JP2005139092A - Cell preparation for preventing/treating renal dysfunction and method for producing the preparation, method for preventing/treating renal dysfunction - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To effectively prevent/treat a patient with a renal dysfunction having a lesion in uriniferous tubules or a renal dysfunction of a patient subjected to the mass chemotherapy of a cancer or an infectious disease. <P>SOLUTION: The subject cell preparation for preventing/treating the renal dysfunction is characterized by containing CD 45 receptor cells and CD 34 receptor cells of leukocyte cells preferably in amounts of ≥45% and ≥1%, respectively, in the cell composition. The method for producing the cell preparation for preventing/treating the renal dysfunction is characterized by administering an anti-cancer agent or a granulocyte-stimulating factor to stimulate the expression of human peripheral blood stem cells and then collecting the CD 45 receptor cells and the CD 34 receptor cells in a blood-collecting operation and in a leukocyte-separating operation. The obtained cell preparation is, for example, used for a patient who is a collection source. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention is for the prevention and treatment of renal dysfunction particularly effective for renal dysfunction, which contains specific white blood cells and is considered to be damaged in renal tubules such as renal failure associated with chemotherapy for cancer and infectious diseases. It relates to cell preparations.

  In addition, the present invention induces the expression of peripheral blood stem cells by administering an anticancer drug or granulocyte stimulating factor to humans according to a substantially harmless fixed administration protocol, and the above-mentioned specific leukocytes are collected by blood collection and leukocyte separation operations. The present invention relates to a method for producing a cell preparation for preventing or treating renal dysfunction, which obtains a cell preparation containing cells.

  Furthermore, the present invention is intended for the prevention of renal dysfunction concomitant, which is a serious problem when performing chemotherapy for cancer and infectious diseases, or for the treatment of renal dysfunction considered to have tubule injury, The present invention relates to a method for preventing / treating renal dysfunction, wherein the above-mentioned cell preparation for preventing / treating renal dysfunction is administered to a patient in the form of autotransplantation or transplantation of another person.

  Among various diseases that cause human death, cancer, infections and cardiovascular disorders are extremely important. In recent years, with the remarkable development of anticancer drugs and antibiotics, the progress of chemotherapy for cancer and infectious diseases is remarkable. On the other hand, however, there is also a problem that renal dysfunction is caused as a side effect of these chemotherapy.

  Such a problem can be said to be the fate of chemotherapy from the fact that anticancer drugs and antibiotics act on normal somatic cells as well as target somatic cells and infectious microorganisms. In particular, recently, in order to pursue a remarkable chemotherapeutic effect, there is a tendency to administer a large amount of anticancer drugs and antibiotics, or to use multiple drugs in combination. Therefore, especially in cancer chemotherapy, renal dysfunction as a side effect tends to increase in frequency and severity.

  For example, in the case of hematopoietic malignant tumors, breast cancer, ovarian tumors, testicular tumors, etc., the development of new anticancer drugs and multi-drug combination chemotherapy and their clinical application have led to the possibility of long-term survival and cure. At the same time, however, there is an increased risk of causing damages due to the cytotoxicity of anticancer drugs to normal cells and secondary damages through the bloodstream. Among complications associated with chemotherapy, renal dysfunction is a serious problem that cannot be left untreated because many serious symptoms that continue chemotherapy and life threaten patients are seen.

  Currently, the only way to deal with such problems is to prevent renal dysfunction by adjusting the infusion for infusions or using diuretics, or waiting for spontaneous recovery of renal dysfunction. More specifically, it is necessary to perform sufficient renal function evaluation before administration of the anticancer agent and to perform renal function evaluation at least once a week after administration of the anticancer agent. In patients with already impaired renal function, care should be taken to avoid administration of highly nephrotoxic anticancer drugs and to avoid large doses of anticancer drugs as much as possible. When large doses of anticancer drugs cannot be avoided, consideration must be given to prevent large tubular transfusions and diuretics from causing tubule injury. In addition, when using methotrexate, which is an anticancer agent, it is also important to make the urine alkaline. Ultimately, however, these measures do not actively cure or ameliorate renal dysfunction, but merely preventive or symptomatic treatment of renal dysfunction.

  On the other hand, several methods for improving secondary renal dysfunction caused by various immune dysfunctions have been reported.

"Treament of severe systemic lupus erythematosuswith high-dose chemotherapy and haemopoietic stem-cell transplantation: a phase 1 study", Traynor AE, Schroeder J, Rosa RM, Cheng D, StefkaJ, Mujais S, Baker S, Burt RK, Lancet. 2000 Aug 26; 356 (9231): 701-7. "High-dose chemotherapy and autologous peripheralblood stem cell transplantation in patients with multiple myeloma andrenal insufficiency", Ballster OF, Tummala R, Janssen WE, Fields KK, Hiemenz JW, Goldstein SC, Perkins JB, Sullivan DM, Rosen R, SacksteinR, Zorsky P, Saez R, Elfenbein GJ, Bone Marrow Transplant. 1997 Oct; 20 (8): 653-6 "Rapid reversal of nephrotic syndrome due to primary systemic AL amyloidosis after VAD and subsequent high-dosechemotherapy with autologous stem cell support", Sezer O, Schmid P, Shweigert M, Heider U, Eucker J, Harder H, Sinha P, Radtke H, Possinger K, Bone Marrow Transplant. 1999 May; 23 (9): 967-9 "Stem cells in renal biology: Bone marrow transplantation for the treatment of IgA nephropathy", Imasawa T, Utsunomiya Y, Exp. Nephrol. 2002; 10 (1): 51-8 For example, Have reported methods for improving secondary renal dysfunction caused by SLE nephropathy, myeloma, and amyloid nephropathy by the administration of peripheral blood stem cells, respectively. In Non-Patent Document 4 described above, a method of improving secondary renal dysfunction caused by IgA nephropathy by bone marrow transplantation is reported.

  However, in the said nonpatent literature 1-nonpatent literature 4, all target renal dysfunction based on an immune dysfunction. Kidney dysfunction caused by an immunological mechanism specifically means glomerular disease, that is, glomerulonephritis, which is inflammation, and nephrotic syndrome in which the basement membrane is damaged and blood substances are leaked.

  On the other hand, renal dysfunction that develops with cancer chemotherapy and the like is a tubular disorder caused by the direct action of cytotoxicity of anticancer drugs, and is not a mere functional disorder, It is considered a tissue “injury” involving cell / tissue destruction or shedding.

  That is, the glomerular disease targeted by Non-Patent Literature 1 to Non-Patent Literature 4 and the tubular injury caused by chemotherapy or the like are both renal disorders, but the cause of the onset, the damaged tissue and the contents of the disorder are Completely different. Therefore, the methods of Non-Patent Document 1 to Non-Patent Document 4 are not helpful for improving renal dysfunction (tubule injury) that develops with chemotherapy and the like. Further, the peripheral blood stem cells used in Non-Patent Document 1 to Non-Patent Document 3 are intended to improve glomerular disease and are unlikely to be effective in improving tubular injury.

  Furthermore, in the method of performing bone marrow transplantation as in Non-Patent Document 4, a) general anesthesia is required to collect the amount of bone marrow necessary for transplantation, b) the bone marrow is once emptied, and then intravenously infused. Hematopoietic stem cells of bone marrow fluid are established and proliferated in the bone marrow to recover hematopoiesis, but the recovery (which takes about one month) is slow, c) Leukemia cells are more likely to be in the bone marrow than in peripheral blood Therefore, problems such as a relatively high risk of contamination of tumor cells by bone marrow transplantation are pointed out.

"Bone marrow stem cells contribute to repair of the ischemically injured renal tubule", Kale S, Karihaloo A, Clark PR, Kashgarian M, Krause DS, Cantley LG, J. Clin. Invest. 2003 Using ischemia reperfusion rats as a model of non-immune renal dysfunction mechanism, irradiation bone marrow injury group (BMA), bone marrow transplantation group (BMA-SCT), We compare and evaluate renal function in non-irradiated bone marrow non-injury group (Comtrol). However, in short, it is a bone marrow transplantation method similar to the case of Non-Patent Document 4, and is not related to the improvement of renal function but is related to the elucidation of the pathology.

"Endothelial disfunction in ischemic acute renal failure: Rescue by transplanted endothelial cells", Brodsky SV, Yamamoto T, Tada T, Kim B, Chen J, Kajiya F, Goligorsky MS, Am. J. Physiol. Renal Physiol. 282: F1140-F1149 (2002) Further, in the above non-patent document 6, as in the case of non-patent document 5, the subject is an ischemic reperfusion rat, but human umbilical cord vascular endothelial cells are injected from the carotid artery, and after reperfusion As a 24-hour renal function, blood flow in capillaries around tubules was measured, and it was reported that blood flow disturbance was attenuated. That is, it is pointed out that human umbilical vascular endothelial cells, which are differentiated cells, are used, renal circulation disorders are targeted, and the like.

Furthermore, in the above-mentioned patent document 1, in order to regulate the stem cell cycle and promote peripheral blood cell regeneration after chemotherapy, as a stem cell regulator, “C-terminal hydrophobic domain is substituted or Disclosed are methods for isolating and using a “polypeptide comprising a deleted hemoglobin α chain”. In other words, it focuses on the regeneration of peripheral blood cells after chemotherapy, but specifically relates to the use of protein preparations, and does not specifically focus on renal dysfunction.

  By the way, as a general theory in the current state of medicine, it is said that a glomerulus having a complicated configuration and shape cannot be regenerated, but a tubule having a relatively simple lumen form is reproducible. Therefore, it is possible to foresee the possibility that the above-described non-immune and directly cytotoxic renal dysfunction (tubule injury) can be improved by using suitable stem cells. However, apart from methods such as bone marrow transplantation, where stress and various risks in patients are large and require a long period of improvement, as seen in the various non-patent documents and patent documents mentioned above, non-immune and direct cytotoxicity No specific means has yet been proposed which can improve the renal dysfunction in a short period of time by a simple method with less patient burden.

  Accordingly, the present invention provides a cell preparation for preventing or treating renal dysfunction that can be expected to have such effects, a method for producing the same, and a method for preventing and treating renal dysfunction. To do. The present inventor has elucidated specific types of stem cells (white blood cells) that are effective in improving non-immune and directly cytotoxic renal dysfunction, and has further developed a substantially harmless and efficient collection method. The above technical problems have been solved.

(Configuration of the first invention)
The configuration of the first invention of the present application for solving the above-described problem is a cell preparation for preventing or treating renal dysfunction containing CD45 receptor cells and CD34 receptor cells, which are white blood cells.

(Configuration of the second invention)
The structure of the second invention of the present application for solving the above problems is that the composition of the number of cells of the cell preparation according to the first invention comprises 45% or more of CD45 receptor cells and 1% or more of CD34 receptor cells. It is a cell preparation for preventing or treating dysfunction.

(Configuration of the third invention)
The structure of the third invention of the present application for solving the above problems is that the composition of the number of cells of the cell preparation according to the first invention is such that the CD45 receptor cells are 95% or more and the CD34 receptor cells are 2% or more. It is a cell preparation for preventing or treating dysfunction.

(Configuration of the fourth invention)
In order to solve the above problems, the fourth invention of the present application is the first invention in which CD45 receptor cells and CD34 receptor cells are collected by blood collection and leukocyte separation operations under the stimulation of human peripheral blood stem cell expression. A method for producing a cell preparation for preventing or treating renal dysfunction, which comprises producing a cell preparation according to any one of the third invention.

(Structure of the fifth invention)
The structure of the fifth invention of the present application for solving the above-mentioned problem is that the cell preparation according to the fourth invention is used for a patient who is a collection source thereof, a cell preparation for preventing or treating renal dysfunction It is a manufacturing method.

(Structure of the sixth invention)
In order to solve the above problems, the sixth invention of the present application is such that the means for stimulating the expression of peripheral blood stem cells according to the fourth or fifth invention is administration of an anticancer agent and / or granulocyte stimulating factor. And a method for producing a cell preparation for preventing or treating renal dysfunction.

(Structure of the seventh invention)
In order to solve the above-mentioned problems, the seventh aspect of the present invention is the administration of the granulocyte stimulating factor over an appropriate number of days after the administration of the anticancer agent according to the sixth aspect over an appropriate number of days, and after the recovery period of the white blood cell count. Is a method for producing a cell preparation for preventing or treating renal dysfunction.

(Configuration of the eighth invention)
The configuration of the eighth invention of the present application for solving the above-mentioned problem is that the diuretic management is performed with the target of 2000 to 3000 mL / day during the administration period of the anticancer agent according to the sixth or seventh invention, and the kidneys are at appropriate intervals. This is a method for producing a cell preparation for prevention / treatment of renal dysfunction, wherein the function (Ccr) is checked.

(Structure of the ninth invention)
The structure of the ninth invention of the present application for solving the above-described problem is the same as the blood collection and leukocyte separation operation according to any of the fourth to eighth inventions, wherein the white blood cell count (WBC) in blood exceeds 4000 / mm ³ . Alternatively, it is a method for producing a cell preparation for the prevention / treatment of renal dysfunction, which starts when the platelet count (PTL) exceeds 1,000,000 / mm ³ .

(Configuration of the tenth invention)
The structure of the tenth invention of the present application for solving the above problem is that apheresis (blood extracorporeal circulation) using at least a blood component separator in the blood collection and leukocyte separation operations according to any of the fourth to ninth inventions. Method for producing cell preparation for prevention / treatment of renal dysfunction, comprising collecting mononuclear cell fraction by HPLC, removing platelet-rich plasma and red blood cells, and counting CD45 receptor cells and CD34 receptor cells by flow cytometry It is.

(Structure of 11th invention)
The constitution of the eleventh invention of the present application for solving the above-mentioned problems is that the renal dysfunction according to any one of the first to third inventions is applied to a patient corresponding to the following (1) and / or (2): This is a method for preventing / treating renal dysfunction by administering a cell preparation for prevention / treatment.
(1) Renal dysfunction with tubule injury.
(2) High-dose chemotherapy for cancer or infectious diseases.

(Configuration of the twelfth invention)
The configuration of the twelfth invention of the present application for solving the above-mentioned problems is that diuresis management is performed for a patient who performs high-dose chemotherapy according to the eleventh invention with a target of 2000 to 3000 mL / day, and renal function (Ccr). This is a method for the prevention and treatment of renal dysfunction, in which the administration timing of a cell preparation for the prevention and treatment of renal dysfunction is determined using the check result of the above.

(Effect of the first invention)
The cell preparation for preventing / treating renal dysfunction according to the first invention is very effective for preventing or treating renal dysfunction. The type of renal dysfunction is not particularly limited. For example, secondary renal dysfunction caused by various immune dysfunctions (glomerulonephritis which is inflammation, basement membrane is damaged and substances in blood are It is considered effective for leaking nephrotic syndrome and the like. However, particularly noteworthy is that it is effective for the prevention or treatment of renal dysfunction, which is considered to have tubule damage.

  Recently, renal dysfunction has tended to become more frequent and serious as a side effect of large doses of anticancer drugs and antibiotics, combined use of multiple drugs, etc., and these renal dysfunctions are non-immune and directly cytotoxic renal functions The disorder is thought to be due to tubular injury. Conventionally, as countermeasures against such renal dysfunction, there have been only symptomatic means such as adjustment of infusion for infusion and use of diuretics, so that the cell preparation for preventing and treating renal dysfunction according to the first invention is effective. And appropriate use is desired.

  The reason why the cell preparation for the prevention and treatment of renal dysfunction according to the first invention is effective for renal dysfunction, particularly tubule injury has not yet been fully elucidated, but it is presumably CD45 receptor cells and CD34 which are stem cells. It is thought that the receptor cells exhibit a specific synergistic action and heal the injury of the tubule having a relatively simple lumen form to regenerate a healthy tubule.

(Effect of the second invention)
The cell preparation for preventing / treating renal dysfunction according to the first invention can be expected to be effective as long as it contains CD45 receptor cells and CD34 receptor cells as active ingredients. When the CD45 receptor cells are 45% or more and the CD34 receptor cells are 1% or more, the preventive and therapeutic effects of renal dysfunction are particularly remarkable.

(Effect of the third invention)
In the cell number composition of the cell preparation for preventing or treating renal dysfunction, when the CD45 receptor cells are 95% or more and the CD34 receptor cells are 2% or more, the effect of preventing and treating renal dysfunction is particularly remarkable.

(Effect of the fourth invention)
The cell preparations of the first to third inventions can be efficiently produced by the method for producing a cell preparation for preventing or treating renal dysfunction according to the fourth invention. In addition, this manufacturing method basically includes a process of stimulating human peripheral blood stem cell expression and a process of blood collection and leukocyte separation. It is possible to avoid the stress and risk burden on humans, such as risk of contamination and tumor cell contamination.

(Effect of the fifth invention)
The cell preparation produced by the method for producing a cell preparation for preventing or treating renal dysfunction according to the fourth invention described above is intended for humans other than the human being from which it is collected under consideration of blood type adaptation and the like. Of course, it is possible to use it, but it is more preferable to use it for the person from whom it is collected.

(Effect of the sixth invention)
The means for stimulating the expression of peripheral blood stem cells is not limited as long as the object can be safely achieved. For example, administration of an anticancer agent and / or administration of granulocyte stimulating factor can be preferably exemplified. It is particularly preferable to administer the anticancer agent and the granulocyte stimulating factor together.

  The administration protocol for anti-cancer drugs and granulocyte stimulating factors is such that the subject is not subject to unnecessary stress or risk depending on whether the subject is a healthy person or a patient with a disease. It is desirable to follow a harmless protocol.

(Effect of the seventh invention)
In the sixth invention described above, when both the administration of the anticancer agent and the granulocyte stimulating factor are performed, the anticancer agent is first administered over an appropriate number of days, and then the recovery period of the white blood cell count is grasped and the granulocyte stimulating factor is appropriately controlled. It is particularly preferable to follow the protocol of administration over a period of days from the viewpoint of avoiding stress and risk for the subject and from the viewpoint of efficient production of the intended CD45 receptor cells and CD34 receptor cells.

(Effect of the eighth invention)
The administration of anticancer drugs in this method for producing cell preparations for the prevention and treatment of renal dysfunction is intended to produce cell preparations, not for the prevention or treatment of cancer. There is. Therefore, during the administration period of the anticancer agent, it is preferable to check the renal function by performing diuretic management and measuring creatine clearance (Ccr) at appropriate intervals as in the eighth invention.

(Effect of the ninth invention)
In certain protocols in which peripheral cancer stem cells are stimulated by administering anticancer agents, granulocyte stimulating factors, etc. to humans, it has been found that the expression level of peripheral blood stem cells varies with time. Therefore, in order to know the timing at which a larger amount of CD45 receptor cells and CD34 receptor cells can be collected more efficiently, it is reasonable to use the number of white blood cells (WBC) or platelets (PTL) in blood as an index. It is.

More specifically, blood collection and leukocyte separation operations are started when the number of white blood cells in blood exceeds 4000 / mm ³ or the number of platelets in blood exceeds 1000000 / mm ³ as in the ninth invention. It is preferable.

(Effect of the tenth invention)
Blood collection and leukocyte separation operations may be performed, for example, by laboratory manual operation, but various blood component separation devices that can automatically perform these series of operations are provided on the market. Is convenient. The normal blood collection and leukocyte separation operations include a mononuclear cell fraction collection process and a platelet-rich plasma and red blood cell removal process. These can be easily performed by apheresis using a blood component separation device.

After obtaining a fraction containing CD45 receptor cells and CD34 receptor cells, the number of CD45 receptor cells and CD34 receptor cells in the fraction (/ mm ³ ), for example, by flow cytometry, and / or Alternatively, it is preferable to calculate the cell number composition (percentage of the cell number in the total cell number).

(Effect of the 11th invention)
The eleventh invention is a method for preventing or treating renal dysfunction using the cell preparations according to the first to third inventions, wherein the subject patient has renal dysfunction with tubule injury. Has and / or is undergoing high-dose chemotherapy for cancer or infection. With this prevention / treatment method, it is possible to expect a remarkable improvement effect of renal dysfunction with tubule injury or a significant prevention effect of renal dysfunction associated with high-dose chemotherapy for cancer or infection.

(Effect of the twelfth invention)
For patients who receive high-dose chemotherapy in the eleventh invention described above, as in the twelfth invention, while performing diuretic management, measuring creatine clearance at appropriate intervals to check renal function is more preferable.

  Next, modes for carrying out the first invention to the twelfth invention of the present application will be described including the best mode. In the following, the term “present invention” refers to each invention of the present application collectively.

[Cellular preparations for the prevention and treatment of renal dysfunction]
As is well known, a CD45 receptor cell is a white blood cell that shows a positive response to CD45, an antibody that stains white blood cells, and a CD34 receptor cell is a CD4 that is an antibody that stains peripheral blood stem cells. White blood cells showing a positive reaction. These CD45 receptor cells and CD34 receptor cells are known to be a kind of stem cells.

  A cell preparation for preventing or treating renal dysfunction according to the present invention is a cell preparation containing at least the above-mentioned CD45 receptor cell and CD34 receptor cell. The method of the tenth invention is preferred, but includes those produced or prepared by other methods.

  In the cell preparation, the cell number composition of the CD45 receptor cell and the CD34 receptor cell, that is, the percentage of the cell number in the total cell number in the unit amount of the cell preparation is not limited. However, CD45 receptor cells are preferably 45% or more, CD34 receptor cells are preferably 1% or more, CD45 receptor cells are preferably 95% or more, and CD34 receptor cells are more preferably 2% or more. Therefore, in the cell preparation of the present invention, white blood cells other than the above-mentioned two types of white blood cells, other types of blood cells or somatic cells other than blood systems are mixed at a constant composition ratio. Have Red blood cells, granulocytes, platelets, and the like have the problem of increasing the viscosity of cell preparations, so it is better to exclude them as much as possible.

  In order to improve the storage stability or stability of the cell preparation, or to protect the white blood cells that are active ingredients, for example, when adding a human albumin-added CP-1 solution to the cell preparation according to the present invention, Optional ingredients can be added. Other examples of such additives include 5% DMSO and 6% hydrodiethyl starch. In addition, the cell preparation of the present invention is preferably stored in a cool and dark place where the temperature is constant, as in the case of other blood preparations.

  The use of the cell preparation according to the present invention is not limited. In general, it is used for the prevention or treatment of any renal dysfunction. Renal dysfunction includes secondary renal dysfunction caused by various immune dysfunctions such as SLE nephropathy, myeloma, amyloid nephropathy, and non-immune and directly cytotoxic kidneys. Also included are dysfunctions (eg, renal dysfunction resulting from tubular injury). The most preferred use is the treatment or prevention of non-immune and directly cytotoxic renal dysfunction (eg, prevention of renal dysfunction associated with high-dose chemotherapy such as cancer).

[Method for producing cell preparation for prevention / treatment of renal dysfunction]
The method for producing a cell preparation for preventing or treating renal dysfunction according to the present invention comprises at least a step of stimulating human peripheral blood stem cell expression, and a step of collecting CD45 receptor cells and CD34 receptor cells by blood collection and leukocyte separation operations. Is included. Here, the “human” as the subject is preferably a healthy person, but in the case of “self-use” in which the produced cell preparation is administered to the subject himself / herself, even if he / she has a certain disease Unavoidable. In that case, particularly when the subject has a renal dysfunction, special careful consideration is required for designing the protocol described later for the subject.

  The means for stimulating peripheral blood stem cell expression is not particularly limited in consideration of medical stress and risk to the subject. However, administration of an anticancer agent and / or granulocyte stimulating factor is particularly effective. The types of anticancer agents and granulocyte stimulating factors are not limited, and can be appropriately selected based on examination of necessary considerations.

  As general anticancer agents, there are carboplatin (Carboplatin: provided under the trade name such as valaplatin, CBDCA, antineoplastic platinum agent, etc.) and etoposide (Etoposide: plant alkaloid Podophylotoxine anticancer agent. For example, VIP16, Cyclophosphamide (an alkylating agent Nitrogenmutard anticancer agent, for example, offered under a trade name such as endoxatin), mitoxantrone (MIT: Mitoxantrone) ), Doxorubicin (DXR: Doxorubicin), vincristine (VCR: Vincristine), prednisolone (PSL: Prednisolone), cytarabine (CA: Cytarabine), daunorubicin (DNR: Daunorubicin), 6-thioguanine (6-TG: 6-Thioguanine) Aclarubicin (ACR: Aclarubicin), 5-fluorouracil (5-FU: 5-Fluorouracil), methotrexate (MTX: Methotrexate), cisplatin (CDDP: Cisplatin), etc. can be illustrated.

  Examples of general granulocyte stimulating factors include G-CSF, GM-CSF, IL-3, and SCF.

  When administering an anticancer agent or granulocyte stimulating factor as a means for stimulating the expression of peripheral blood stem cells, the administration protocol is not particularly limited, but preferably, after the administration of the anticancer agent over an appropriate number of days, recovery of the white blood cell count It is preferable to administer the granulocyte-stimulating factor over an appropriate number of days in accordance with the period. Here, the administration period of the anticancer agent can be, for example, about 3 to 7 days, and the recovery period of the white blood cell count can be, for example, 1 to 2 weeks after the end of the administration of the anticancer agent. The administration days of the stimulating factor can be about 5 to 10 days, for example.

  During the period of administration of an anticancer drug as a means of stimulating the expression of peripheral blood stem cells, diuresis management is carried out with a target of 2000 to 3000 mL / day by adjusting the infusion for infusion and the use of a diuretic, and creatine clearance at appropriate intervals It is desirable to check renal function by measuring (Ccr).

  After peripheral blood stem cells are expressed in the blood of the subject, blood collection and leukocyte separation are performed, and CD45 receptor cells and CD34 receptor cells are collected. The timing of starting this blood collection and leukocyte separation operation is a problem that should be arbitrarily and appropriately determined on a case-by-case basis. As one effective index, the white blood cell count (WBC) in the subject's blood is 4000. A point in time when the number of platelets (mL) exceeds / 1,000,000 or mL can be illustrated. This is because these values in WBC or PTL suggest sufficient expression of peripheral blood stem cells.

  The method of blood collection and leukocyte separation operation may be arbitrarily determined within the possible range.For example, the collection of mononuclear cell fraction by apheresis using a blood component separation device, the removal of platelet-rich plasma and red blood cells Preferably, CD45 receptor cells and CD34 receptor cells are counted by flow cytometry.

  Apheresis (blood extracorporeal circulation) generally refers to extracorporeal circulation treatment for various diseases in which high molecular weight proteins that cannot be removed by hemodialysis are pathogenic substances. What is apheresis? In this method, the blood of a subject is circulated extracorporeally and passed through a blood component separation device, and the target CD45 receptor cells and CD34 receptor cells are collected efficiently and in large quantities.

  The calculation method of CD45 receptor cells and CD34 receptor cells by flow cytometry is a well-known matter, but its contents will be briefly explained below. The cell size can be calculated in two dimensions using the Radiofrequency / Direct Current method. When the cells are expanded, immature cells are expanded downward, and the above-mentioned CD34 receptor cells and the like are shown in the lower region.

[Prevention and treatment of renal dysfunction]
The method for preventing / treating renal dysfunction according to the present invention comprises the above-mentioned renal dysfunction for patients who have renal dysfunction with tubule injury and / or are undergoing high-dose chemotherapy for cancer or infection. This is a method of administering a cell preparation for preventing or treating dysfunction. Unlike the case of administering an anticancer agent or the like as a stimulation means for peripheral blood stem cell expression in the method for producing a cell preparation for preventing or treating renal dysfunction, in this case, the type of anticancer agent or antibiotic used, the dosage, The administration period is determined according to the protocol for chemotherapy.

  With respect to such a chemotherapy protocol, how to set the timing and dose of administration of the cell preparation and incorporate it into the protocol is appropriately determined on a case-by-case basis. However, in general, administration of a cell preparation immediately after the high-dose chemotherapy to several days later is considered to be most effective, including cost performance. The frequency and dose of administration of cell preparations for the prevention and treatment of renal dysfunction are matters to be determined as necessary and cannot be defined uniformly.

In the method for preventing / treating renal dysfunction according to the present invention, during the period of high-dose chemotherapy for cancer or infectious disease, diuresis with a target of 2000-3000 mL / day by adjusting the infusion for infusion or using a diuretic, etc. It is desirable to check renal function by performing management and measuring creatine clearance (Ccr) or urinary NAG or β ₂ MG at appropriate intervals.

  Next, Example 1 and Example 2 according to the present invention will be described. Of course, these examples do not limit the technical scope of the present invention.

  As Example 1, an anticancer drug and granulocyte stimulating factor were administered to cancer patients according to the protocol of FIG. The direct purpose of Example 1 is not to treat cancer itself, but to prepare the cell preparation for preventing or treating renal dysfunction according to the present invention for self-use. Therefore, in Example 1, the dose of anticancer agent or the like is relatively reduced. Example 2 is a high-dose chemotherapy of an anticancer agent for the same cancer patient, and as shown in FIG. 1 (b), the administration of the cell preparation prepared in Example 1 is incorporated into the protocol.

(Example 1: Production of cell preparation for prevention / treatment of renal dysfunction)
The target cancer patients are 18 patients who have a primary cancer site in the testis or the like, or have metastasized cancers to the lung, liver, peritoneum or the like. To these cancer patients, Cis (anticancer agent: cisplatin described above) and VIP16 (anticancer agent: etoposide described above) were administered from day 1 to day 5. The dosage per day is Cis is 100mg ^/ m 2, VIP16 was 20 mg / ^{m 2.} Separately, bleomycin, an anticancer agent, was administered at 30 mg / body from day 2 to day 16. In addition, 2000 to 3000 mL of diuresis was secured every day from the first day to the seventh day.

  When the transition of white blood cell count (WBC) in each patient's blood has been traced since the start of administration of the anticancer drug on the first day, the presentation of detailed data is omitted, but overall the first few days the WBC Although it is in a decreasing trend, the recovery period of the white blood cell count has reached the 12th to 13th days. Therefore, administration of granulocyte stimulating factor G-CSF (trade name: Neuup) was started from the 14th day. The dose is 5 mg / kg i.v.

  When the WBC of each patient was further traced after the start of administration of G-CSF, the WBC started to show a significant increase tendency from around the 18th day, and the WBC exceeded 4000 / μL on the 21st day. Also came out. For this reason, administration of G-CSF was terminated on the 21st day, and at this time (indicated as “PBSCH” in the illustrated protocol), blood was collected from the subject and a leukocyte separation operation was performed.

  In the blood collection and leukocyte separation operations, apheresis was performed twice for each patient using a continuous blood component separation apparatus AS104, AS.TEC204 manufactured by Fresenius HemoCare Inc. In a single apheresis, 10-15 L of blood is processed over about 3 hours to collect a mononuclear cell fraction, and platelet-rich plasma and red blood cells are removed by back centrifugation, and Buffy Coat is recovered. Dimethyl sulfoxide (DMSO) was added. An equal amount of 25% human albumin-added CP-1 solution was added thereto, and then dispensed as a cell preparation into a freezing bag and stored at −80 ° C.

(Example 2: Prevention / treatment of renal dysfunction)
In the protocol of FIG. 1 (b) showing Example 2, for convenience, the day on which the cell preparation prepared in Example 1 is administered is indicated as the reference day (Day 0). Therefore, the notation “−6”, for example, in FIG. 1B means “the sixth day”, that is, 6 days before the reference date. Moreover, the 8th day which is the first administration date of the anticancer agent in the protocol of FIG. 1B is appropriately spaced from the day of “PBSCH” in the protocol of FIG.

First, diuresis management was started to secure 2000 to 3000 mL of diuresis every day from around -10th to -9th days for each patient. Then, 500 mg / m ² CBDCA (anticancer agent: carboplatin) and 400 mg / m ² of VIP16 were administered on days -8, -6, and -4, respectively. In addition, Cyc (an anticancer drug cyclophosphamide) was administered at 500 mg / kg on day -6 and day -4, respectively.

  Next, on day 0, the autologous cell preparation prepared and stored in Example 1 was administered to each patient. The dosage form is intravenous infusion, and the dosage is the total amount prepared in Example 1.

  The renal function of each patient was checked by measuring creatine clearance (Ccr) at a predetermined time. As shown in the protocol of FIG. 1 (b) by “Ccr”, the predetermined time is immediately before the start of high-dose chemotherapy (before HDCT), immediately after the end of high-dose chemotherapy (after HDCT), and administration of cell preparations. Within 1 week after (within 1 week after PBSCT) and 2 weeks after the day of administration of the cell preparation (2 week after PBSCT). The measurement results of these Ccr are shown in the bar graph of FIG. In FIG. 2, the vertical axis represents the measured value of Ccr, and each bar graph represents the average value and standard deviation of the measured Ccr value of each patient at each of the above times.

As is clear from FIG. 2, renal function tended to decrease immediately after administration of the anticancer agent compared with before the start of high-dose chemotherapy, and significantly improved from 1 to 2 weeks after administration of the cell preparation. . Furthermore, although presentation of detailed data is omitted, β ₂ MG as a tubular urinary marker showed a marked improvement in several patients. For example, there were patients who showed an improvement of 108 μg / L and patients who showed an improvement of 135 μg / L.

The above results confirm that high-dose chemotherapy for cancer has cytotoxic effects on tubule cells, and against such deterioration of renal function (non-immune and direct cytotoxic renal dysfunction) It has been found that administration of the cell preparation according to the present invention is very effective. In addition, it is estimated that the cell preparation of this invention acted on the glomerulus by improving Ccr, and it was estimated that it acted on the tubule by improving urinary β ₂ MG.

  The present invention provides a novel cell preparation for preventing and treating renal dysfunction and a method for producing the same, and a method for preventing and treating renal dysfunction using the cell preparation for preventing and treating renal dysfunction. These have an effective effect on non-immune and direct cytotoxic renal dysfunction, for which there has been no effective coping measure in the past.

FIG. 1 (a) shows an example of a protocol in a method for producing a cell preparation for prevention / treatment of renal dysfunction, and FIG. 1 (b) shows protocol 1 in a method for prevention / treatment of renal dysfunction.

It is a graph which shows the improvement of the renal function in an Example by the measured value of Ccr.

Claims (12)

A cell preparation for preventing or treating renal dysfunction, comprising a CD45 receptor cell and a CD34 receptor cell, which are white blood cells. 2. The cell preparation for preventing or treating renal dysfunction according to claim 1, wherein the cell preparation contains 45% or more of CD45 receptor cells and 1% or more of CD34 receptor cells. The cell preparation for preventing or treating renal dysfunction according to claim 1, wherein the cell preparation contains 95% or more of CD45 receptor cells and 2% or more of CD34 receptor cells. The CD45 receptor cell and the CD34 receptor cell are collected by blood sampling and leukocyte separation operation under stimulation of human peripheral blood stem cell expression, and the cell preparation according to any one of claims 1 to 3 is produced. A method for producing a cell preparation for preventing or treating renal dysfunction characterized by the above. The method for producing a cell preparation for preventing or treating renal dysfunction according to claim 4, wherein the cell preparation is used for a patient as a collection source. 6. The cell preparation for preventing or treating renal dysfunction according to claim 4 or 5, wherein the means for stimulating the expression of peripheral blood stem cells is administration of an anticancer agent and / or administration of a granulocyte stimulating factor. Manufacturing method. The renal dysfunction prevention / treatment according to claim 6, wherein the granulocyte-stimulating factor is administered over an appropriate number of days after the recovery period of the white blood cell count after the administration over the appropriate number of days. A method for producing a cell preparation. 8. The kidney according to claim 6, wherein during the administration period of the anticancer agent, diuresis management is performed with a target of 2000 to 3000 mL / day, and renal function (Ccr) is checked at an appropriate interval. A method for producing a cell preparation for preventing or treating dysfunction. The blood collection and leukocyte separation operations are started when the white blood cell count (WBC) in blood exceeds 4000 / mm ³ or the platelet count (PTL) exceeds 1000000 / mm ^3. A method for producing a cell preparation for prevention / treatment of renal dysfunction according to any one of claims 4 to 8. In the blood collection and leukocyte separation operations, at least collection of a mononuclear cell fraction by apheresis using a blood component separation device, removal of platelet-rich plasma and red blood cells, and CD45 receptor cells and CD34 receptor cells by flow cytometry The method for producing a cell preparation for preventing or treating renal dysfunction according to any one of claims 4 to 9, wherein: A kidney characterized by administering the cell preparation for preventing or treating renal dysfunction according to any one of claims 1 to 3 to a patient corresponding to the following (1) and / or (2): How to prevent or treat dysfunction.
(1) Renal dysfunction with tubule injury.
(2) High-dose chemotherapy for cancer or infectious diseases. Perform diuretic management with the goal of 2000-3000 mL / day for patients undergoing high-dose chemotherapy, and determine the administration time of the cellular preparation for prevention / treatment of renal dysfunction using the check result of renal function (Ccr) as an index The method for preventing / treating renal dysfunction according to claim 11.

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