RU2283113C2 - Method for treating the cases of chronic diffuse hepatic diseases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves administering hepatoprotector means and transplanting spinal marrow stem cells. The stem cells are selected from aspirated spinal marrow sample taken from the patient. The portion to be introduced is calculated so that the number of CD34-position cells is equal to 40-240x10⁶ cells. Then the portion is divided into two halves of which the first one is intrahepatically introduced, and the second one is intravenously introduced.

EFFECT: enhanced effectiveness of treatment; reduced risk of hepatic cell insufficiency syndrome.

2 cl

Description

The present invention relates to medicine, in particular to methods for treating chronic diffuse liver diseases accompanied by the development of hepatic cell failure, including toxic, viral and mixed etiology of liver cirrhosis, chronic hepatitis, but not limited to primary sclerosing cholangitis. The cause of hepatic cell failure in patients with chronic diffuse liver diseases is the death of hepatocytes due to the toxic effects of various agents and the replacement of dead cells with connective tissue. The only effective method for treating the final stages of hepatic cell failure is liver transplantation. However, a shortage of donor organs is a limiting factor. In this regard, technologies of cell replacement therapy, which are an alternative to organ transplantation, are of great interest. It is known that the high regenerative ability of the liver is due to the proliferation of hepatocytes. Therefore, one of the directions of cell replacement therapy is transplantation of mature hepatocytes. Isolated hepatocytes can be introduced into the subcutaneous tissue, spleen, and liver parenchyma [1]. Moreover, not only adult hepatocytes, but also fetal liver cells can be used as donor material [2, 3].

Thus, a method has been described according to which autologous hepatocytes isolated from liver fragments were injected into the spleen in patients with liver cirrhosis and chronic hepatitis in order to stop liver-cell insufficiency [4]. Hepatocyte transplantation was not accompanied by the development of any complications, however, due to the use of autologous material and the production of a small number of hepatocytes, no noticeable clinical effect was noted. There is also known a method according to which allogeneic cells (in the amount of about a billion cells) obtained from the liver of 9-17-week-old human fetuses were administered to patients under a capsule of the rectus abdominis muscle. The introduction of such cells was not accompanied by the development of transplant reactions and in some patients was accompanied by a decrease in the manifestations of hepatic cell failure [5].

The disadvantage of the above methods is that the introduction of hepatocytes can only temporarily improve liver function, without significantly affecting the repair processes of damaged hepatic parenchyma. The big problem is the isolation of donor hepatocytes and the maintenance of their functional activity during storage, the risk of infection through the use of allogeneic donor material and the possibility of developing transplant reactions. In the case of the use of embryonic liver cells, there are moral and ethical limitations, a high risk of contamination of the material with bacterial and / or viral microflora. In addition, the possibility of malignant transformation of embryonic cells is discussed in the literature.

Subsequent studies have shown that, along with hepatocytes, an important role in liver regeneration, especially with chronic damage to liver cells, is played by liver stem cells that can differentiate into hepatocytes and cholangiocytes. So, there is a method of obtaining liver stem cells for the treatment of degenerative liver diseases [Patent US 6,129,911, publ. 10/10/2000]. According to this method, stem cells (SCs) or stem cell doublets with hepatocytes that are administered to patients are obtained from liver tissue. The procedure for isolation of SCs includes the disaggregation of connective tissue by enzymatic treatment, filtration through pores of nylon fiber, centrifugation in a density gradient of percoll, negative selection on magnetic beads, enzymatic treatment with trypsin, and positive selection of cells expressing stem cell markers — SK19, CCAM, dipeptidyl peptidase 4 using magnetic beads or flow cytofluorimetry. The resulting stem cells are injected either through the portal vein, or deposited in the spleen, pancreas. The cell source may be its own liver tissue obtained by biopsy or partial hepatectomy; cadaveric liver tissue of donors; liver tissue of fruits. Significant disadvantages of this approach are the complexity and cost of isolating liver stem cells, the limitation in obtaining a sufficient number of autologous stem cells and the lack of evidence for the effectiveness of treatment for human liver pathology.

In addition to liver stem cells, bone marrow SC can also be involved in liver regeneration. Thus, it was shown that, when allogeneic bone marrow transplantation of male donors is performed, liver cells with a Y chromosome are found in the liver of female recipients [6]. In addition, it was shown that bone marrow transplantation in mice in a model of lethal pathology associated with severe liver failure due to a deficiency of the enzyme fumaryl acetate acetate hydrolase is accompanied by the survival of 30% of the animals. These data served as the basis for using bone marrow SC to stimulate liver regeneration [7].

So, Goff and Peterson have proposed a method for transplantation of bone marrow cells in order to stimulate the regeneration and restoration of the liver [Patent No. WO 0050048, publ. 08/31/2000]. According to this method, in order to stimulate regeneration, the bone marrow cells are introduced into the recipient in a dose sufficient to ensure the formation of new hepatocytes, cholangiocytes and / or oval liver cells. An enriched fraction of oval liver cells isolated using antibodies recognizing the Thy 1 antigen can also be used as a source of stem cells. Bone marrow cells or oval cells can be genetically modified to express a functional active protein, transplanted on a matrix, or processed by growth factors before transplantation. Before transplantation, bone marrow cells can be enriched by isolation of cells expressing stem cell markers (Thy 1, CD34, Flt-3 ligand, c-kit), using magnetic bead separation methods, affinity chromatography, or flow cytometry. Moreover, the appearance in the liver of cells (hepatocytes, cholangiocytes, oval cells) of donor bone marrow origin was proved in experimental models of rats by the appearance of hepatocytes with markers of donor stem cells.

However, a significant drawback of this method is the lack of data on its clinical efficacy in the treatment of human liver pathology. It is not clear how the appearance of hepatocytes of bone marrow origin will affect the course of the pathological process in the liver. Moreover, the model of liver damage in experimental animals is fundamentally different from the real clinical situation in terms of etiological factors, the timing of the development of the pathological process and the mechanisms of its formation. Another disadvantage is the lack of data on the volume of bone marrow harvest in humans, sufficient to generate new liver cells.

There is also a method of treating chronic hepatitis (application RU No. 2003105379, published September 20, 2004), which consists in hepatoprotective and immunosuppressive effects by the introduction of heptral, legalon, LIV-52, prednisolone, azathioprine and amino acid mixtures, as well as the introduction of the cytokine G-CSF , causing migration of hematopoietic stem cells from the bone marrow to the periphery, capable of repopulating the liver and differentiating into hepatocytes. However, the administration of G-CSF in patients with chronic hepatitis, as with other chronic diffuse liver diseases, can lead to the activation of the inflammatory process due to the ability of G-CSF to cause an increase in the content of neutrophils and their production of pro-inflammatory cytokines. In addition, the use of immunosuppressants can, on the one hand, provoke activation of virus replication in patients with chronic hepatitis of viral etiology, and on the other hand, reduce the efficiency of stem cell migration into the liver and their differentiation into liver epithelial cells. Finally, by its definition, the method is designed to treat chronic hepatitis and does not include the category of patients with outcomes in cirrhosis, in which hepatic cell failure is the leading pathogenetic factor.

An object of the present invention is to provide a method for the treatment of chronic diffuse liver diseases, including the treatment of a wide range of diseases accompanied by hepatic cell failure, including cirrhosis.

The problem is solved by the use of hepatoprotectors and stem cell transplantation, directly isolated from the patient’s bone marrow aspirate, which are administered intravenously and intrahepally under ultrasound control, containing a sufficient number of CD34-positive cells to achieve a clinical effect.

The application of the claimed method allows to achieve a significant improvement in clinical and laboratory parameters by reducing the manifestations of hepatic cell failure in patients with chronic diffuse liver diseases, including patients with toxic and viral etiology of liver cirrhosis. Moreover, treatment using the proposed method is well tolerated and does not cause the development of any adverse or toxic reactions.

The method is as follows. The patient receives course treatment with hepatoprotectors in the form of preparations of ursodeoxycholic acid (ursosan or ursofalk at a dose of 10-15 mg / kg body weight / day and / or heptral (800 mg, iv, 5-10 injections). After that, the patient in the operating room with anesthetic support, a trepanobiopsy procedure is performed by a hematologist, during which 200-300 ml of bone marrow aspirate is taken from the iliac wing into a sterile vial with a heparin solution (10000-15000 Units) in a laboratory block, the vial is incubated at 37 ° C in 40 m After gravitational separation of the whole bone marrow, the “upper” fraction is collected in a separate vial, consisting of leukemia cells, which are then centrifuged and precipitated (1200 rpm, 15 min) .The supernatant containing autoplasma is collected in a separate sterile vial, and the sediment suspension cells are resuspended in 40-50 ml of phosphate-buffered saline containing 10 IU / ml heparin (PBS / heparin). Then the suspension cells are layered on the density gradient of ficoll-verographin (1.078 g / l) and centrifuged for 20 minutes When 3000 rev / min. The bone marrow mononuclear cells (MNC KM) collected from the interphase are washed three times in PBS / heparin. In order to characterize the isolated cells and count the hematopoietic stem cells, the total number of CMC MNCs selected, their viability by trypan blue staining, and the relative content of CD34 ⁺ stem cells among them by flow cytometry (FACSCallibur, Becton Dickinson) are determined. Half of the isolated cells are resuspended in 2-3 ml of physiological saline supplemented with 10% (v / v) autoplasma (first portion). The other half is in 50-100 ml of physiological saline supplemented with 10% (v / v) autoplasma (the second portion). The first portion is administered intrahepatic at 3-5 points from a single injection under ultrasound guidance. The second portion is intravenous drip.

This method of treating chronic diffuse liver diseases was tested in clinical conditions on the basis of the immunological department of the immunopathology clinic of the Scientific Research Institute of Clinical Immunology SB RAMS, Novosibirsk. The proposed method was treated 14 patients (8 men and 6 women) aged 27 to 67 years. According to a histological study of liver biopsy, liver cirrhosis was detected in all patients. According to the Child-Pugh classification, cirrhosis in stage A was diagnosed in 43% (6/14) cases, in stage B in 50% (7/14) cases, stage C in 7% (1/14). The cause of cirrhosis in 4 patients was toxic hepatitis, in 6 - chronic viral hepatitis (B, C or combined variants - B + C, B + D), in 4 - hepatitis of mixed etiology (viral + toxic). The number of MNCs extracted from bone marrow aspirate varied from 0.65 × 10 ⁹ to 5.3 × 10 ⁹ cells and averaged 1.77 ± 0.2 × 10 ⁹ . The number of introduced SCs in terms of CD34-positive cells averaged 107 ± 26 × 10 ⁶ cells, varying in the range of minimum and maximum values from 40 to 240 × 10 ⁶ cells.

Stem cell transplantation was characterized by good tolerance, in particular, the absence of adverse reactions. Low-grade fever was recorded in only 2 of 14 patients. According to a prospective observation, 1-3 months after transplantation, 43% of patients noted a subjective improvement in the form of a decrease in the severity of asthenovegetative syndrome, improvement of working capacity and vitality. Objectively, a decrease in edema, pain, dyspeptic and hemorrhagic syndromes was recorded in 57, respectively; 86; 93 and 43% of cases. According to biochemical examination, 1-3 months after transplantation, an increase in serum albumin was observed in 64.3% of patients, a decrease in ACT and ALT in 71.4% of patients. Deterioration in the form of an increase in hepatic cell failure 2 months after transplantation was recorded in only 1 patient.

Thus, the proposed method for the treatment of chronic diffuse liver diseases using hepatoprotectors and transplantation of autologous bone marrow stem cells allows us to achieve positive dynamics of clinical and laboratory parameters, including reducing cytolysis and improving the protein-synthesizing function of the liver, which indicates a decrease in the severity of hepatic cell insufficiency. Moreover, treatment using the proposed method is well tolerated and does not cause the development of any adverse or toxic reactions.

The following are clinical examples that explain this method of treatment in patients with cirrhosis.

Example 1. Patient A., 41 years old.

He was admitted to the clinic of immunopathology on October 15, 2003 with complaints of severe general weakness, decreased working capacity, heartburn, constant bitterness in the mouth, sometimes vomiting of blood, aching pains in the right hypochondrium, not associated with eating, skin itching.

The patient has been abusing alcohol for many years. The diagnosis of cirrhosis was first made in 1994, when there was weakness, an increase in the abdomen, weight loss, pain in the right hypochondrium and epigastrium. At the same time, duodenal ulcer was diagnosed. Markers for hepatitis B, C, and D have always been negative. Repeatedly treated in gastroenterological departments.

On examination: the patient's condition is moderate. The skin is dry, grayish yellow in the axillary regions of hyperhidrosis. On the skin of the breast there are single vascular asterisks, gynecomastia, in the umbilical region, the expansion of the veins of the anterior abdominal wall, the presence of palmar erythema. The tongue is wet, "raspberry", at the root is covered with a whitish-yellow coating, in the tongue there are isolated small "cracks".

On palpation, the abdomen is soft, an enlarged liver protruding by 5 cm is palpated, the edge is dense, rounded. The size of the liver according to Kurlov is 14 × 15.5 × 16 cm. The spleen is not palpable. Free fluid in the abdominal cavity is not determined. The result of a histopathological study: Chronic hepatitis of moderate degree of activity (IGA = 18 points), formed cirrhosis of the liver. In the analyzes: PTI - 84%, platelet count 177 thousand / μl; AST - 0.73 mM / L; ALT - 0.60 mM / L; De Ritis index - 1.2; GGTP - 53.3; KFK - 373 U / L; cholesterol - 6.69 mM / l; beta-lipoproteins - 64.5 units; total protein - 77 g / l, albumin - 49 g / l. According to ultrasound: the size of the liver is increased - the right lobe is 14 × 13 cm, the left is 6.0 × 5.4 cm, the spleen is S = 58 cm ² . Echoscopically diffuse changes in the liver, splenomegaly, signs of portal hypertension.

Diagnosis after examination: cirrhosis of the liver of ethanol etiology, active phase, moderate degree of activity. Stage A by Child Pugh classification.

The patient underwent a course of treatment with hepatoprotectors (heptral 400 mg, iv × 5 days). Trepanobiopsy was performed on 10.22.03 and 200 ml of bone marrow aspirate were obtained from the iliac wing, of which 650 × 10 ⁶ MNCs containing 60 × 10 ⁶ CD34 ⁺ cells were isolated. Of these, half of the cells in 2 ml of physiological saline was administered under the control of ultrasound intrahepatic, and the other half in a volume of 50 ml of saline was administered intravenously, calendally. Stem cell transplantation was not accompanied by the development of side effects. When viewed after a month, an improvement was noted: a significant reduction in weakness; the disappearance of pain; heartburn, vomiting is not noted; bitterness in the mouth does not bother. On physical examination, the size of the liver decreased and amounted to 12.5 × 14 × 15 cm according to Kurlov.

A year later, on November 1, 2004, he was hospitalized in the clinic for re-examination. Complaints: rarely occurring pain in the right hypochondrium associated with movement, a tendency to constipation. On examination: palmar erythema is absent, the number of spider veins has decreased (there is only one telangiectasia in the neck), there is a decrease in the severity of gynecomastia. The compacted left lobe of the liver is palpated, painless. The size of the liver is not clinically increased.

According to a histopathological study: chronic hepatitis of a weak degree of activity (IGA = 10 points). Fibrosis of the 4th stage (cirrhosis). In blood tests, platelets are 162 thousand / μl; ALT - 0.67 mM / L; AST - 0.46 mM / L; De Ritis Index 0.68; albumin - 44 g / l, CPK - 307 U / l, cholesterol - 6.12 mmol / l; beta-lipoproteins - 60.1 units.

Ultrasound results: liver - normal sizes: right lobe - 12 × 11 cm, left - 7 × 5.7 cm, portal vein - 14 mm; spleen - S = 50 cm ² . Echoscopically diffuse changes in the liver, moderate signs of portal hypertension.

Thus, 12 months after treatment with the proposed method, a clear positive dynamics of clinical and laboratory indicators was noted in the form of the disappearance of severe weakness, dyspeptic disorders, increased performance and tolerance to physical activity, normalization of the size of the liver and spleen, and a decrease in the activity of the inflammatory process from 18 to 10 points according to the results of histological examination of liver biopsy samples, improvement of blood biochemical parameters.

Example 2. Patient A., 36 years old.

Was admitted to the immunopathology clinic on 06/15/2004 with complaints of severe general weakness, an increase in the abdomen, swelling of the legs and feet, decreased urine output on days free of diuretics, yellowness of the skin and sclera, dry skin, dry mouth, thirst, decreased appetite and bloating, pain in the right hypochondrium, shortness of breath with slight exertion, palpitations, subcutaneous hemorrhages on the legs and abdomen.

The patient considers himself since 1994. After the operation "cesarean section" for a long time remained general weakness, subfebrile condition. In 1995, yellowness and itching of the skin appeared. Diagnosed with chronic cholangiohepatitis. Treatment included hepatoprotectors, infusion therapy, and vitamins. In 1999, she began to note an increase in body weight due to an increase in the volume of the abdomen, and a decrease in diuresis. At the next examination in 2000, antibodies to HCV were detected, with ultrasound - signs of liver cirrhosis, ascites, portal hypertension, based on which liver cirrhosis of viral etiology, chronic viral hepatitis C were diagnosed. Additionally, diuretics with an initial good effect were prescribed - signs of ascites were stopped . Since the fall of 2003, despite the constant intake of diuretics, ascites persisted. After discharge from the hospital, there was practically no improvement; in 2004, swelling of the legs joined. In May 2004, during a regular hospitalization on FGS, signs of varicose veins of the esophagus were revealed, in blood tests - mild anemic syndrome, hypoproteinemia with hypoalbuminemia, hepatosplenomegaly.

An objective examination of the patient's condition of moderate severity, contact, speech is somewhat slowed down. There is ictericity of the skin and sclera, "raspberry tongue", expansion of the veins of the anterior abdominal wall, a sharp increase in the volume of the abdomen, tension of the anterior abdominal wall due to ascites. On palpation, the abdomen is moderately painful along the intestines, swollen. The liver and spleen are not palpable, percussion - the size of the liver is reduced, splenomegaly.

According to laboratory examinations, anemia (Hb = 101 g / l), leukopenia (leukocytes 3.2 × 10 ⁹ / l) and thrombocytopenia (platelets 77 thousand / μl) were detected. Total protein - 55.8 g / l; albumin - 26.2 g / l; thymol test - 15.7; total bilirubin - 60.6 μm / l, ALT - 190 U / l; ACT - 80 U / L; in the urine, the presence of acetone and urobilin was determined. According to the results of ultrasound - a decrease in the size of the liver, cirrhosis, ascites, portal hypertension, splenomegaly. The results of a histopathological study dated 06/17/2004 confirm the signs of moderate hepatitis with a moderate degree of activity, the forming cirrhosis of the liver. PCR for viral hepatitis C in a liver biopsy is negative. When chest x-ray revealed right-sided hydrothorax.

Diagnosis after examination: Cirrhosis of the liver of viral etiology. Chronic viral hepatitis C, active phase (moderate activity). Stage B according to the classification of Child Pugh. Symptoms of portal hypertension - varicose veins of the esophagus and anterior abdominal wall. Ascites. Hepatic cell failure syndrome. Hypersplenism syndrome (cytopenic syndrome). Right-sided hydrothorax.

The patient underwent a course of treatment with hepatoprotectors, including ursofalk (10 mg / kg / day for 10 days) and heptral (800 mg iv for 10 days). 06/26/2004, an autologous stem cell transplant procedure was performed. MNCs extracted from bone marrow aspirate containing 240 million CD34 ⁺ cells were introduced in two portions - 500 million in a volume of 2 ml intrahepatic and 500 million in a volume of 50 ml iv. Adverse reactions with the introduction of stem cells were not observed.

During dynamic observation, after 1 month, an improvement was noted: increased fatigue stopped, body weight decreased due to a decrease in ascites, swelling on the legs, yellowness of the skin, flatulence, bruises on the legs, icteric coloring of the sclera disappeared. According to a laboratory examination: anemia was stopped (Hb - 130 g / l), the platelet count increased to 290 thousand / μl. In the urine analysis, the presence of acetone and urobilin was not observed, ALT - 73 U / L, ACT - 118 units, total bilirubin 24.8 μM / L, total protein 75 g / L, albumin - 32.1 g / L, thymol sample - 12 units

After 3 months - in September 2004, a further decrease in body weight was noted due to a decrease in the severity of ascites, and relief of dyspeptic syndrome. On examination: Hb - 120 g / l, leukocytes - 2.8 × 10 ⁹ / L, platelets - 190 thousand / μl, ALT - 60 U / ml, ACT - 87 U / ml, total bilirubin 9 μM / l, total protein - 66 g / l, albumin - 30.8 g / l, thymol test - 11.65 units. The data obtained indicate that 1-3 months after treatment by the proposed method, the patient showed a positive dynamics of clinical and laboratory parameters in the form of a decrease in the severity of asthenovegetative syndrome, ascites, anemic and thrombocytopenic syndromes, and improvement of the protein-synthetic function of the liver (increase in albumin ), a decrease in cytolysis (decrease in thymol test and ALT), normalization of urine analysis (disappearance of acetone and urobilin), which generally indicates a decrease in the severity of the liver night cell failure.

Thus, verification of the proposed approach under clinical conditions allows us to conclude that this method, including the use of hepatoprotectors and transplantation of autologous bone marrow stem cells, can achieve a significant improvement in clinical and laboratory parameters by reducing the manifestations of hepatic cell failure, primarily cytolysis syndromes, mesenchymally -inflammatory, jaundice, portal hypertension and, to a slightly lesser extent, cholestasis, in patients with chronic diffuse disease iyami liver, including in patients with liver cirrhosis toxic and viral etiology. Moreover, treatment using the proposed method is well tolerated and does not cause the development of any adverse or toxic reactions.

Literature

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Claims (2)

1. A method of treating chronic diffuse liver diseases, including the use of hepatoprotectors and bone marrow stem cell transplantation, characterized in that they use stem cells directly isolated from a patient’s bone marrow aspirate, the portion of which is calculated so that the number of CD34-positive cells in it is from 40 to 240 × 10 ⁶ cells, which are divided into two halves, the first is administered intrahepatic, and the second intravenously. 2. The method according to claim 1, characterized in that the chronic diffuse liver disease is cirrhosis.