 Due to the appearance of hypoglycemia on the fifth day, the daily dose of insulin was reduced to 34 units.

 The patient was discharged home in satisfactory condition.

 Example 7. Patient B., born in 1957, was admitted to the diabetology department of the Kiev Research Institute of Endocrinology and Metabolism on September 4, 1992 with complaints of severe weakness, thirst, dry mouth, excessive urination, weight loss, poor appetite, and headaches.

 Considers herself a patient over the past three months, when the condition gradually began to worsen. Not examined and not treated. A sharp deterioration in the condition was the reason for seeking medical help.

 The diagnosis was established: Type 1 diabetes mellitus, first detected, severe, decompensation state, diabetic ketoacidosis. Upon receipt, glycemia - 20.2 mmol / l, glucosuria - 51.2 g / l, acetonuria - / ++++ /. The treatment was prescribed: insulin therapy - 62 units per day, vitamin therapy, potassium preparations.

After the subcompensation of the condition was achieved, native hematopoietic cells of the human embryonic liver were transplanted / sample З038С - 20HL /: the age of the embryo was 7 weeks, the number of nucleated cells was 25 • 10 ⁶ / ml, the volume of introduced tissue was 1 ml, CFU GM - 19.3 • 10 ³ / ml, CFU bl, - 2.1 • 10 ³ / ml, CD ₃₄ - 1.8 • 10 ⁶ / ml. The route of administration is intravenous. The volume of transplanted cells from the total tissue mass of this sample located in the bank of cryopreserved cultures was 50%.

 The patient underwent transplantation satisfactorily. On the first day, an syndrome of early post-transplant improvement of the general condition was observed: a decrease in weakness, headaches, an improvement in appetite, and sleep. From the third day after transplantation, in connection with the onset of hypoglycemia, they began to reduce the dose of daily insulin with a maximum decrease after 2.5-3 months / Table. eleven/.

 Positive dynamics of immunological parameters of patient B. are given in Table. 12.

 The dynamics of changes in the number of red blood cells containing fetal hemoglobin indicates the substitution effect of transplanted cells.

 Remission of the disease lasts more than twelve months.

 Thus, the proposed drug and method for the treatment of diabetes mellitus with the aim of using the drug allows: to terminate autoimmune aggression against islets of Langerhans; reduce the dose of insulin by removing cell blockade from the preserved β-cells; reduce or remove the lability of the flow; reduce immunoresistance; restore hematopoiesis; reduce such complications of diabetes as infectious, micro- and macroangiopathies, neuropathies.

 In addition, cell suspensions of the claimed composition can be stored in drug stores-banks of cryopreserved tissues and, given the absence of the need to determine histocompatibility antigens, suggest an easier application if indicated than, for example, blood transfusion.

Claims (6)

1. An immunocorrective drug based on a cell suspension prepared from native or cryopreserved hematopoietic cells of the embryonic liver and / or spleen, characterized in that the number of nucleated cells in such a suspension is from 5 to 90 • 10 ⁶ ml ^-1 , the number of colony forming units granulomonocytic series from 20 to 80 • 10 ³ ml ^-1 , the number of colony forming blast units - from 0.5 to 9 • 10 ³ ml ^-1 , the number of early hematopoietic precursors from 1 to 9 • 10 ⁶ ml ^-1 .  2. The drug according to claim 1, characterized in that during cryopreservation, the cell suspension additionally contains dimethyl sulfoxide in an amount of from 3 to 10%.  3. A method for the treatment of diabetes mellitus, which consists in the introduction of biological material with immunocorrective action, characterized in that the drug according to claim 1 is used as the material of immunocorrective action.  4. The method according to claim 3, characterized in that the said preparation is administered in a dose of 0.5 to 5 ml.  5. The method according to claim 4, characterized in that the indicated drug is administered before or after therapy with cytostatic drugs.  6. The method according to any one of claims 3 to 5, characterized in that the drug is selected from the formed tissue bank of various samples, taking into account the individual characteristics of the patient, and when re-introduced using the same sample.

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