RU2413524C1 - Method of treating mental and neural disorders - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to neurology and psychiatry, and concerns the use of stem cells of umbilical blood for treating mental and neural disorders. That is ensured by introduction of nuclear cells of umbilical blood prepared by sedimentation or gradient centrifugation of sampled umbilical blood with separating the nuclear cells in the form of packed cells to be resuspended in cooled plasma with a cryoprotector added, in an amount 1×10⁸ to 5×10⁸ nuclear cells per one introduction.

EFFECT: invention provides effective therapy of neurodegenerative diseases and schizophrenia with lower risk of immune-associated diseases due to the fact that the method for preparing the nuclear cells of umbilical blood eliminates cultivation thereof with a foreign serum added.

5 cl, 11 ex, 1 tbl

Description

FIELD OF THE INVENTION

The invention relates to medicine and relates to the use of cord blood cells for the treatment of mental and neurological disorders.

State of the art

Cord blood stem cells are a unique cell population that differs from cells obtained from other sources, including embryonic, fetal and “adult” stem cells. The uniqueness of cord blood cells lies in the fact that this is the only type of cells of postnatal origin, capable of supporting blood formation during transplantation to experimental animals and forming a full-fledged human immune system, due to the formation of B and T lymphocytes and dendritic cells, the formation of primary and secondary lymphoid organs and production functional immune responses [Traggai E, Chicha L, Mazzuchelli L, et al. Development of a human adaptive immune system in cord blood cell-transplanted mice. Science 2004; 304: 14-107].

Hematopoietic stem cells (CD34 +) and endothelial progenitor cells (CD 133+) are the most represented cell populations found in cord blood. The high content of hematopoietic progenitor cells allows the use of cord blood cell transplantation as an alternative to bone marrow transplantation in acute and chronic leukemia, lymphomas, Fanconi anemia and other hematological diseases [Barker J. Umbilical cord blood (UCB) transplantation: An alternative to the use of unrelated volunteer donors? Hematology Am Soc Hematol Educ Program. 2007: 55-61].

In addition to hematopoietic, cord blood contains other types of stem cells, including embryo-like cells, epithelial progenitor cells, mesenchymal stem cells and unlimited somatic stem cells [Harris David T., Rogers I. Umbilical Cord Blood: A Unique Source of Pluripotent Stem Cells for Regenerative Medicine, Current Stem Cell Research & Therapy, 2007, 2, 301-309].

The pluripotent properties of cord blood stem cells have been demonstrated in a number of in vitro and in vivo studies. Under cultivation, cord blood stem cells are able to differentiate into osteoblasts, chondroblasts, adipocytes and nerve cells, as well as endothelial and muscle cells, hepatocytes and insulin-producing cells [Kogler, G., Sensken, S., & Wernet, P. ( 2006) Comparative generation and characterization of pluripotent unrestricted somatic stem cells with mesenchymal stem cells from human cord blood. Experimental Hematology, 34 (11), 1589-95 and Tondreau T, Meuleman N, Delforge A, et al. Mesenchymal stem cells derived from CD133-positive cells in mobilized blood and cord blood: proliferation, expression and plasticity. Stem Cells, 2005; 23: 1105-1112].

Cord blood stem cells are capable of homing and, during transplantation, experimental animals can improve the course of a number of diseases, including diabetes, cardiovascular, and neurological and ophthalmic diseases, diseases of the musculoskeletal system, etc.

Type 1 diabetes occurs on average in one in 300 newborns and is found in more than 8 million adults in Russia. Type 1 diabetes is caused by immune destruction of beta cells in pancreatic islets responsible for insulin production. The result is an uncontrolled blood glucose level. Complications of diabetes include cardiomyopathy, damage to the coronary arteries and peripheral vessels, neurological disorders, etc. In attempts to treat type 1 diabetes, surgical methods have been developed for transplantation of the pancreas or individual islets, which have had limited success due to immune rejection, the absence of compatible donors and numerous complications. In this regard, special hopes were placed on the use of cellular technologies based on the use of stem cells from various sources and aimed at restoring the function of insulin-producing pancreatic tissue. A study by Haller et al. Found a significant improvement in controlled glucose levels and longer insulin production compared to other children with a similar prognosis in response to the use of cord blood cells [Haller MJ, Cooper SC, Brusco T, et al. Autologous cord blood transfusion associated with prolonged honeymoon in a child with type 1 diabetes. Diabetes 2005; 53 (Suppl 1): Poster A485]. Clinical trial protocols were developed on the basis of data obtained on a type 1 diabetes model, according to which experimental animals had a lower level of glucose and insulin in the blood and an increased life span compared to control animals [Ende N, Chen R, Reddi AS. Effect of human umbilical cord blood cells on glycemia and insulinitis in type 1 diabetic mice. Biochem Biophys Res Commun 2004; 325: 665-669 and Ende N, Chen R, Mack R. NOD / LtJ type I diabetes in mice and the effect of stem cells (Berashis) derived from human umbilical cord blood. J. Med 2002; 33: 181-187].

Although the mechanisms of the action of cord blood stem cells in treating diabetes are unknown, it is suggested that stem cells differentiate into pancreatic islet-secreting insulin cells during in vivo infusion [Haller MJ, Cooper SC, Brusco T, et al. Autologous cord blood transfusion associated with prolonged honeymoon in a child with type 1 diabetes. Diabetes 2005; 53 (Suppl 1): Poster A485], which was confirmed in in vitro studies [Sun B, Roh K-H, Lee S-R, Lee Y-S, Kang K-S. Induction of human umbilical cord blood-derived stem cells with embryonic stem cell phenotypes into insulin producing islet-like structures. Biochem Biophys Res Commun 2007, 354: 919-923].

The use of umbilical cord blood mononuclear cells in the treatment of chronic hepatitis or liver cirrhosis is described in patent RU 2,295,351. The disadvantage of this method is mandatory HLA typing and, moreover, in order to achieve a clinical effect, the cord blood stem cells of the donor must differ from the recipient cells by no less than four antigens from the set of antigens HLA-A, HLA-B, HLA-DR.

As for the possibility of using cord blood stem cells in the repair of bone and cartilage tissue, unlike bone marrow stem cells, their potential role in orthopedics has not been studied. The most common orthopedic disorders are joint damage, including damage to cartilage, ligaments and / or tendons, as well as fractures. Cord blood stem cells are capable of differentiating into bone cells in vitro upon shock wave induction [Wang FS, Yang KD, Wang CJ, et al. Shockwave stimulates oxygen radical-mediated osteogenesis of the mesenchymal cells from human umbilical cord blood. J Bone Mineral Res 2004; 19: 973-979]. The ability to use cord blood cells for fractures has been demonstrated in animal models with hip fractures. The ability of these cells to differentiate into cartilage cells was also shown [Szivek JA, Wiley D, Cox L, Harris D, Margolis DS, Grana WA. Stem cells grown in dynamic culture on micropatterned surfaces can be used to engineer cartilage-like tissue. Abstract, Orthopedic Research Society meeting, San Diego, CA, 2006].

The ability of cord blood stem cells to give rise to epithelial tissue opens up the possibility of their clinical use in regenerative medicine for diseases of the eyes (cornea), skin (wound healing) and other organs (for example, the lungs and digestive tract). The outer layer of the eye consists of a central cornea, limbus and sclera. The corneal epithelium is capable of rapid regeneration and can be used as an own source of stem cells during regeneration. Deficit of corneal epithelial cells is an important cause of vision loss resulting from chemical or thermal damage, Stevens-Johnson syndrome, cicatricial changes, aniridia, chronic rosacea, keratoconjunctivitis. If the corneal epithelium is disturbed, a clear image cannot be focused on the retina. Autologous transplantation of corneal epithelial stem cells has been successful in patients with unilateral disease. However, obtaining cells from a functional healthy eye poses a risk of loss of vision for a healthy eye. Additionally, autologous transplantation is not available under bilateral conditions, and corneal epithelial stem cell allotransplantation requires immunosuppressive therapy with possible systemic side effects, as a result of which the average survival time of such an allograft does not exceed 2 years. Severe corneal lesions requiring intervention are no exception, accounting for up to 37% of all cases of vision loss [Germain L, Auger FA, Grandbois E, et al. Reconstructed human cornea produced in vitro by tissue engineering. Pathobiology 1999, 67: 140-147 and Germain L, Carrier P, Auger FA, Salesse C, Guerin S L. Can we produce a human corneal equivalent by tissue engineering? Prog Retinal Eye Res 2000; 19 (5): 497-527].

Nichols Group Researchers [Harris DT, He X, Camacho D, Gonzalez V, Nichols JC. The Potential of Cord Blood Stem Cells for Use in Tissue Engineering of the Eye, Stem Cells & Regenerative Medicine, Jan 23-25, 2006, San Francisco, Abstract and Nichols JC, He X, Harris DT. Differentiation of Cord Blood Stem Cells Into Corneal Epithelium. Invest Ophthalmol Vis Sci 2005; 46: E-Abstract 4772] used CD34-positive cord blood stem cells as a source of tissue for reconstructing the surface of the eye. Histological and immunohistochemical studies of the epithelium formed from umbilical cord blood cells revealed that the layer of differentiated cells was morphologically indistinguishable from the corneal epithelium. Newly formed cells were also able to express a specific marker of corneal epithelium - cytokeratin k3. During stem cell transplantation, the cornea was restored to rabbits to form an optically clear surface. The ability of cord blood stem cells to differentiate into corneal epithelial cells suggests the possibility of using them to accelerate wound healing of various etiologies, for example, diabetic ulcers. According to this hypothesis, in 2004 it was shown that allogeneic cord blood stem cells are able to initiate the restoration of wound skin lesions [Valbonesi M, Giannini G, Migliori F, Dalla Costa R, Dejana AM. Cord blood (CB) stem cells for wound repair. Preliminary report of 2 cases. Transfus Apher Sci 2004; 30 (2): 153-156]. Precursor cells were mixed with an autologous fibrin matrix and introduced at a concentration of 3x10 ⁴ into the area adjacent to the lesions. 3-7 months after the injection, significant healing of skin lesions was observed in both patients.

Cardiovascular diseases are one of the main causes of mortality and disability in the population. Heart tissue cells have a limited ability to regenerate, and the use of stem cells could be an effective substitute for traditional therapies.

In animal models, data have been obtained indicating the migration of stem cells to damaged myocardial tissue, an increase in the density of capillaries in the lesion site and an improvement in heart function [Botta R, Gao E, Stassi G, et al. Heart infarct in NOD-SCID mice: therapeutic vasculogenesis by transplantation of human CD34 + cells ad low dose D34 + KDR + cells. FASEB J 2004; 18: 1392-1394; Henning RJ, Abu-Ali H, Balis JU, et al. Human umbilical cord blood mononuclear cells for treatment of acute myocardial infarction. Cell Transplant 2004; 13: 729-739; Chen HK, Hung HF, Shyu KG, et al. Combined cord blood cells and gene therapy enhances angiogenesis and improves cardiac performance in mouse after acute myocardial infarction. Eur J Clin Invest 2005; 35: 677-686; Hirata Y, Sata M, Motomura N, et al. Human umbilical cord blood cells improve cardiac function after myocardial infarction. Biochem Biophys Res Commun 2005; 327: 609-614; Kim BO, Tian H, Prasongsukarn K, et al. Cell transplantation improves ventricular function after a myocardial infarction: a preclinical study of human unrestricted somatic stem cells in a porcine model. Circulation 2006; 112 (9 suppl): 196-204 and Leor J, Guetta E, Feinberg MS, et al. Human umbilical cord blood-derived CD133 + cells enhance function and repair of the infracted myocardium. Stem Cells 2006; 24 (3): 772-780].

Gaballa et al. [Furfaro MEK, Gaballa MA. Do adult stem cells ameliorate the damaged myocardium? Is human cord blood a potential source of stem cells? Curr Vascular Pharm 2007; 5: 27-44] on a model of myocardial infarction in rats showed that CD34-positive cells of umbilical cord blood induce the formation of blood vessels, reduce the size of the heart attack, restore heart function. It is believed that these effects are due to the release of angiogenic factors and growth factors (for example, VEGF, EGF, angiopoietin-1, 2, etc.) induced by hypoxia.

The ability of cord blood pluripotent cells to form blood vessels and restore myocardium has been shown in studies on the restoration of cardiac function after myocardial infarction using CD34 +, CD133 + CD45 cells in various animal models [Botta R, Gao E, Stassi G, et al. Heart infarct in NOD-SCID mice: therapeutic vasculogenesis by transplantation of human CD34 + cells ad low dose CD34 + KDR + cells. FASEB J 2004; 18: 1392-1394; Kim BO, Tian H, Prasongsukarn K, et al. Cell transplantation improves ventricular function after a myocardial infarction: a preclinical study of human unrestricted somatic stem cells in a porcine model. Circulation 2006; 112 (9 suppl): 196-204; Leor J, Guetta E, Feinberg MS, et al. Human umbilical cord blood derived CD133 + cells enhance function and repair of the infracted myocardium. Stem Cells 2006; 24 (3): 772-780 and Bonnano G, Mariotti A, Procoli A, et al. Human cord blood CD133 + cells imunoselected by a clinical-grade apparatus differentiate in vitro into endothelial-and cardiomyocyte-like cells. Transfusion 2007; 47: 280-289].

Cerebrovascular diseases are in one of the first places due to mortality, not including individuals who survived after a stroke, but with consequences that manifest over the remaining period of life.

Cerebral ischemia is the most prevalent cause of strokes. At a relatively young age, due to the plasticity of the brain, a sufficiently large part of the brain can be removed (in the case of tumors or hemispherectomy with serious damage) without neurological damage or with a short recovery period. These data suggest that the younger the nerve cells that could be produced by differentiation from stem cells, the greater the possibility of regeneration of the damaged brain.

Throughout a person’s life there is always a risk of neurological diseases and injuries. So, the incidence of cerebral palsy in newborns is high. Both children and adults suffer from spinal cord injuries. The risk of embolic and hemorrhagic strokes increases among individuals over the age of 40. And finally, after 40 years and in old age, the incidence of neurodegenerative diseases is high, such as Parkinson's disease [Harris, D. T., Badowski, M., Ahmad, N., & Gaballa, M. (2008). The potential of cord blood stem cells for use in regenerative medicine. Expert Opinion on iological Therapy, 7 (9), 1311-1322 and Harris, D.T., & Rogers, I. (2007). Umbilical cord blood: a unique source of pluripotent stem cells for regenerative medicine. Current Stem Cell Research & Therapy, 2, 301-309]. Traditional therapies used in relation to neurological diseases and injuries are ineffective, which places a burden on society. The use of stem cells in the event of neurological diseases or injuries would open up the possibility of replacing or regenerating damaged tissues of the spinal cord or brain. However, success depends on the choice of stem cell source and their appropriate application.

Unlike many other pathological conditions that were evaluated for the possibility of cord blood stem cell therapy, the restoration of neurological damage has a much more complicated etiology.

However, in animal models with neurological damage caused by brain injuries, cord blood stem cell infusion led to an improvement [Chen J, Sanberg PR, Li Y, et al. Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats. Stroke 2001; 32: 2682-2688; Willing AE, Lixian J, Milliken M, et al. Intravenous versus intrastriatal cord blood administration in a rodent model of stroke. J Neurosci Res 2003; 73 (3): 296-307; Borlongan CV, Hadman M, Sanberg CD, Sanberg PR. Central Nervous System Entry of Peripherally Injected Umbilical Cord Blood Cells Is Not Required for Neuroprotection in Stroke. Stroke 2004; 35: 2385-2389; Newman MB, Willing AE, Manressa JJ, Sanberg CD, Sanberg PR. Cytokines produced by cultured human umbilical cord blood (HUCB) cells: implications for brain repair. Exp Neurol 2006, 199 (1): 201-208; Vendrame M, Cassady J, Newcomb J, et al. Infusion of human umbilical cord blood cells in a rat model of stroke dose dependently rescues behavioral deficits and reduces infarct volume. Stroke 2004; 35: 2390-2395; Xiao J, Nan Z, Motooka Y, Low WC. Transplantation of a novel cell line population of umbilical cord blood stem cells ameliorates neurological deficits associated with ischemic brain injury. Stem Cells Dev 2005; 14: 722-733; Newcomb JD, Ajrno CT, Sanberg CD, et al. Timing of Cord Blood Treatment After Experimental Stroke Determines Therapeutic Efficacy. Cell Transplant 2006; 15: 213-223; Nan Z, Grande A, Sanberg CD, Sanberg PR, Low WC. Infusion of Human Umbilical Cord Blood Ameliorates Neurologic Deficits in Rats with Hemorrhagic Brain Injury. Ann NY Acad Sci 2005, 1049 (1): 84 - 96; Bliss T, Guzman R, Daadi M, Steinberg GK. Cell transplantation therapy for stroke. Stroke 2007; 38: 817-826; Saporta S, Kim JJ, Willing AE, et al. Human umbilical cord blood stem cells infusion in spinal cord injury: engraftment and beneficial influence on behavior. J Hematother Stem Cell Res 2003; 12: 271-278; Kuh SU, Cho YE, Yoon DH, et al. Functional recovery after human umbilical cord blood cells transplantation with brain derivedneurotropic factor into the spinal cord injured rats. Acta Neurochir (Wein) 2005; 14: 985-992; Kang KS, Kim SW, Oh YH, et al. Thirty-seven-year old spinal cord-injured female patient, transplanted of multipotent stem cells from human UC blood with improved sensory perception and mobility, both functionally and morphologically: A case study. Cytotherapy 2005; 7: 368-373; Lu D, Sanberg PR, Mahmood A, et al. Intravenous administration of human umbilical cord blood reduces neurological deficit in the rat after traumatic brain injury. Cell Transplant 2002; 11: 275-281; Meier C, Middleanis J, Wasielewski B, et al. Spastic paresis after perinatal brain damage in rats is reduced by human cord blood mononuclear cells. Ped Res 2006; 59: 244-249; Ende N, Chen R. Parkinson's Disease Mice and Human Umbilical Cord Blood. J Med 2002; 33: 173-80; Gaebuzova-Davis S, Willing AE, Zigova T, et al. Intravenous administration of human umbilical cord blood cells in a mouse model of amyotrophic lateral sclerosis: distribution, migration, and differentiation. J Hematother Stem Cell Res 2003; 12: 255-270].

Jang et al. [Jang YK, Park JJ, Lee MC, et al. Retinoic acid-mediated induction of neurons and glial cells from human umbilical cord-derived hematopoietic stem cells. J Neurosci Res 2004; 75: 573-584] demonstrated that CD 133+ cord blood stem cells, when exposed to retinoic acid, differentiate into nerve (astrocytes and oligodendrocytes) and glial cells with neuromarkers, including tubulin, neurospecific enolase, NeuN, protein-2 associated with microtubulin (MAP2 ) and acidic glial fibrillar protein (GFAP), which is an astrocytospecific marker.

Moreover, non-hematopoietic stem cells found in cord blood can differentiate into astrocytes and oligodendrocytes [Buzanska L, Jurga M, Stachowiak EK, Stachowiak MK, Domanska-Janik K. Neural stem-like cell line derived from a nonhematopoietic population of human umbilical cord blood. Stem Cells Develop 2006; 15: 391-406]. Thus, descendants of non-hematopoietic cord blood stem cells could also be useful in treating brain injuries and neurological disorders.

In a rat spinal cord injury model, umbilical cord blood stem cell infusion resulted in a significant improvement on day 5 after infusion compared to control animals. Stem cells were detected in areas of damage to the nerve tissue, but were not found in the intact areas of the spinal cord [Saporta S, Kim JJ, Willing AE, et al. Human umbilical cord blood stem cells infusion in spinal cord injury: engraftment and beneficial influence on behavior. J Hematother Stem Cell Res 2003; 12: 271-278]. These data supplement the results of studies on the ability of cord blood cells transplanted into the spinal cord of animals with injuries to differentiate into various types of nerve cells, improving axon regeneration and motor function [Kang KS, Kim SW, Oh YH, et al. Thirty-seven-year old spinal cord-injured female patient, transplanted of multipotent stem cells from human UC blood with improved sensory perception and mobility, both functionally and morphologically: A case study. Cytotherapy 2005; 7: 368-373].

In 2001, the most common stroke model (medial carotid obstruction) demonstrated that infusion of cord blood cells into rats could improve various physical and behavioral deficiency conditions associated with the disease [Chen J, Sanberg PR, Li Y, et al. Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats. Stroke 2001; 32: 2682-2688].

Studies demonstrate that direct injection of stem cells into the brain is not required [Willing AE, Lixian J, Milliken M, et al. Intravenous versus intrastriatal cord blood administration in a rodent model of stroke. J Neurosci Res 2003; 73 (3): 296-307] and, in fact, positive effects can be observed even if the stem cells do not reach the target organ (probably through the release of growth and repair factors triggered by anoxia) [Borlongan CV, Hadman M, Sanberg CD , Sanberg PR. Central Nervous System Entry of Peripherally Injected Umbilical Cord Blood Cells Is Not Required for Neuroprotection in Stroke. Stroke 2004; 35: 2385-2389 and Newman MB, Willing AE, Manressa JJ, Sanberg CD, Sanberg PR. Cytokines produced by cultured human umbilical cord blood (HUCB) cells: implications for brain repair. Exp Neurol 2006; 199 (1): 201-208].

It is significant that, unlike the use of pharmaceuticals that require use in the first few hours after a stroke, cord blood stem cell therapy is effective up to 48 hours after a thrombotic incident [Newcomb JD, Ajmo CT, Sanberg CD, et al. Timing of Cord Blood Treatment After Experimental Stroke Determines Therapeutic Efficacy. Cell Transplant 2006; 15: 213-223]. The benefits of cord blood cell therapy are also shown in a hemorrhagic stroke model [Nan Z, Grande A, Sanberg CD, Sanberg PR, Low WC. Infusion of Human Umbilical Cord Blood Ameliorates Neurologic Deficits in Rats with Hemorrhagic Brain Injury. Ann NY Acad Sci 2005; 1049 (1): 84-96].

A method for treating a stroke based on bone marrow stem cell transplantation is described in RU 2 283 121. A significant drawback of the method is neurosurgical intervention: intracerebral stereotactic administration of stem cells under neuronavigation control to the lesion of functionally important structures (depending on clinical manifestations). If a deficiency of perfusion of the substance of the brain is detected, this operation is supplemented by the application of extra-transcranial microanastamosis.

Although bone marrow is a source of hematopoietic, mesenchymal, and endothelial stem cells, most bone marrow stem cells are obtained from adult donors, which limits their plasticity. Umbilical cord blood cells obtained at birth are a unique source of pluripotent stem cells [Atala et al. Umbilical Cord Blood Current Stem Cell Research & Therapy, 2007, Vol. 2, No. 4 307]. Moreover, umbilical cord blood cells are not exposed to environmental-induced epigenetic effects.

The use of cord blood mononuclear cells in the treatment of coronary heart disease, myocardial infarction, cerebral ischemia caused by atherosclerosis or acute cerebrovascular accident is described in patent RU 2 284 190. The disadvantage of this method is the obligatory HLA typing, and, moreover, to achieve a clinical effect in ischemic conditions, donor cord blood stem cells must differ from the recipient cells in at least four antigens from the set of antigens HLA-A, HLA-B, HLA-DR.

In addition to strokes, animal models have shown the possibility of using cord blood stem cells in the treatment of other forms of neurological damage. Among them [Lu et al. Intravenous administration of human umbilical cord blood reduces neurological deficit in the rat after traumatic brain injury. Cell Transplant 2002; 11: 275-281] in a rat model, it was shown that intravenous administration of umbilical cord mononuclear cells could be useful in treating traumatic brain injuries. Moreover, it was demonstrated that umbilical cord blood cells reach the brain, selectively migrate to the damaged area of the brain, express neuromarkers and reduce neurological damage.

Similarly, cord blood stem cell transplantation could also alleviate the symptoms of cerebral palsy in newborns in a rat model [Meier C, Middleanis J, Wasielewski B, et al. Spastic paresis after perinatal brain damage in rats is reduced by human cord blood mononuclear cells. Ped Res 2006; 59: 244-249].

Regenerative therapy using cord blood stem cells, focusing on the functional restoration of damaged tissues, is especially relevant for diseases of the central nervous system, in particular degenerative diseases.

Alzheimer's disease is the most common cause of dementia in the elderly and senile. The main role in the pathogenesis of the primary neurodegenerative Alzheimer's process is assigned to the disruption of transformations of the protein precursor of β-amyloid (Aβ), accompanied by the accumulation of insoluble Aβ in the brain parenchyma in the form of aggregated clusters - amyloid plaques. Aβ aggregates, in turn, are potential activators of microglia and astrocytes that respond to cerebral amyloidosis by activation of a chronic inflammatory process [Benzing W.C., Wujek J.R., Ward E.K., Shaffer D., Ashe K.H. et al. Evidence for glialmediated inflammation in aged APP (SW) transgenic mice. Neurobiol Aging, 1999, 20, p. 581-589].

Although it was previously believed that activation of microglia and astrocytes is only an epiphenomenon and is not involved in the pathogenesis of Alzheimer's disease, recent studies have established that the Aβ-induced inflammatory cascade is one of the important pathogenetic mechanisms of the Alzheimer's type neurodegenerative process. It has been proven that therapeutic strategies aimed at various components of this inflammatory cascade, including non-steroidal anti-inflammatory drugs, immunization and modulation of microglial activation, reduce Alzheimer-like β-amyloid pathology, cognitive deficit and behavioral disorders in the model of Alzheimer's disease in some transgenic mice, cases also reduce pathology in patients with Alzheimer's disease [Bard F., Cannon C, Barbour R., Burke RL, Games D. et al. Peripherally administered antibodies against amyloid-beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nature Med., 2000, 6, p. 916-919; Lim G.P., Chu T., Yang F., Beech W., Frautschy S.A. et al. The curry spice curcumin reduces oxidative damage and amyloid pathology in an Alzheimer transgenic mouse. J. Neurosci, 2001, 21, p. 8370-8377; Nicoll J.A., Wilkinson D., Holmes C, Steart P., Markham H. et al. Neuropathology of human Alzheimer disease after immunization with amyloid-beta peptide: a case report. Nature Med., 2003, 9, p. 488-452; Scheme D., Barbour R., Dunn W., Gordon G., Grajeda H. et al. Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature, 1999, 400, p. 173-177; Szekely C.A., Thorne J.E., Zandi P.P., Ek M., Messias E. Nonsteroidal antiinflammatory drugs for the prevention of Alzheimer's disease: a systematic review. Neuroepidemiology, 2004, 23, p. 159-169; Veld B.A., Ruitenberg A., Hofman A., Launer L.J., van Duijn CM. Nonsteroidal anti-inflammatory drugs and the risk of Alzheimer's disease. N. Engl. J. Med., 2001, 345, p.1515-1521].

As shown in animal models, human cord blood cells enhance the anti-inflammatory response of type 2 T helper cells, and this response was accompanied by a decrease in the volume of myocardial infarction in the brain tissue [Vendrame M., Cassady J., Newcomb J., Butler T., Pennypacker K.R. et al. Infusion of human umbilical cord blood cells in a rat model of stroke dose-dependently rescues behavioral deficits and reduces infract volume. Stroke, 2004, 35, p. 2390-2395]. Later, this therapeutic effect was confirmed on other models of neuroinflammatory processes [El-Badri N.S., Hakki A., Saporta S., Liang X., Madhusodanan S. et al. Cord for glial-mediated inflammation in aged APP (SW) transgenic mice. Neurobiol blood mesenchymal stem cells: Potential use in neurological disorders. Stem Cells Dev., 2006, 15, p. 497-506; Garbuzova-Davis S., Gografe S.J., Sanberg CD., Willing A.E., Saporta S. et al., Maternal transplantation of human umbilical cord blood cells provides prenatal therapy in Sanfilippo type B mouse model. FASEB J., 2006, 20, p. 485-487; Henning R.J., Burgos J.D., Ondrovic L., Sanberg P., Balis J. et al. Human umbilical cord blood progenitor cells are attracted to infracted myocardium and significantly reduce myocardial infarction size. Cell Transplant., 2006, 15, p. 647-658].

In animal experiments on a model of Alzheimer's disease, this effect was associated with an increase in the lifespan of transgenic mice [Nikolic WV, Hou N., Town T., Zhu Y., Giunta B. Peripherally administered human umbilical cord blood cells reduce parenchymal and vascular β-amyloid deposits in Alzheimer mice. Stem Cells Dev., 2008, 17, p.1-17], reported a marked reduction in the level of Aβ and the number of amyloid plaques in transgenic mice after a series of iv injections of cord blood stem cell concentrate.

Parkinson's disease, the frequency of which is also high in old age, is a neurodegenerative disease with a "point" locus - damage to the cells of the substantia nigra leading to their death. And although the history of the study of this pathology has been going on for almost two centuries, the etiology and genesis of this process are not reliably known. Hypotheses on the exotoxic role of certain substances (for example, 1-methyl-4-phenyl-1,2,3,6-tetrohydroperidine, which is converted under the influence of MAO B into the compound N-methyl-4-phenylpyridine toxic to neurons of the substantia nigra) or genetic factor (association with the 4q21-q23 segment inherited in an autosomal dominant manner) is nevertheless an assumption [Cleeter MWJ et al .: Irreversible inhibition of mitochondrial complex I by 1-methyl-4-phenylpyridinium: Evidence for free radical relaxation. J Neurochem 58: 786, 1992; Polymeropoulos MH et al .: Mapping of a gene for Parkinson's disease to chromosome 4q21-q23. Science 274: 1197, 1996].

The deficiency of dopaminosynthetic function resulting from the death of the above cells leads to a malfunction of the mediator chain of the extrapyramidal system, which leads to inhibition of the motor cortex, which leads to clinical symptoms of Parkinson's disease. The relevance of the study of this problem is also dictated by epidemiological data (the prevalence of Parkinson's disease in the general population is 1-2 per 1000, and for every person over 65 years old, every one in a hundred suffers from this ailment).

Currently used methods of drug correction (drugs containing L-DORA; D2 / D3 agonists of dopamine receptors; MAO inhibitors, etc.) are not effective because they lead to the progression of the process and present a symptomatic approach with a lot of limitations (Aminoff MJ: Treatment of Parkinson's disease. West J Med 161: 303, 1994). Moreover, the use of this therapy has a time limit. Thus, the restoration of normal natural dopamine production by resuscitated (or newly grown) substantia nigra cells is a key problem in the treatment of Parkinson's disease. Given the degenerative nature of the pathological process in Parkinson's disease, the implementation of cell intervention in order to restore the empty area of the nervous tissue is the most attractive.

For amyotrophic lateral sclerosis, Parkinson's disease in animal models, umbilical cord blood stem cell infusion led to a marked improvement in behavior compared to control animals [Ende N, Chen R. Parkinson's Disease Mice and Human Umbilical Cord Blood. J Med 2002; 33: 173-80; Garbuzova-Davis S, Willing AE, Zigova T, et al. Intravenous administration of human umbilical cord blood cells in a mouse model of amyotrophic lateral sclerosis: distribution, migration, and differentiation. J Hematother Stem Cell Res 2003; 12: 255-270].

Attempts have been made to use embryonic stem cells and adult bone marrow stem cells for ischemic strokes and parkinsonism in clinical trials. Thus, methods have been proposed for the treatment of ischemic stroke and parkinsonism using pre-propagated neuronal stem cells in culture obtained from the tissue of the forebrain or periventricular region of the brain of aborted human embryos (patents RU 2258521 and RU 2259836). However, the possibility of using embryonic stem cells and bone marrow stem cells in therapy is limited due to significant disadvantages.

The use of fetal stem cells is associated with ethical problems, difficulties in cell cultivation and is associated with a high risk of bacterial and / or viral microflora contamination of the material. With the introduction of fetal stem cells by infusion without long-term and expensive stages of cultivation and differentiation in vitro, malignant transformation of embryonic cells is possible, the formation of teratoma is the most common threat. Long-term immunosuppression, which is necessary when using embryonic cells due to the threat of immune rejection, is also undesirable. A significant disadvantage of the methods of treating ischemic strokes and parkinsonism described in the patents RU 2258521 and RU 2259836 is the endolumbar method of transplantation.

A known method for the treatment of amyotrophic lateral sclerosis by introducing into the blood of nucleated cells of umbilical cord blood in an amount of not less than 5 × 10 ⁹ (WO 2004/071283). The method described in WO 2004/071283 does not require preliminary suppression of the immune response or HLA typing of the donor and recipient, but extremely large numbers of cells are necessary for its implementation. The number of nucleated cells in one portion of cord blood does not exceed 2.8 × 10 ⁹ . This amount is not enough to implement the method. Thus, not only simultaneous introduction of allogeneic cells into the body is required, but also cells from several different donors. In this case, the risk of allergic, including anaphylactic reactions, as well as the risk of other immunopathological conditions, including immunosuppression, sharply increases.

In addition, the simultaneous immunization with cells of various donors makes it impossible to reapply the method in the same patient. Application of the method in 5 cases out of 10 led to toxic and allergic reactions with the development of immunosuppression in all patients within 7-30 days after administration. An attempt to re-use the method led to toxic-allergic reactions in most patients.

The problem of treating multiple sclerosis, a chronic demyelinating disease of the central nervous system (CNS), remains one of the most urgent in modern neurology, due to the high prevalence of the disease among young working age people and the inevitable development of persistent disability at a certain stage.

Identification of various types of demyelination, from the predominance of inflammatory reactions to oligodendrogliopathy and abnormal remyelination, as well as evidence of axonal damage not only in the active foci, but also in the apparently unchanged white matter brought multiple sclerosis closer to neurodegenerative diseases [Lucchinetti C. et al. Brain Pathol., 1996, V.6, P.25922; Trapp B.D. et al. Neuroscience, 1999, V.5, p. 48].

Immunopathological mechanisms of the development of exacerbations in multiple sclerosis [Zavalishin I.A., Zakharova M.N. Multiple sclerosis and other demyelinating diseases. / Ed. Guseva E.I. et al. M., 2004, S. 60; Multiple sclerosis. Selected questions of theory and practice. / Ed. Zavalishina I.A., Golovkina V.I. M., 2000; Hohlfeld R. Brain, 1997, V.120, P.865] include activation of vigorous, inactive CD4 + T cells outside the central nervous system; their penetration through the blood-brain barrier (BBB); the formation of a trimolecular complex, including the corresponding activated T-cell receptor and autoantigen, bound to class II molecules of the main histocompatibility complex on antigen-presenting cells (macrophages and glia cells act as the latter). Penetrated into the nervous system, autoreactive T cells and secondary activated macrophages and microglia secrete pro-inflammatory cytokines (interferon-gamma, tumor necrosis factor-alpha, lymphotoxin, etc.), which in turn even more induce and support inflammatory reactions and exacerbate disorders BBB permeability. Despite the fact that the clinical course of multiple sclerosis in the initial stages is usually characterized by alternating exacerbations and remissions, the immuno-inflammatory process as a whole is characterized by a constant course.

As a result of the cascade of immunological and biochemical disorders, damage to myelin and oligodendrocytes develops. At later stages of the pathological process, non-specific mechanisms are activated: phagocytosis of damaged structures and proliferation of glial elements. However, in addition to the development of inflammatory reactions, the demyelinating process, and glial disorders, the main role in the development of multiple sclerosis is given to the involvement of axons in the process. It is axonal damage in multiple sclerosis that is considered responsible for the development of irreversible neurological deficiency and the transformation of remitting course with the lability of symptoms into secondary progressive [Trapp B.D. et al. Neuroscience, 1999, V.5, P.48].

Some of the most likely causes of axon destruction in multiple sclerosis are considered. The mechanism of direct immunological attack of axons in multiple sclerosis is the expression of class I molecules of the main histocompatibility complex on them, which makes them vulnerable to the cytotoxic effect of CD8 + type T-lymphocytes [Neumann N. et al. Science, 1995, V.269, P.549]. The development of axonal degeneration due to inflammatory reactions may be associated with various factors [Rieckmann P., Maurer M. Curr. Opin. Neurol., 2002, V.15, p. 361], including an increase in extracellular pressure in inflammatory edema; damage due to exposure to glutamate (produced by activated macrophages and microglia) on oligodendroglial AMPA receptors; the release of pro-inflammatory cytokines.

However, degenerative changes in axons are also possible in a chronic demyelinating process due to a violation of myelin-axonal interactions and the trophic effect of oligodendrogliocytes, in particular the modulating effect of myelin-associated glycoprotein and proteolipid protein. The mechanisms of their trophic effect on the axon are different, however, with a deficiency of both of these proteins, axonal pathology develops in the nodal region with damage to the axonal cytoskeleton [Trapp B.D. et al. Neuroscience, 1999, v. 5, p. 48].

Another important aspect of axonal-glial interactions is the effect of trophic factors. So, in the group of patients with multiple sclerosis with an early onset and severe course of the disease, mutations were identified that led to the absence of a ciliary neurotrophic factor [Ciess R. et al. Arch. Neurol., 2002, v. 59, p. 407]. A similar effect on the course of the disease was also described in patients with the e4 allele of apolipoprotein E, in whom an early onset, a higher frequency of exacerbations, and faster progression of the disease were noted [Fazekas F. et al. Neurology, 2001, v. 57, p. 853]. These data indicate that, regardless of the immune-mediated damage to myelin and axons, the genetically determined structural and molecular integrity of axonal-glial interactions is important in the demyelinating process.

The development of neurological deficiency in multiple sclerosis is considered from the point of view of two pathogenetic mechanisms: reversible acute inflammatory demyelination, which dominates in the exacerbation of the disease in the remitting course of multiple sclerosis, and irreversible axonal degeneration, which prevails in the secondary progressive course [Trapp B.D. et al. Neuroscience, 1999, V.5, P.48].

The same changes determine the development of atrophic processes in the central nervous system in multiple sclerosis. First, demyelination itself leads to the loss of compact myelin and, consequently, the brain parenchyma. Secondly, axonal degeneration secondary to axon damage in acute foci of demyelination.

A special place is occupied by the primary progressive variant of the course of multiple sclerosis, which has clinical, genetic, immunobiochemical, neuroimaging and morphological differences from the remitting and secondary progressive variants [Ge Y. et al. Radiology, 2000, V.214, P.665; Redmond IT. Et al. Acta Neurol. Scand., 2000, V.102, P.99], which suggests the presence of special pathogenetic mechanisms in the primary progressive course of multiple sclerosis. Among them, primary damage of oligodendrogliocytes, secondary demyelination in the absence of remyelination and signs of inflammation, as well as diffuse axonal pathology, which could cause a gradual increase in neurological deficiency from the very beginning of the disease, are considered.

One of the standard regimens in the treatment of multiple sclerosis is currently the use of glucocorticosteroids (intravenous pulse therapy with methylprednisolone, methylprednisolone tablet), which positively affect the rate of recovery of neurological disorders during exacerbation of the disease [Goodin D.S. et al. Neurology, 2002, V.58, P.169]. In addition, pulse therapy is also used when the condition worsens against the background of secondary progression of neurological symptoms. In primary progressive multiple sclerosis, as a rule, it does not give an effect. For relief of severe exacerbations of multiple sclerosis with severe neurological deficiency, especially in case of impaired vital functions, plasmapheresis in combination with intravenous administration of methylprednisolone can be used.

To prevent or reduce the frequency of exacerbations, stabilize the state, prevent transformation into a progressive course (with remitting multiple sclerosis), as well as slow down the growth of disability in the secondary progressive course, interferons (betaferon and rebif for subcutaneous administration, avo-nex for intramuscular injection) and glatiramer acetate (Copaxone-Teva).

Of the side effects associated with treatment with interferons, there are flu-like (fever, headache and muscle-joint pain), local (hyperemia, soreness), cardiovascular (hypotension, tachycardia and arrhythmias) and hematological (leukopenia, thrombocytopenia) reactions; changes in liver function (increased levels of bilirubin, transaminases); neurological symptoms (increase in spasticity, less often - aggravation of other neurological symptoms against the background of flu-like phenomena). The most common side effects when using glatiramer acetate are local reactions, but general systemic side effects may also develop, including clinical manifestations of vasodilation, chest pain, shortness of breath, palpitations, and anxiety.

When using traditional therapy for both remitting and secondary progressive multiple sclerosis, the need for a continuous course of treatment is indicated. In addition, given the immunogenicity and side effects of each of the drugs that modulate the course of multiple sclerosis, an individual approach is required when prescribing this therapy.

In recent years, the results of tests of intravenous administration of immunoglobulin G in limited groups of patients have been published. They indicate a possible decrease in the number of exacerbations in the remitting course of the disease. However, at present, the lack of clinical trials and a clearly established administration schedule at a high cost of the drug are the main problems in the use of immunoglobulin G in multiple sclerosis.

At the heart of all the problems of mental and neurological deficit in diseases of the brain is a violation of the functioning of brain neurons. Unfortunately, the clinic currently does not yet have effective methods for solving this problem. In light of the foregoing, the use of cell therapy in order to enhance the regeneration of nerve tissue, stimulating the biological activity of damaged neurons can be very fruitful.

Improving the effectiveness of treatment of patients with schizophrenia is one of the most urgent tasks of modern psychiatry, which is associated with the serious consequences of this disease, as well as the problematic and debatable principle of the possibility of reverse reduction of part of the symptoms with compensation for some lost functions and, finally, with pharmacogenic deficiency due to side effects of therapy antipsychotics.

As a significant characteristic of remissions in schizophrenia in modern psychiatry, the parameters of cognitive functioning are highlighted, the study of which has received the status of an independent scientific direction [Green M.F. What are the functional consequences of neurocognitive deficits in schizophrenia? Am. J. Psychiat. 1996, vol. 153, p.p.321-330; Green M.F., Olivier B., Crawley J.N., Perm D.L., Silverstein S. Social Cognition in Schizophrenia: Recommendations from the Measurement and Treatment Research to Improve Cognition in Schizophrenia New Approaches Conference. Schizophr Bull. Oct 2005, vol. 31, p.p. 882-887; Green M.F., Perm D.L., Bentall R., Carpenter W.T., Gaebel W., Gur R.C., Kring A.M., Park S., Silverstein S.M., Heinssen R. Social Cognition in Schizophrenia: An NIMH Workshop on Definitions, Assessment, and Research Opportunities. Schizophr Bull. Nov 2008, vol. 34, p.p.1211-1220].

Signs of cognitive decline in patients with schizophrenia, which are at least 10 times more common than in healthy people (85-94% versus 7%, respectively) [Saykin A.J., Shtasel D.L., Gur R.E. et al. Neuropsyhological deficits in neuroleptic naive patients with first-episode schizophrenia. Arch. Gen. Psyhiatry 1994, vol. 51, p.p.124-131], precede the development of manifest psychosis, intensifying with exacerbation and returning to the “base” level after passing it [Spaulding W.D., Fleming S.K., Reed D et al. Cognitive functioning in schizophrenia: implications for psychiatric rehabilitation. Schizophr. Bull, 1999, vol. 25, p. P. 275-289]. Moreover, disorders defined by the term "neurocognitive deficit" correlate with the severity of negative symptoms [Harvey P.D., Keefe S.E. Cognition and new antipsychotic. J. Adv. Schizophr. Brain. Res., 1998, vol. 1, p. 2-8].

Neurocognitive deficit (poverty of the associative process, impaired attention, memory and productivity of thinking, speed of processing information and executive / executive functions) is considered as a precursor of not only the outcome of the disease, but also the adaptive capacity of patients [Gold J.M., Harvey P.D. Cognitive deficits on schizophrenia. Psychiatr. Clin. Norm. Am., 1993, vol. 16, p. P. 295-312; Gallhofer B. Preserving cognitive function - optimizing integration into the community. Schizophr. Rew. 1995, vol. 3, Suppl. 2, No. 8, p. P. 3-4; Stratta P., Daneluzzo E., Bustini M. et al. Schizophrenic deficit in the processing of contex. Arch. Gen. Psychiat. 1998, vol. 55, p.p.186-187; Weickert T.W., Goldberg T.E. The course of cognitive impairment in patients with schizophrenia. Cognition in schizophrenia. Impairments, Importance and Treatment Strategies. Sharma T., Harvey Ph. (eds.), Oxford University Press, Oxford-New York, 2000, p. 3-16; Breier A.F. Cognitive deficit in schizophrenia and its neurochemical basis. Br. J. Psychiat. 1999, vol. 174, Suppl. 37, p.p.16-18]. According to several researchers [Green M.F. What are the functional consequences of neurocognitive deficits ni schizophrenia? Am. J. Psychiat. 1996, vol. 153, p.p.321-330; Goldberg T.E., Weinberger D.R. The effects of clozapine on neurocognition: an overview. J Clin Psychiatry. 1994, vol. 55, Suppl B, p. P. 88-90; Harvey P.D., Keefe S.E. Cognition and new antipsychotics. J. Adv. Schizophr. Brain. Res. 1998, vol. 1, p.p. 2-8; Sharma T., Harvey Ph. Cognition in Schizophrenia. Impairments, Importance and Treatment Strategies. Oxford University Press, Oxford - New York, 2000, p. 363; Mueser K.T. Cognitive functioning social adjustment and long-term outcome in schizophrenia. Cognition in Schizophrenia. Impairments, Importance and Treatment Strategies, Sharma T., Harvey Ph. (eds.), Oxford University Press, Oxford-New York, 2000, pp157-177], the phenomena of neurocognitive deficiency persist even with the complete reduction of clinical symptoms in remission and are even more informative for predicting the functioning of patients, since they may impede their reintegration into society .

Therapeutic strategies aimed at optimizing the cognitive functioning of patients can help maintain stability in remission, improve the functioning of patients and their quality of life. However, as shown in a number of studies, remissions with a predominance of negative symptoms and cognitive impairment (including hypochondriacal ones) are highly resistant to drug effects, even with drugs of the latest generations [Mosolov S.N. Psychopharmacotherapy resistance and methods for overcoming it. Psychiatry and psychopharmacotherapy, 2002, No. 4, 132-136; Smulevich A.B. The non-manifest stages of schizophrenia are psychopathology and therapy. Zhurn. neuropath, and psychiatrist them. S. S. Korsakova, 2005, No. 5].

In cerebral palsy (cerebral palsy), clinical manifestations are also persistent and resistant to the use of traditional methods of treatment. In the pathogenesis of the clinical manifestations of cerebral palsy, the leading place is occupied by structural damage to the brain, which is the result of damage to the central nervous system (CNS), discirculatory disorders. The persistence of intellectual-mnestic, emotional-volitional, motor, sensitive disorders and other focal signs of brain damage is due, in particular, to the low activity of CNS reparative processes in such patients.

The term “cerebral palsy” (Cerebral Palsy) combines the syndromes resulting from brain damage in the early stages of ontogenesis and manifested by the inability to maintain a normal posture and perform arbitrary movements. Movement disorders (paralysis, paresis, impaired coordination, violent movements) can be combined with changes in the psyche, speech, vision, hearing, convulsive seizures, and sensitivity disorders. As the child develops, especially at an early age, clinical symptoms may change. This is due to the age-related dynamics of the morphofunctional relationships of a pathologically developing brain, an increase in decompensation, due to the growing discrepancy between the capabilities of the nervous system and the requirements of the environment for a growing organism. In addition, in the case of the addition of pathological syndromes such as hydrocephalus, convulsions, autonomic disorders, as well as infectious diseases, intoxications, repeated brain injuries, the process progresses.

The frequency of cerebral palsy according to different authors in different years and in different countries ranges from 1.5 to 3.0 per 1000 newborns.

Currently, there is no specific therapy conducive to the restoration of dystrophically altered brain tissue. Existing methods of treatment, including neurosurgical interventions, are purely palliative, symptomatic.

Thus, the urgency of the treatment of patients with cerebral palsy and / or hydrocephalus with persistent neurological deficit is due to the high prevalence of these pathological conditions, the severity of clinical manifestations and the low effectiveness of traditional methods of treatment. The urgent need to solve this problem is also dictated by the fact that we are talking about a large number of children for whom modern treatment methods are not effective, which leads to low family and social adaptation of such patients. The use of cord blood cells could help accelerate the recovery processes in the central nervous system by replacing, regenerating or enhancing the biological activity of damaged cells, leading to the restoration or improvement of brain function.

The use of cord blood cells, which are supposed to have the ability to induce stem cells that persist in the brain and differentiate brain neurogenesis due to paracrine effects (exposure to various cytokines and transcription factors), can be an innovative strategy for the treatment of diseases that occur with degenerative changes in the brain.

Disclosure of invention

The authors of the present invention for the first time proposed a method of treating mental and neurological disorders by intravenous infusion of nucleated cells of human cord blood without the risk of immunopathological conditions, not requiring HLA typing, and without limiting the re-use of the method in case of clinical indications.

The present invention provides a method of treating mental and neurological disorders, comprising administering to a patient nucleic acid cells of umbilical cord blood in a therapeutically effective amount, which, depending on the weight of the patient and the clinical picture, is from 1 × 10 ⁸ to 5 × 10 ⁸ and preferably from about 2.2 × 10 ⁸ to about 2.8 × 10 ⁸ nucleated cells per administration. Thus, the effective number of nucleated cells in accordance with the proposed method is at least an order of magnitude less than the required number of nucleated cells in cord blood according to the method for treating amyotrophic lateral sclerosis and multiple sclerosis disclosed in WO 2004/071283.

Moreover, by the unexpected discovery of the authors of the present invention it turned out that the claimed method is highly effective in a single injection of cord blood cells to patients. In certain specific embodiments of the method, re-administering the drug is contemplated; in one of the specific embodiments, the drug is administered up to four times to achieve persistent remission in patients with schizophrenia. Thus, the effectiveness of the claimed method significantly exceeds, for example, the effectiveness of the method of treating cerebral ischemia caused by atherosclerosis or acute cerebrovascular accident described in patent RU 2284190, according to which the re-administration of cord blood mononuclear cells to patients up to 10 times is provided.

In specific embodiments, the invention is directed to the treatment of mental and neurological disorders, including multiple sclerosis, amyotrophic lateral sclerosis, age-related loss of cognitive function, Alzheimer's disease, Parkinson's disease, dementia, including immunodeficiency syndrome, memory loss, schizophrenia, cerebral infant paralysis, hydrocephalus and other pathological conditions associated with degenerative changes in the brain.

In one specific aspect of the invention, the method is used to treat patients with Alzheimer's disease with varying degrees of dementia.

Another specific aspect of the invention relates to the use of nucleated umbilical cord blood cells in Parkinson's disease with various forms of rigidity.

Another specific aspect of the invention relates to the use of nucleated cells of umbilical cord blood for schizophrenia in remission with predominant negative symptoms in combination with severe cognitive impairment.

Another specific aspect of the invention relates to the use of nucleated cells of umbilical cord blood in cerebral palsy and hydrocephalus.

Another specific aspect of the invention relates to the use of nucleated cells of cord blood in multiple sclerosis.

The implementation of the invention

The present invention is based on the unexpected discovery that nucleated umbilical cord blood cells can be administered to patients with mental and neurological disorders without the risk of immune rejection and lead to a significant improvement in degenerative disorders.

The proposed method for the treatment of mental and neurological disorders involves administering to a patient by intravenous infusion of nucleated cord blood cells in a therapeutically effective amount. The number of introduced cells, depending on the weight of the patient and the clinical picture, is from 1 × 10 ⁸ to 5 × 10 ⁸ and, preferably, from about 2.2 × 10 ⁸ to about 2.8 × 10 ⁸ nucleated cells per administration.

In one embodiment of the invention, a single administration of the drug to the patient is carried out.

In specific embodiments of the invention, the introduction of umbilical cord blood cells is repeated (up to 3 times) depending on the clinical picture with an interval of 1-4 weeks in an amount of from 1 × 10 ⁸ to 5 × 10 ⁸ and, preferably, from about 2, 2 × 10 ⁸ to about 2.8 × 10 ⁸ nucleated cells per administration.

In yet another embodiment, the introduction of nucleated cells of cord blood is carried out up to four times with an interval of 1-4 weeks in an amount of from 1 × 10 ⁸ to 5 × 10 ⁸ and, preferably, from about 2.2 × 10 ⁸ to about 2.8 × 10 ⁸ nucleated cells per administration.

To obtain a cord blood cell preparation for use according to the method, cord blood samples are sedimented or gradient centrifuged to select nucleated cells, followed by resuspension in chilled plasma with the addition of dimethyl sulfoxide. The resulting material is distributed in cryovials, subjected to program freezing, and then transferred to a quarantine Duar in pairs of liquid nitrogen. At the end of the quarantine shelf life, subject to negative test results for blood-borne infections and inoculation under aerobic / anaerobic conditions, the samples are transferred for permanent storage to a Dewar vessel with liquid nitrogen.

The technology used to obtain umbilical cord blood stem cell concentrate is stably reproducible and highly effective, which ensures high cell viability both immediately after isolation and after freezing and subsequent thawing.

The preparation is a sterile suspension of not less than 100 million live human cord-containing cells of umbilical cord blood washed from a cryoprotectant in 100 ml of physiological saline supplemented with reopoliglyukin and human serum albumin and is intended for intravenous infusion through a system for blood transfusion and blood substitute solutions (dropper), and also in a jet way.

In one specific embodiment of the method, umbilical cord blood cells washed from a cryoprotectant are used.

In another specific embodiment of the method, umbilical cord blood cells are used without washing from the cryoprotectant.

In one specific embodiment, the proposed method for the treatment of cord blood cells is directed to the treatment of patients with Parkinson's disease who have a stable neurological deficit amid ongoing anti-Parkinsonian therapy.

Patient neurological deficiency was assessed using standard tests for the disease using the Columbian Rating Scale (CRS), Yahr MD, et al. Treatment of parkinsonism with levodopa. Archives of Neurology, 1967, v.21, P.343- 354; Lang AET, Fagn S. Assessment of Parkinson's disease In: Quantification of neurologic deficit (ed. TL, Munsat). Butterworths, Stoneham, 1989, Ch. 21, P.285-309; Wade DT Measurement in neurological rehabilitation. Oxford Univercity Press. 2000) and the Life Quality Assessment System for Parkinson's Disease (PDQ-39 Scoring System, Loma Linda University Surgery Medical Group).

The study included patients aged 48 to 86 years with Parkinson's disease of stages 2 and 3 of moderate type of progression with a rigid-trembling form and an akinetic-trembling form with a disease duration of 3 to 10 years.

All patients included in the study did not reveal any undesirable side effects during the treatment of the claimed method, or after three months.

As a result of treatment by the claimed method, an increase in functional activity was noted in all patients. The range of everyday, everyday activity, a motivational degree has expanded, the emotional component of the patients' condition has improved. A positive effect developed during the first month and was characterized by an improvement in the condition of the skin in the form of a decrease in sebum, seborrhea, and a decrease in the severity of the clinical manifestations of the disease.

The test also revealed statistically significant differences by the end of the third month from the start of treatment in terms of rigidity, bradykinesia, and functional capabilities. A decrease in tremor in patients with tremor forms was clearly observed by the end of the first month of the study. Rigidity underwent a clear regression with a gradual decrease in severity by the third month. Bradykinesia, as the most disabling symptom, underwent a more distinct regression in patients with the prevalence of the akinetic component. The expansion of the range of functional capabilities was clearly registered in all patients by the end of the first month.

Thus, based on the data obtained when testing the PDQ-39 Scoring System by the end of the third month, all patients showed a positive trend.

It should be noted that an unconditional positive result of the claimed method of treatment was also a reduction in the dose of replacement therapy with L-DOPA drugs during the observation process.

The use of umbilical cord blood cells in Parkinson's disease is well tolerated by patients and has no negative side effects. According to the assessment of clinical data, the use of the claimed method positively affects the course of the pathological process in the form of moderate regression of the main symptoms of the disease. A reduction in the dose of L-DOPA preparations taken by patients against the background of a stable state is also an unconditional important technical result achieved using the claimed method. Ease of use in combination with efficiency provide the advantages of the claimed method as a new and promising direction in the treatment of Parkinson's disease.

In another specific embodiment, the proposed method is directed to the treatment of Alzheimer's disease. The study included patients aged 50 to 85 years with moderate severity of dementia according to the criteria of CDR [Morris J.C. The Clinical dementia rating (CDR). Current version and scoring rules. Neurology, 1993, Vol. 43, p.2412-2413].

After preliminary somatic, neurological and laboratory examinations (clinical analysis of blood and urine, ECG), umbilical cord blood cells were introduced to all patients by intravenous drip infusion in an amount of from 1 × 10 ⁸ to 5 × 10 ⁸ and, preferably, from about 2, 2 × 10 ⁸ to about 2.8 × 10 ⁸ nucleated cells per administration with an interval of 1 week.

Assessment of the status of cognitive abilities (MMSE scale, Mini-Mental-State Examination, Folstein MF, Folstein SE, McHugh PR Mini-Mental State. A practical method of grading the cognitive state of patients for the clinical. Journal of Psychiatry Research, 1975, Vol .12, p.189-198), cognitive impairment (ADAS-cog scale), everyday activity disorders (Bristol-ADL scale), quantitative assessment of the state of higher cortical functions (according to the adapted method of A.R. Luria) and general clinical impression (CGI scale) was performed 7, 14 and 90 days after the start of therapy of the claimed method. At the same time, the main indicators of the state of the body (pulse rate, clinical analysis of blood and urine, ECG) were evaluated before and after the study.

During the study period, 4 out of 5 patients continued to take the drugs traditionally used to treat Alzheimer's disease, one of the acetylcholinesterase inhibitors or memantine in stable doses.

The primary criteria for evaluating the effectiveness of the claimed method were the dynamics of indicators of cognitive impairment on the ADAS-cog scale and the change in indicators of impaired daily functioning on the Bristol-ADL scale. The secondary criteria for evaluating effectiveness were the dynamics of cognitive functioning indicators on the MMSE scale and the assessment of the overall clinical impression (CGI scale).

All patients showed positive dynamics in assessing the state of cognitive functions according to the ADAS-cog and MMSE scales on the 7th and 14th day of the study, i.e. a week after the 1st and 2nd injection of cord blood cells.

Positive dynamics was also noted in the daily functioning of patients (according to the Bristol ADL scale) on the 14th day of the study, i.e. one week after the 2nd injection of cord blood cells. On the 90th day of the study, 3 out of 5 patients maintained positive dynamics and stabilization of the state achieved by the 14th day of the study, according to the ADAS-cog, and Bristol ADL, and MMSE scales.

According to the General Clinical Impression Scale (CGI) on the 14th day of the study, 2 out of 5 patients showed a pronounced improvement in cognitive and everyday functioning, accompanied by an improvement in memory, orientation and speech, as well as the restoration of lost personal hygiene skills and the expansion of daily activities, for example in cleaning the premises, in preparing drinks.

After 90 days, the condition of 2 patients was assessed as moderate or minimal improvement compared with the assessment before treatment.

The advantages of the claimed method, manifested in the positive prolonged dynamics of cognitive and everyday functioning in the absence of side effects, indicate the promise of its use in the treatment of Alzheimer's disease.

In yet another specific embodiment, the proposed method for the treatment of cord blood cells is directed to treating patients with multiple sclerosis in relapsing, primary-progressive and secondary-progressive course of the disease. Patients were administered twice with blood group and Rh factor cells of cord blood in an amount of from 1 × 10 ⁸ to 5 × 10 ⁸ and, preferably, from about 2.2 × 10 ⁸ to about 2.8 × 10 ⁸ nucleated cells for one administration with an interval of 14 days. After repeated administration within 1 month of observation, all patients showed a positive trend.

In yet another embodiment, the proposed method is directed to the treatment of patients with schizophrenia.

The structure of remission in schizophrenia is dominated by negative symptoms (emotional fencing, difficulty communicating, loss of energy and volitional promptings) combined with severe cognitive impairment (slippage, sperrunga, diverse thinking) and subdepressive disorders of the negative affectiveness circle (apathy) Smulevich A.B. The non-manifest stages of schizophrenia are psychopathology and therapy. Zhurn. neuropath, and psychiatrist them. S.S. Korsakova. 2005, No. 5].

The treatment by the claimed method was carried out in relation to 10 patients with an average age of 33.6 ± 10.6 years in a state of hypochondria remission with a predominance of cognitive disorders in the structure of the defect, the average duration of the disease 17.3 ± 4.1 years, the average number of seizures suffered - 4.4 ± 2.2 and an average remission duration of 36.7 ± 24.8 months. In the control sample (10 patients with the same diagnosis, comparable by sex, age, disease parameters), a placebo was used.

Umbilical cord blood cells were introduced by intravenous drip infusion in an amount of from 1 × 10 ⁸ to 5 × 10 ⁸ and, preferably, from about 2.2 × 10 ⁸ to about 2.8 × 10 ⁸ nucleated cells per administration for 4 injections with an interval of 14 days. against the background of maintenance therapy (stable doses of antipsychotics).

The patients' condition was evaluated clinically, psychometrically and pathopsychologically before each injection, and then after 3, 5 and 7 weeks after the last procedure and after 3 months in the pilot phase, as well as after 9, 11 and 13 weeks in the clinical phase.

Pathopsychological evaluation was carried out using the well-known testing methodology - the MCCB cognitive battery (The MATRICS Consensus Cognitive Battery) [Nuechterlein K.H., Green M.F., Kern R.S., Baade L.E., Barch D.M., Cohen J.D. et al. The MATRICS Consensus Cognitive Battery, Part 1: Test Selection, Reliability, and Validity. Am. J. Psychiatry, 2008, Vol. 165, p. P. 203-213].

The battery includes 10 tests evaluating 7 cognitive areas and aimed at assessing the following parameters: conscious control of emotions for personal growth and improving interpersonal relationships (social intelligence), hand-eye coordination (attention, spatial orientation, motor coordination, speed of intellectual processes); verbal memory and verbal learning; verbal associative productivity and violation of the lexical system; executive / executive functions (planning, programming, modeling and control of mental activity); ability to symbol coding (attention, working memory and speed of intelligent processes); visual memory and visual learning; attention (volume, concentration, switching and alertness); non-verbal (spatial) working memory; the ability to carry out analytical and synthetic operations and the associative process.

For a psychometric assessment of the positive and negative symptoms of schizophrenia, the “Positive and Negative Symptom Rating Scale” (PANSS) was used [Kay S.R., Fiszbein A., Opler L.A. The Positive and Negative Syndrome Scale (PANSS) for Schizophrenia. Schizophr Bull., 1987, Vol.13, p. P. 261-276; Mosolov C.H. Scales for the psychometric assessment of the symptoms of schizophrenia and the concept of positive and negative disorders. M., 2001, p.238].

In all cases, the introduction of a cord blood cell concentrate was not accompanied by adverse events.

Identified in 4 patients transient episodes of subfebrile condition (t ° to 37.1-37.2 ° C) did not require additional interventions and stopped on their own within 1-2 days.

Improvement of the patients' condition was recorded during a pathological examination (MSSV indicators) already 3 weeks after a single infusion of nucleated cord blood cells, returning to the initial level after 5-7 weeks.

A series of 4 consecutive infusions of the drug has a positive effect on cognitive functioning.

At the pathopsychological level with high statistical certainty (p <0.001), a significant improvement is recorded. 3 weeks after the 4th infusion, the speed of mental processes significantly increases (dynamic characteristics of attention, hand-eye coordination, associative thinking productivity, regulatory functions, the speed of skill formation). Attention / alertness (volume, concentration, switching, distribution) and memory (verbal, non-verbal) are improved. The ability to learn - visual (memorization of material in visual modality) and verbal (memorization of material in auditory-speech modality) is increasing. Opportunities related to executive (executive) functions are expanding (the processes of planning, programming, modeling and monitoring mental activity are optimized).

The effect achieved in accordance with the claimed method is not only persistent, but also long (the positive dynamics of the parameters of cognitive functioning is recorded 3 and 5 weeks after the last infusion, and from 7 to 13 weeks it remains at the plateau level).

A statistically significant (p <0.05) improvement in psychometric indicators, assessed using the PANSS scale, was registered by the parameter “impaired spontaneity and smoothness of speech” (the initial score was 3.0 ± 0.71), and after a series of 4 infusion decreased to 2.3 ± 0.27; according to the “depression” parameter, the total score decreased to 2.2 ± 0.45 from the initial values of 3.3 ± 0.71; according to the parameter “motor inhibition”, the total score after treatment decreased to 2.3 ± 0.45 from the initial values of 3.7 ± 0.71.

The obtained psychometric data are consistent with clinical ones (the improvement of the patients' condition is limited by the appearance of an optimistic attitude, the revival of facial expressions and pantomimics, signs of interest in communication, concern for their appearance).

The cognitive effects achieved by the introduction of cord blood cell concentrate to patients in a state of hypochondria remission according to the claimed method lead to pronounced metabolic (nootropic) and psychostimulating actions, which is manifested in the activation of intellectual activity, acceleration of thinking processes, correction of memory functions, and increase of the level of wakefulness. The effectiveness of the claimed method in terms of improving cognitive functions is not only stable, but also prolonged.

In yet another embodiment, the proposed method is directed to the treatment of patients with cerebral palsy and hydrocephalus.

Morphologically, cerebral palsy is characterized by structural changes in the central nervous system that are diverse in nature, severity and localization. The features of these disorders depend mainly on the time of action of pathogenic factors and, to a lesser extent, on the specific properties of the latter. Malformations of the cerebral hemispheres in cerebral palsy can be combined with hydrocephalus, due to either a congenital anomaly in the development of cerebrospinal fluid circulation pathways, or a change in the structure of the meninges during subarachnoid hemorrhages, inflammatory processes, etc. Changes in the brain during fetal hypoxia of the fetus and asphyxiation of newborns with discirculatory origin. Their severity depends on the duration and severity of hypoxia. Vascular disorders and edema create conditions for secondary ischemia of the nervous tissue. In the white matter of the hemispheres, especially in the subependymal regions, foci of intense edema are formed, which can later be transformed into foci of softening and porencephaly. With severe prolonged hypoxia, neurons undergo degenerative changes in the form of chromatolysis, central acidophilia, acute swelling. Ischemic changes in neurons result in cell lysis (“shadow cells”).

Thus, deep circulatory disorders (edema, hemorrhage, thrombosis, ischemia) that occur under the influence of adverse external influences in the ant and intrapartum periods lead to dystrophic changes in the brain tissue that underlie cerebral palsy. In immature children, the white matter of the brain is more often damaged. The increased vulnerability of the white matter of the developing brain is associated with a high level of its metabolism, compared with the cortex with relatively poor blood supply (C. Kennedy et al., 1979).

Other areas may also undergo necrosis - the subcortical white matter, the corpus callosum, and the internal capsule.

The treatment of the claimed method was carried out in relation to 19 patients aged 1 year, 3 months to 10 years, 17 of whom were disabled from birth, with the nature of the pathological process, including hydrocephalus (in 2 patients), cerebral palsy (4 patients) hydrocephalus in combination with cerebral palsy and epileptic syndrome (4 patients), hydrocephalus with epileptic syndrome (7 patients), hydrocephalus with cerebral palsy (2 patients). All patients were treated in various specialized clinics from 3 to 8 hospitals without a positive effect. In all cases, the pathological process during treatment had a progressive course.

The main clinical manifestations of the disease were spastic hemiparesis (in 8 patients), tetraparesis (4 patients), epileptic syndrome (11 patients), lag in psychomotor and physical development was noted in all 19 patients.

Given the severity of the clinical picture of the disease and the failure of drug therapy, a single transfusion (8 patients), two-time (10 patients) and three-time (1 patient) transfusion of a concentrate of cord blood cells and Rh factor of umbilical cord blood cells in a total dose of from about 100 to about 600 was performed million cells.

When treating the proposed method, positive dynamics was observed in all patients. In 5 patients, a decrease in spasticity and hyperkinesis was noted. In one patient, on the 2nd day after the repeated administration of stem cells, a regression of right-sided hemiparesis was noted (from 2 to 4 points), a decrease in the frequency of clonic twitches in the arm. Another patient with congenital amaurosis noted an improvement in visual functions: the boy began to describe the objects that are around him and name the objects that are shown in the pictures, fine motor skills also improved and self-service skills appeared. In patients with a diagnosis of cerebral palsy or hydrocephalus in combination with epileptic syndrome, a decrease in the frequency of seizures was observed without changing the dose of anticonvulsants received. In 9 patients, the condition stabilized.

It should be noted that in the implementation of all specific variants of the claimed method, the introduction of nucleated cells of umbilical cord blood was not accompanied by any reactions from the cardiovascular, respiratory or immune systems.

In embodiments of the proposed method aimed at treating mental and neurological disorders, a clear tendency was observed in all patients after the introduction of cells to rapidly reduce the structural components of degenerative disorders (symptoms), the level of mental activity was significantly increased.

Thus, in patients with various neurological symptoms, suffering from degenerative pathological disorders, there was an obvious improvement. The data obtained indicate that therapy with cord-containing cells of cord blood for mental and neurological disorders in accordance with the proposed method unexpectedly leads to rapid nootropic and psychostimulating effects, accompanied by accelerated recovery of cognitive and "instrumental" cortical functions.

The following examples of the implementation of the proposed method for the treatment of mental and neurological disorders illustrate, but do not limit the claimed scope of claims.

Example 1. Harvesting cord blood and the selection of nucleated cells

Harvesting of cord blood was carried out in sterile containers (systems for the collection of donated blood) containing an isotonic anticoagulant, with the observance of precautions that exclude infection of the material during collection and transportation. The material was delivered to the laboratory-production complex in an isothermal transport container at room temperature (+ 18 ... + 22ºС).

All procedures for the isolation of umbilical cord blood cells were performed in accordance with the national standard of the Russian Federation GOST R 52249-2004 "Rules for the production and quality control of drugs" using medical technology developed by the inventors.

After measuring the volume of umbilical cord blood, aliquots are selected for hematological analysis (the content of the formed elements before isolation, blood type, Rh factor).

To obtain nucleated cells by sedimentation, a 6% solution of hydroxyethyl starch (HES) is added to the blood container at the rate of 1 volume of HES to 4 volumes of blood and mixed for 10-15 minutes. The contents of the container are transferred into 50 ml tubes, centrifuged for 5 minutes at 90 g with the braking disabled, and left for 10-15 minutes to form a visible section between the white blood-rich plasma and the erythrocyte sediment. The plasma enriched with leukocytes is collected in separate tubes and centrifuged for 10 minutes at 600 g to precipitate the cell elements. The resulting cell pellets are combined and resuspended in plasma. The remainder of the plasma is transferred to a separate tube and placed in an ice container.

When using the SEPAX automatic cell separator, after adding the calculated amount of HES, the blood bag is connected to the separation kit through a sterile port and the separation is carried out in accordance with the instructions for the device.

To obtain nucleated cells by gradient centrifugation, the blood is distributed into 50 ml tubes and centrifuged for 10 minutes at 600 g to precipitate the cells. The resulting plasma is collected in a clean tube and placed on ice. Hensk or Earl phosphate-saline is added to the cell pellets and resuspended. In sterile 50 ml tubes add 20 ml of ficoll solution. Cell suspensions are layered on a ficoll solution and centrifuged for 30 minutes at 400-500g. The interphase mononuclear ring is selected and transferred to clean tubes. Up to 50 ml of phosphate-saline solution is added to each tube, resuspended and centrifuged for 10 minutes at 600g. The washing procedure is repeated twice. Cell pellets are subsequently resuspended in chilled plasma.

Aliquots of cell suspensions are used for staging sterility tests, identifying pathogens of blood-borne infectious diseases, counting the number of cells and their viability using the trypan blue test, and determining the content of CD34 / CD45-positive cells by flow cytometry.

Dimethyl sulfoxide (DMSO) is added to the remaining plasma volume to a final concentration of 20%. An equal volume of plasma with DMSO is added dropwise to the cell suspension with constant stirring. The resulting material is distributed in cryovials, subjected to program freezing, and then transferred to a quarantine Duar in pairs of liquid nitrogen. At the end of the quarantine shelf life, subject to negative test results for blood-borne infections and inoculation under aerobic / anaerobic conditions, the samples are transferred for permanent storage to a Dewar vessel with liquid nitrogen.

Samples of cord / placental blood are tested to identify pathogens of blood-borne infectious diseases: human immunodeficiency virus (HIV-1/2, antigen / antibodies), hepatitis B viruses (HBs Ag, anti-HBc-total) and C (anti-HCV-total ), T-cell leukemia virus (anti-HTLV-1/2), herpes simplex viruses type 1 and 2 (anti-HSV IgM), cytomegalovirus (anti-CMV IgM), toxoplasmosis pathogens (anti-Toxo IgM), syphilis ( Syphilis PRP), bacterial and fungal agents.

A statistical analysis of the results of the isolation of cord-containing cord blood cells (Table) indicates the high efficiency and reproducibility of the technology used, including the high viability of the isolated cells before and after the trial freezing / thawing.

The results of the analysis of nucleated cells of human umbilical cord blood during isolation by gradient centrifugation, sedimentation or automatic separation No. p.p. Indicator Mean + SD Gradient Sedimentation Automatic separation one The number of samples included in the analysis 500 500 500 The content of nuclear cells before isolation, × 10 ⁹ 1.15 ± 0.5 2 The output of nuclear cells,% of the original 50.5 ± 12.2 81.03 ± 2.99 83.0 ± 11.5 3 The degree of release from red blood cells,% 97.82 ± 0.19 71.94 ± 3.33 79.2 ± 12.0 four Cell viability before freezing,% 99.9 99.9 99.9 5 The ratio of cell populations according to flow cytometry,% 5.1 lymphocytes 51.3 ± 10.9 30.6 + 1.98 38.0 + 9.0 5.2 monocytes 13.8 ± 3.8 10.04 ± 0.45 11.7 ± 2.0 5.3 granulocytes 25.1 ± 11.0 55.93 ± 1.97 43.6 ± 8.6 5.4 hematopoietic precursors (CD34 + / CD45 +, ISHAGE protocol),% 0.46 ± 0.26 0.28 ± 0.03 0.36 ± 0.22 6 The output of hematopoietic precursors,% 85.0 ± 23.0 93.0 ± 19.1 91.4 ± 18.9 7 Cell viability after thawing,% 90.8 ± 10.1 87.12 ± 4.7 83.7 ± 7.1

Example 2. Preparation of a dosage form of a preparation of cryopreserved umbilical cord blood cells

The preparation is a sterile suspension of at least 100 million live human cord-containing cells of umbilical cord blood washed from a cryoprotectant in 50-100 ml of physiological saline supplemented with reopoliglyukin and human serum albumin and is intended for intravenous infusion through a system for blood transfusion and blood substitute solutions (dropper) as well as in an inkjet manner.

In the process of thawing the cell suspension, the required number of cryovials with frozen cells is removed from the cryogenic storage and left for 5 minutes at room temperature.

Without removing the protective packaging, cryovials are placed in a water bath (+ 37 ° C) and the contents are thawed until ice crystals disappear. Stir the contents regularly by turning the tubes. Open cryovials by unscrewing the cap with a female thread.

Using a pipette dispenser or a dispenser with a 1 ml tip, transfer the contents of each cryovial to 50 ml tubes.

Dropwise with stirring (the first 10 ml), then a cooled solution is washed down to wash to a final volume of 45 ml. Resuspend the contents by repeated pipetting and close the tubes with caps.

Centrifuge for 10 minutes at 600 g and remove the supernatant using a pipette dispenser or an aspiration pipette and a medical aspirator. About 1 ml of liquid is left at the bottom of each tube.

The cell pellets are subsequently resuspended in 10 ml of chilled wash solution, avoiding foaming of the suspension. Using a microdoser, 100-200 μl of the cell suspension is transferred into a microtube for subsequent assessment of viability and counting the number of cells. Using a 20 or 50 ml syringe equipped with a needle, the remaining cell suspension is transferred to a vial or polymer container. Using a 50 ml syringe, physiological saline with reopoliglucin and albumin is added to the final volume of 100 ml in a vial (polymer container). Determine the content and viability of cells in suspension using microtube material.

The time from the preparation of the dosage form (transfer of the washed cells to the vial or polymer container) to the start of the infusion should not exceed 4 (four) hours. Transportation of the vial (polymer container) to the place of infusion is carried out at a temperature of + 2-4 ° C (on ice) in a thermal container.

Example 3. Treatment of Alzheimer's disease

A 68-year-old patient has been observed since 65 for Alzheimer's disease. Memory disorders first appeared at the age of 60. An MRI scan of the head revealed an increase in the lateral ventricles and subarachnoid spaces in the projection of the frontal, parietal and temporal lobes with a diagnosis of external and internal communicating hydrocephalus. The patient's orientation in time and place is broken. Disturbances of memory on events of the recent and distant past are expressed, the dating of events is violated.

Memorizing a series of simple words is difficult. Speech with paraphases and single logoclonies. Clock recognition is not available. Remembering the appearance of people is difficult. Recognizing realistic objects with errors. Confuses the sequence of poses when performing tests on the study of dynamic praxis. Spatial-constructive activity is disturbed to a pronounced degree (when redrawing three-dimensional geometric figures, it makes significant errors). Actually, the intellectual functions are moderately disturbed: confused in the interpretation of logical and grammatical constructions, specifically understands the figurative meaning of many proverbs, does not fully understand the plot pictures. The counting function is violated - it makes mistakes during sequential subtraction, in addition examples with two-digit numbers. The letter is broken, the letters are written separately, large. Reads correctly, reads memorized fragmentary. Treatment with excelon (rivastigmine) 1.5 mg 2 times a day did not lead to improvement.

Against the background of continuing excelon treatment, the patient was given two drops of intravenous infusion washed from a cryoprotectant, compatible with the blood group and Rh factor of umbilical cord blood cells in an amount of 2.5 × 10 ⁸ for each injection with an interval of two weeks. After the first injection of the drug, the patient became more energetic, better remembered the words during the conversation, the ability to independently perform some household chores returned to her. During the examination, on the 7th day after 1 introduction, an improvement in memorization and recognition of words, an improvement in the ability to speak, and an improvement in orientation over time were noted.

On the 14th day after the start of treatment, the claimed method noted an improvement in orientation in place and time, better memorization of a series of words and naming of objects and fingers, further improvement in recognition of words, marked ease of searching for words in random speech and an increase in the number of executed commands. In everyday life, I began to properly prepare drinks, do housework. Compared to the initial assessment of cognitive functioning, the MMSE score improved by 7 points, the ADAS score by 14 points.

3 months after the start of treatment, the patient's condition remained at the level reached by the 14th day of the study.

Example 4. The treatment of Parkinson's disease

A 64-year-old patient with an akinetic-rigid form 2 of Parkinson's disease constantly took 250 mg of madopar in a daily dose of 1250 mg, midcalm - 100 mg 4 times a day, pronoran - 50 mg 3 times a day, PK-Merz - 100 mg 3 times a day.

The number of points on the Columbian Evaluation Scale before treatment by the claimed method was 32, according to the Life Quality Assessment System for Parkinson's Disease-39 corresponded to 30 points.

The patient was intravenously infused according to the invention with cord blood at a dose of 259 million cells. At 4 weeks after administration, a positive trend appeared in the form of a decrease in muscle stiffness, a positive cosmetic effect. The dosage of antiparkinsonian drugs was significantly reduced: madopar to 500 mg per day, pronoran to 100 mg per day, PK-Merz to 300 mg per day. Three months later, the number of points on the Columbian Evaluation Scale decreased to 16, according to the Life Quality Assessment System for Parkinson's Disease-39 to 28 points.

Example 5. The treatment of Parkinson's disease

Patient 58 years old. Weakness and tremor were noted in the left hand, weakness in the left leg and right leg, stiffness in the whole body, slowdown of movements, inability to hold urine for a long time. For continuous use, Madopar was recommended at a dose of 125 mg, 1 capsule 4 times a day every 6 hours. I went on my own for short distances with the body tilted forward. When walking, propulsions were noted. The number of points on the Columbian Evaluation Scale was 44, according to the Life Quality Assessment System for Parkinson's Disease-39, it corresponded to 100 points.

The patient in accordance with the proposed method by intravenous infusion were introduced blood group and Rh factor cells of umbilical cord blood at a dose of 249 million cells. After the introduction, positive dynamics were noted: the manifestations of seborrheic dermatitis on the face decreased, the mood background improved, the stiffness in the body decreased, the exercise tolerance increased, and postural instability decreased. The number of points on the Columbian Evaluation Scale decreased to 18, according to the Life Quality Assessment System for Parkinson's Disease-39 to 56 points.

Example 6. The impact on negative symptoms and cognitive deficit in the framework of incomplete remission in schizophrenia

A 24-year-old patient after a 5-month inpatient treatment with a diagnosis of “Schizophrenia is paranoid, an episodic type of course with an increasing defect, paranoid syndrome” (acute manifest paranoid psychosis with delusions of greatness, Kandinsky-Clerambo syndrome), where he received aminazine, haloperidol, ziprexp, rippi, , cyclodol, experienced fatigue, lethargy, apathy, depression, taking haloperidol (10 mg / day) as a maintenance therapy, then risplept (6 mg / day), in the status of incomplete remission in the framework of paranoid schizophrenia with predominantly negative symptoms and cognitive impairment was subjected to treatment according to the proposed invention.

Before treatment in accordance with the invention, positive symptoms corresponded to 9 points on the PANSS scale, negative symptoms - 20 points, general psychopathology corresponded to 40 points. When testing with the MATRICS cognitive battery, violations in such cognitive areas as attention - 25 T-points (0.65%), visual learning - 21 T-point (0.2%), mental process speed - 34 were in the foreground T-score (5%) and regulatory functions - 34 T-points (5%). Working memory (verbal and visual-spatial) - 45 T-points (31%) and verbal learning - 48 T-points (42%) are also scarce.

Against the background of a fixed intake of rispolept at a dose of 6 mg / day, a series of infusion (4 administrations) of umbilical cord blood cells was carried out with an interval of 14 days), compatible by blood group and Rh factor, in an amount of about 250 million per administration (total dose of about 1 billion cells).

Measurement of cognitive indicators 9 weeks after a series of infusion of cord blood cell concentrate with high statistical reliability led to an improvement in the following parameters of cognitive functioning: the rate of mental processes was 51 T-points (54%>), working memory - 63 T-points (90%) , verbal and visual learning were 78 T-points (99.7%) and 60 T-points (84%), respectively, and executive (regulatory) functions - 59 T-points (82%). The most pronounced effect is manifested in such cognitive areas as learning (visual and verbal), which consists in the ability to memorize material in the visual and auditory-speech modalities, and the executive functions responsible for the processes of regulation, planning, programming, and control of mental activity. When evaluated according to the PANSS scale, 9 weeks after a series of infusion of umbilical cord blood concentrate, the positive symptoms were 9 points, the negative symptoms were 14 points, and the general psychopathology corresponded to 36 points. An improvement in mood (reduction of depression), a decrease in the severity of fatigue and lethargy, and the appearance of an optimistic attitude were noted.

The degree of restoration of executive functions, visual and verbal learning, as well as the memory and speed of mental processes in the post-therapeutic period (9 weeks) reaches the level of the average statistical norm, i.e. exceeds the average scale score of 50 T-points and 50 percentiles, respectively, for the MATRICS cognitive battery.

Thus, the patient after 9 weeks showed a clear tendency to restore all of the above cognitive functions. Treatment with the method according to the proposed invention, leading to optimization of the cognitive functioning of patients, helps to maintain stability of remission, increase the level of functioning of patients and their quality of life.

Example 7. Impact on negative symptoms and cognitive deficit in the framework of incomplete remission in schizophrenia

A 47-year-old patient suffers from schub-like schizophrenia from the age of 31 with predominantly affective-paranoid psychoses (mainly depressive-paranoid episodes with exposure syndrome, delirious ideas of guilt and self-abasement), suffered 6 attacks with hospitalizations in psychiatric hospitals, and a history of 2 suicidal attempts.

Inoperative, in the picture of remission hypochondria, apathy, fatigue predominate, seasonal depressive phases with a feeling of vital longing, circadian rhythm, weight loss are observed annually. He received maintenance therapy: rispolept 8 mg / day, carbamazepine 200 mg n / n, amitriptyline 50 mg / day.

In the status with incomplete remission in the framework of fur-shaped schizophrenia, determined mainly by negative symptoms, depressive disorders and severe cognitive impairment, he was treated with the claimed method. Before treatment in accordance with the invention, positive symptoms corresponded to 12 points on the PANSS scale, negative symptoms - 24 points, general psychopathology corresponded to 44 points. When testing with the MATRICS cognitive battery, violations in such cognitive areas as the speed of mental processes - 32 T-points (3.6%), working memory (verbal and visual-spatial) - 37 T-points (10%) were in the foreground ), verbal learning - 34 T-points (5%) and executive functions - 36 T-points (8%). Violations of the volume and dynamic characteristics of attention were also detected - 41 T-points (18%).

Against the background of fixed doses of maintenance therapy, a series of infusion (4 administrations) of cord blood cells was carried out with an interval of 14 days, compatible by blood group and Rh factor, in an amount of about 2.5x10 ⁸ per administration.

Measurement of cognitive parameters 9 weeks after a series of infusion of cord blood cell concentrate with high statistical reliability led to an improvement in all parameters of cognitive functioning, namely: the speed of mental processes up to 69 T-points (97.1%), attention to 67 T-points ( 96%), working memory up to 66 T-points (95%>), verbal and visual learning up to 61 T-points (86%) and 72 T-points (98.6%), respectively, as well as executive (regulatory) functions up to 64 T-points (92%). The most pronounced effect was manifested in such cognitive areas as the speed of mental processes, executive functions, direct memory (verbal and nonverbal) and verbal learning. When evaluated according to the PANSS scale, 9 weeks after a series of infusion of umbilical cord blood concentrate, the positive symptoms were 10 points, the negative symptoms were 18 points, and the general psychopathology was 39 points. Depression, anxiety was noticeably reduced, the patient became more sociable, began to monitor the appearance.

The degree of restoration of executive functions, visual and verbal learning, as well as the memory and speed of mental processes in the post-therapeutic period (9 weeks) reaches the level of the average statistical rate, i.e. exceeds the average scale score of 50 T-points and 50 percentiles, respectively, for the MATRICS cognitive battery.

Thus, the patient after 9 weeks showed a clear tendency to restore all of the above cognitive functions. Treatment with the method according to the proposed invention, leading to optimization of the cognitive functioning of patients, helps to maintain stability of remission, increase the level of functioning of patients and their quality of life.

Example 8. Cerebral Palsy with Epileptic Seizures

A 9-year-old patient with a diagnosis of chronic progressive focal Rasmussen encephalitis, epilepsy with frequent simple partial motor attacks, myoclonic and secondary generalized attacks, right-sided hemiparesis. Keppra took 750 mg 2 times a day, Pagluferal-3 1/2 tablets. 2 times a day, Trileptal 150 mg 3 times a day. Despite the ongoing therapy, there was a progressive worsening of the condition, an increase in seizures with unchanged or partial impaired consciousness, lead to the right and tension of the right extremities, with the development of Todd's post-attack paralysis, not stopped by the introduction of seduxen.

In the study by echoencephalography (EEG), epileptiform activity was recorded. When analyzing magnetic resonance imaging, a pattern of changes was recorded that was characteristic of the manifestations of the atrophic process in the hippocampus on the left, pathological foci in the cortical areas of the left temporal and parietal regions.

Given the duration and failure of conservative therapy, the presence of frequent epileptic seizures and a lag in psycho-speech development, the child was given twice intravenous administration of a human cord blood concentrate in a total dose of about 500 million cells (246 and 254 million, respectively) with an interval of 1 month.

On the next day after repeated administration of the cord blood cell preparation, the girl showed positive dynamics: a decrease in right-sided hemiparesis, especially in the arm (up to 4 points), the girl began to raise her right arm vertically. The number of twitches in the right hand decreased.

During the follow-up examination after 5 months: while taking antiepileptic drugs, the seizures completely disappeared. The degree of hemiparesis decreased to 3 points. On the EEG marked positive dynamics. The quality of life indicators have improved significantly: speech has improved, the child is able to fully serve himself, he dances a lot.

Example 9. Cerebral Palsy with hydrocephalus

The patient is 7 years old and 5 months old with a diagnosis of mixed hydrocephalus, delayed psychomotor and speech development, congenital bilateral amaurosis, with a sharp decrease in visual functions to light perception, the condition did not improve against the background of the ongoing drug therapy. EEG data indicated widespread changes involving stem formations in the process with impaired cortical-subcortical relationships.

Given the severity of the clinical picture of the disease, the data of interoscopy, the progressive course of the disease, the failure of drug therapy, three infusions were made to the child with an interval of 1 month of cord blood cell concentrate in a total dose of about 600 million cells (about 103, 250 and 250 million per administration, respectively )

3 months after the last injection of cells, the boy showed persistent positive dynamics. He became calmer, hyperkinesis decreased, complex sentences appeared in the speech, self-service skills appeared - he eats independently. An improvement in visual functions is noted: the boy began to say that he sees the sun, the sky, sees the outlines of objects, names some objects in the pictures.

Example 10. Cerebral Palsy

A patient of 3.5 years with a diagnosis of cerebral palsy, spastic tetraparesis, symptomatic focal epilepsy, delayed psycho-speech development, was in the stage of drug remission for 1.5 years.

The child was speechless with the ability to reproduce only individual vowels, articulation was grossly impaired. Eyes are almost always closed for centuries. Marked tetraparesis was noted, mainly in the lower extremities; there was no ability to move independently. The patient poorly kept himself upright, even with reliance on objects and poorly held objects. The degree of spasticity of the hands was estimated from 2 to 3 points, the degree of plasticity of the legs was 3 points. The muscle strength of the limbs was 3 points.

Considering the complaints, the clinical picture of the disease, the progressive course of the disease and the failure of the previous therapy, the child was given twice, with an interval of 14 days, intravenous administration of umbilical cord blood cells in a total dose of about 500 million cells (about 240 million and 260 million for one administration, respectively) .

6 months after the administration, a marked improvement in the clinical picture was noted. The child began to fix his gaze on others, came out of the delay in speech development, impressive speech began to correspond to age, indicated pictures of all thematic groups. The restoration of the articulation apparatus was noted - switchability and posture of the posture improved, crawling on all fours, walking with support or resting on objects, taking and holding a toy, a pencil. The degree of spasticity decreased by 1 point, the strength of the muscles of the limbs increased by 1 point.

Perception of color and subject has become age-appropriate. Quantitative representations and the ability to compare and generalize began to take shape. The patient became able to understand speech in full and build phrases.

Example 11. Multiple sclerosis

In a 37-year-old patient with primary progressive course of multiple sclerosis and a disease duration of 8 years, a pronounced lower spastic paraparesis with muscle strength in the legs of 2 points, a pronounced cerebellar syndrome, and pelvic disorders were noted in neurological status.

The level of violations on the EDSS scale was 6.5 points. The level of cognitive impairment on the MMSE scale corresponded to 30, on the PASAT scale - 32 points. MP spectroscopy data: NAA \ Cr = 1.62 ± 0.34; Cho \ Cr = 1.09 ± 0.17.

The patient was twice injected with cord and rhesus factor-compatible cord blood cells with a difference of 14 days in the amount of about 250 million cells for each injection (the total dose of the introduced cells was about 500 million).

After repeated administration and within 1 month there was an improvement in the severity of the pyramidal syndrome with a decrease in the transit time of 7.2 meters (before the introduction - 45 seconds, after the introduction of 40 seconds). EDSS level remains the same. MMSE level is 30, PASAT is 41. MP spectroscopy data 3 months after therapy: NAA \ Cr = 1.66 ± 0.25; Cho \ Cr = 0.96 ± 0.17. Thus, a tendency towards an increase in the level of the NAA marker was observed in brain metabolites, which indicates an improvement in the state of neurons.

Claims (5)

1. A method for the treatment of neurodegenerative diseases and schizophrenia by intravenous infusion of nucleated umbilical cord blood cells, characterized in that the patient is injected with umbilical cord blood cells obtained by sedimentation or gradient centrifugation of cord blood samples with the selection of nucleated cells in the form of a cell pellet followed by resuspension in cell suspension with the addition of cryoprotectant, in an amount of 1 · 10 ⁸ to 5 · 10 ⁸ nucleated cells per administration. 2. The method according to claim 1, characterized in that the number of introduced cells is preferably from about 2.2 · 10 ⁸ to about 2.8 · 10 ⁸ nucleated cells per administration. 3. The method according to claims 1 and 2, characterized in that the neurodegenerative disease is selected from the group including multiple sclerosis, age-related loss of cognitive function, Alzheimer's disease, Parkinson's disease, dementia, memory loss, and the introduction of cord blood cells with an interval of 1-4 weeks. 4. The method according to claims 1 and 2, characterized in that the treatment of schizophrenia is carried out, the nucleated cells of cord blood being administered up to 4 times with an interval of 1-4 weeks. 5. The method according to PP.1 and 2, characterized in that the disease is cerebral palsy and / or hydrocephalus, and the introduction of nucleated cells of umbilical cord blood is carried out up to 3 times.