JP2016114606A - Use of erythrocytic adma as biomarker for renal anemia - Google Patents

Abstract

A biomarker capable of properly using ESA is provided. Use of asymmetric dimethylarginine (hereinafter referred to as ADMA) as a biomarker for monitoring the following (1) to (3): (1) Onset of renal anemia in mammals; ) ESA hyporesponsiveness in mammals undergoing treatment with erythropoiesis stimulating agent preparation (ESA); or (3) Onset of heart disease in mammals. The same use, wherein the ADMA is ADMA present in red blood cells of a blood sample obtained from a mammal or said mammal undergoing ESA treatment. [Selection figure] None

Description

  The present invention relates to the use of asymmetric dimethylarginine (hereinafter referred to as ADMA) as a biomarker for renal anemia.

  Anemia is not a disease name, but is defined as a state in which the amount of hemoglobin (Hb) in the blood unit volume is reduced. According to the WHO criteria, adult males are less than 13 g / dl, adult girls and children are less than 12 g / dl, pregnant women and infants Is defined as less than 11 g / dl. Usually, as the Hb concentration decreases, the number of red blood cells and the hematocrit value also decrease. However, since the main physiological function of red blood cells is oxygen transport from the lungs to whole body tissues by Hb, the Hb concentration is the most important index for the living body. .

  Anemia is based on MCV (mean corpuscular volume), microcytic (iron deficiency anemia, anemia associated with chronic diseases, ironblastic anemia, thalassemia, atransferrinemia), normocytic ( Renal anemia, hemolytic anemia, aplastic anemia, erythroblastic fistula, myelodysplastic syndrome, anemia associated with chronic disease, leukemia) and macrocytic (renal anemia, megaloblastic anemia (vitamin B12 deficiency) Folic acid deficiency), liver damage, hypothyroidism, aplastic anemia, myelodysplastic syndrome, DNA synthesis disorder caused by drugs). Renal anemia is said to indicate orthocytic or macrocytic anemia.

  Red blood cell production in the bone marrow is stimulated by erythropoietin (EPO: a peptide hormone consisting of 165 amino acids) produced in the kidney. That is, EPO produced in the kidney, exiting into the bloodstream and reaching the bone marrow binds to receptors on erythroid progenitor cells and promotes their differentiation and proliferation. Thus, since EPO is mainly produced in the kidney, EPO is not produced or production is extremely reduced in renal failure patients whose kidneys are devastation. Therefore, in patients with renal failure, the production of red blood cells is suppressed and severe anemia occurs. Anemia due to this mechanism is renal anemia. That is, renal anemia refers to anemia caused by a decrease in EPO production ability in the kidney due to kidney damage. Renal anemia also includes factors such as shortened red blood cell life, reduced EPO responsiveness of hematopoietic cells, nutritional disorders, and residual blood in the circuit in hemodialysis (HD) patients.

  Thus, a recombinant human erythropoietin preparation (rHuEPO) has been developed to supplement EPO for patients with renal anemia, such as dialysis patients. A recombinant human erythropoietin preparation is produced by incorporating a human EPO-producing gene into hamster cells and culturing in large quantities. Currently available recombinant human erythropoietin preparations are epoetin α and epoetin β. Thereafter, the second generation such as darbepoetin alfa appears in rHuEPO, and the name of the erythropoiesis stimulating factor preparation (hereinafter referred to as ESA) including the second generation is used instead of the name of human erythropoietin preparation. It became so. The first-line drug for renal anemia is ESA.

  ESA is used in about 80% of end-stage renal failure patients. In Japan, more than 300,000 patients on maintenance dialysis and patients with renal failure in preservation stage are currently using ESA. In addition to the large number of patients, the EPO market forecast for FY2015 is expected to exceed $ 143 billion worldwide ($ 1.5 billion in Japan), and companies are also actively researching new drugs.

  In chronic kidney injury (CKD), endogenous EPO production decreases as kidney disease progresses, and may cause anemia in combination with decreased nutrition, iron deficiency, tendency to bleed, shortened erythrocyte life span, and the like. Usually, the proportion of anemia patients increases from CKD stage 3, but depending on the primary disease of CKD, there are cases where anemia is present even at an early stage. It is said that anemia is checked every 6 months in stage 3, every 3 months in stage 4, and every month in stage 5 (Non-patent document 1; Non-patent document 2; Non-patent document 3).

  Anemia is an independent exacerbation factor of heart failure (Non-patent document 4; Non-patent document 5), and improvement of life prognosis can be expected by treatment of anemia. Furthermore, in patients with CKD, cardio-renal-anemia syndrome has been proposed in which kidney disease, anemia, and heart disease affect each other (Cardio-Renal-Anemia syndrome) (Non-patent document 4; Non-patent document 5; Non-patent document) Reference 6; Non-patent reference 7; Non-patent reference 8), anemia treatment is recommended.

  Anemia is an independent cause of CKD progression, and it has been reported that progression of CKD is suppressed by improving anemia early by ESA (Non-Patent Document 8; Non-Patent Document 9; Non-Patent Document 10). When anemia is observed in CKD patients, it is claimed that it is necessary to treat actively using ESA or the like.

  However, in the large-scale randomized intervention trial CREATE study (Non-patent document 11) and CHOIR study (Non-patent document 12) reported in the fall of 2006, results that did not match these target values were reported. That is, when comparing the group that maintained the Hb value within the normal range (13.0 to 15.0 g / dL) and the group that maintained the Hb value slightly lower (10.5 to 11.5 g / dL) in the former, In the group improved to the normal range, the incidence of cardiovascular complications did not decrease, and the rate of renal function decline did not differ. In the latter group, the incidence of death, myocardial infarction, hospitalization for heart failure, and stroke increased in the group that maintained Hb at a high level (13.5 g / dL) than the group that maintained it at a low level (11.3 g / dL). Later, even in the results of meta-analysis of 9 RCTs for dialysis patients and patients with chronic renal failure during storage (Non-patent Document 13), there were many total deaths in the high Hb group, poor control of hypertension, occlusion of shunts The rate was reported to be high. The reason why the prognosis worsens in the group in which the Hb value is maintained at a high value is presumed to be due to the exacerbation of hypertension / thrombosis or the direct action due to high dose administration of ESA (Non-patent Document 11; Non-patent document 13). Based on these results, the FDA's instructions were revised, the K / DOQI guidelines were revised, and the target Hb value is now set to 11.0 to 12.0 g / dL (Non-Patent Document 1). Non-patent document 2; Non-patent document 3).

  Thus, it is known that renal anemia is an onset / progress factor of independent CKD and cardiovascular disease (CVD). Therefore, although renal anemia treatment with ESA is expected to contribute to the suppression of CKD progression and the improvement of life prognosis, no clear effect on the renal protective effect or complication prevention has been found. From this, it was suggested that it is not appropriate to evaluate the therapeutic effect of renal anemia only by increasing the Hb value by the 2010 US FDA (Non-patent Document 14).

  ADMA is a modified amino acid in which two methyl groups are added to arginine, which is a kind of amino acid. Arginine methylation is performed on proteins synthesized by translation, one methyl each at the guanidino group nitrogen of the arginine residue incorporated into the protein by protein arginine methyltransferases (PRMT). A group is added. PRMT has type I that asymmetrically methylates arginine residues and type II that symmetrically methylates, and ADMA is produced by type IPRMT (FIG. 1).

  The methyl group of the arginine residue in the protein is introduced by introducing a methyl group into the ω-nitrogen atom present in the δ-guanodino group of the arginine side chain with PRMT using S-adenosylmethionine (AdoMet or SAM) as a methyl group donor. It is produced by catalyzing. PRMT is a highly conserved protein from yeast to human, with four characteristic motifs (motif followed by postI, II, III) and a THW loop. Motif I, postI, and THW loop form a binding pocket with SAM. PRMT is classified into type I and type II due to the difference in the mode of methyl group transfer (FIG. 1). PRMTs belonging to type I are known as PRMT 1, 2, 3, 4, 6, 8 and monomethylate and asymmetrically dimethylate arginine residues in proteins. On the other hand, PRMTs belonging to type II are known as PRMT5 and 7 (there are reports that PRMT7 is a type III arginine methyltransferase that catalyzes only monomethylation), which are monomethylation of arginine residues. And catalyzing symmetrical dimethylation. Both reactions produce S-adenosylhomocysteine (AdoHcy) as a byproduct of the transfer of the (di) methyl group of the protein.

  Proteins with methylated arginine residues are degraded with turnover (FIG. 2). Methylarginines released by this proteolysis are always present in cells and body fluids at a constant concentration. The clearance pathway is excreted in the urine or metabolized in the liver, but monomethylarginine (MMA) and ADMA are hydrolase specific to them, dimethylarginine dimethylamino hydrolase (DDAH). Is decomposed into citrulline and methylamines. When this metabolic balance is disrupted and the ADMA concentration in the body rises, it inhibits the synthesis enzyme (NOS) activity of nitric oxide (NO), which is a vascular relaxation factor derived from vascular endothelium, and reduces the amount of NO production, resulting in vascular endothelial damage. It is known to be involved in the onset and development of various diseases such as arteriosclerosis, hypertension, diabetes, cardiovascular disease and kidney disease (Non-patent Document 15).

  In recent years, the present inventors have clarified that the entire ADMA metabolic system exists in erythrocytes themselves based on previous studies that erythrocytes are rich in methylated proteins and free ADMA (FIG. 3; Patent Literature 16; Non-Patent Literature 17; Non-Patent Literature 18).

  Although methylated arginine derivatives have been identified and isolated over 30 years ago, their physiological role has long remained unknown. Over the past 10 years, PRMT identification and analysis technology to detect methylation has led to methylation of arginine residues involved in a variety of cellular functions such as signal transduction, RNA processing, transcriptional control and DNA repair. It has been shown that

  So far, the use of ADMA includes screening for preeclampsia (Patent Document 1), biomarker for evaluating liver function (Patent Document 2), and determination of chromosome dominant polycystic kidney disease (Patent Document 3). Evaluation of dialysis membrane (Patent Document 4) and biomarker of depression (Patent Document 5) are known, but the correlation with renal anemia is not known.

JP-T-2006-524325 JP 2010-502979 gazette JP 2012-112784 A JP 2012-112785 A JP 2012-221908 A

Japanese Society for Dialysis Therapy, 2008 edition "Guidelines for the Treatment of Renal Anemia in Chronic Kidney Disease" Dialysis Society Journal 41; 2008: 661-716 National Kidney Foundation. K / DOQI clinical practice guidelines and clinical practice recommendations for anemia in chronic kidney disease, Am J Kidney Dis 2006; 47 (Suppl 3): S11-S145 National Kidney Foundation, K / DOQI clinical practice guidelines and clinical practice recommendations for anemia in chronic kidney disease: 2007 Update of Hemoglobin Target, Am J Kidney Dis 2007; 50: 471-530 Vlagopoulos PT, et al., J Am Soc Nephrol 2005; 16: 3403-3410 Al-Ahmad A, et al., J Am Coll Cardiol 2001; 38: 955-962 Hayashi T, et al., Am J Kidney Dis 2000; 35: 250-256 Roth D, et al., Am J Kidney Dis 1994; 24: 777-784 Silverberg DS, et al., J Am Coll Cardiol 2001; 37: 1775-1780 Kuriyama S, et al., Nephron 1997; 77: 176-185 Gouva C, et al., Kidney Int 2004; 66: 753-760 Drueeke TB, et al., N Engl J Med 2006; 355: 2071-2084 Singh AK, et al., N Engl J Med 2006; 355: 2085-2098 Phrommintikul A, et al., Lancet 2007; 369: 381-388 Unger EF, et al. N Engl J Med, 2010, 362 (3); 189-192 Ueda S, et al., J Nephrol., 2010, 23; 377-386 Kang ES, Kimoto M, et al., Free Radic Res, 2001, 35 (6); 693-707 Billecke SS, Kimoto M, et al., Am J Physiol Heart Circ Physiol, 2006, 91; H1788-H1796 Yokoro M, Kimoto M, et al., Biosci Biotechnol Biochem, 2012, 76 (7); 1334-1342

  Until now, there has been no monitoring method for administration suitable for the use of ESA for renal anemia, and the ESA dose is adjusted only at the Hb concentration. As a result, problems such as overdose of ESA in a short period of time, the emergence of ESA hyporesponsive patients, and long-term use of ESA in ESA hyporesponsive patients have arisen. Overdose of ESA may cause hypertension due to an increase in body fluid volume due to an increase in Hb and peripheral vasoconstriction. Moreover, it is used for ESA hyporesponsive patients without knowing, resulting in overdose.

  The number of patients with chronic renal failure is steadily increasing, and the number of patients receiving ESA is also increasing. The demand for ESA is increasing all over the world, and it always occupies the top in the statistics of drug sales. For this reason, not only in Japan but also in many countries, the burden on the medical economy is large, and the establishment of a biomarker that can properly use ESA and the creation of a monitoring method that can grasp renal anemia treatment are urgent issues. In addition, when developing a new therapeutic agent for renal anemia, an appropriate index other than Hb is required.

  The present inventors considered that ESA can be properly used by establishing a biomarker for renal anemia, and focused on erythrocyte ADMA as a factor causing renal anemia. As a result, there is a correlation between the erythrocyte ADMA concentration and the Hb value. Therefore, erythrocyte ADMA can be used as a marker for renal anemia in CKD patients, and erythrocyte ADMA concentration can be used as an index to determine proper use of ESA. I found it applicable. Also, erythrocyte ADMA levels in patients already undergoing ESA treatment show a significant positive correlation with ESA low responsiveness index, thus erythrocyte ADMA levels reflect ESA low responsiveness and erythrocyte ADMA levels decrease Has been found to be a useful indicator of improving ESA low reactivity. Furthermore, since the red blood cell ADMA concentration showed a strong positive correlation with NT-proBNP, which is known as a risk factor for heart disease, the red blood cell ADMA concentration can predict not only the degree of anemia but also the risk of heart disease complications. The present inventors have found that it is a useful marker for the determination of anemia treatment with an effect of preventing disease complication.

  That is, the present invention relates to the use of ADMA as a biomarker for monitoring the onset of renal anemia based on the correlation between erythrocyte ADMA and renal anemia. According to the present invention, ADMA as a biomarker can be used for (i) an indicator of proper use of ESA, (ii) discrimination of ESA hyporesponsive patients, and (iii) prediction of risk of heart disease complications.

The present invention includes the following.
[1] Use of ADMA as a biomarker to monitor the following (1) to (3):
(1) development of renal anemia in mammals;
(2) ESA hyporesponsiveness in mammals undergoing ESA treatment; or (3) Onset of heart disease in mammals.
[2] Use of ADMA as a biomarker of [1] above, wherein ADMA is ADMA present in erythrocytes of a blood sample obtained from a mammal or the mammal undergoing ESA treatment.
[3] The method according to (1) to (3) below, comprising measuring or monitoring the ADMA concentration in erythrocytes of a blood sample obtained from a mammal:
(1) Test method for renal anemia in mammals;
(2) A method for monitoring ESA hyporesponsiveness in mammals treated with ESA; or (3) A method for examining heart disease in mammals.
[4] The monitoring kit according to the following (1) to (3), including means for measuring the ADMA concentration in erythrocytes of a blood sample obtained from a mammal:
(1) a kit for monitoring renal anemia in mammals;
(2) A kit for monitoring low ESA reactivity in mammals treated with ESA; or (3) a kit for monitoring the onset of heart disease in mammals.
[5] The kit for monitoring renal anemia in a mammal according to [4] above, a means for receiving a blood sample from the mammal and a means for measuring the ADMA concentration in erythrocytes contained in the blood sample were measured. The method according to [4] above, comprising means for comparing the ADMA concentration with a reference value, and means for determining whether the mammal has developed renal anemia and / or the degree of renal anemia from the comparison with the reference value. Kit for monitoring the onset of renal anemia in mammals.
[6] The kit for monitoring low ESA reactivity in mammals treated with ESA as described in [4] above is obtained from a means for receiving a blood sample from a patient undergoing ESA treatment and a patient undergoing ESA treatment. Means for monitoring the ADMA concentration in red blood cells contained in a blood sample, means for comparing the measured ADMA concentration with a reference value, and means for determining whether the patient exhibits low ESA reactivity from a comparison with the reference value A kit for monitoring low ESA reactivity in mammals treated with ESA according to the above [4], comprising:
[7] The monitor kit for developing heart disease in a mammal according to [4] above, a means for receiving a blood sample from the mammal, and a means for measuring the ADMA concentration in red blood cells contained in the blood sample were measured. The kit for monitoring the onset of heart disease in a mammal according to the above [4], comprising means for comparing the ADMA concentration with a reference value, and means for determining whether the mammal has developed a heart disease from the comparison with the reference value. .
[8] The monitoring method according to (1) to (3), comprising screening for an increase in ADMA concentration in erythrocytes of a blood sample obtained from a mammal:
(1) A method for monitoring the onset of renal anemia in mammals;
(2) A method for monitoring low ESA reactivity in mammals treated with ESA; or (3) a method for monitoring the onset of heart disease in mammals.
[9] The method for monitoring the onset of renal anemia as described in [8] above, wherein an increase in ADMA concentration correlates with a decrease in hemoglobin (Hb) concentration.
[10] The method for monitoring the onset of renal anemia as described in [8] above, wherein an increase in ADMA concentration correlates with a decrease in iron availability for hematopoiesis.
[11] The method for monitoring the onset of renal anemia as described in [8] above, wherein an increase in ADMA concentration correlates with a decrease in serum iron and / or transferrin saturation (TSAT).
[12] The method for monitoring the onset of heart disease according to [8] above, wherein an increase in ADMA concentration correlates with an increase in NT-proBNP concentration.
[13] A method for diagnosing renal anemia or heart disease in a mammal, comprising screening for an increase in the concentration of ADMA in the red blood cells of the mammal.
[14] An excess of ESA in a mammal comprising screening for an increase in ADMA concentration in a red blood cell of a mammal and reducing the dosage of ESA to an appropriate value if the ADMA concentration in the red blood cell exceeds a reference value A method of administering ESA to prevent administration.
[15] The method for monitoring low ESA responsiveness of a mammal treated with ESA as described in [8] above comprises determining ESA treatment resistance, wherein ESA low in a mammal treated with ESA as described in [8] How to monitor reactivity.
[16] The use, method or kit according to any one of [1] to [8], or [13] to [15] above, wherein the mammal is a human.

  The first aspect of the present invention relates to the use of ADMA as a biomarker to monitor the following (1) to (3): (1) Development of renal anemia in mammals; (2) ESA ESA hyporesponsiveness in mammals receiving treatment; or (3) development of heart disease in mammals. Here, ADMA is ADMA present in erythrocytes of a blood sample obtained from a mammal or said mammal undergoing ESA treatment.

  A second aspect of the present invention relates to a method according to the following (1) to (3), comprising measuring or monitoring the ADMA concentration in erythrocytes of a blood sample obtained from a mammal: (1) A method for examining renal anemia in mammals; (2) a method for monitoring ESA hyporeactivity in mammals treated with ESA; or (3) a method for examining heart disease in mammals.

  The third aspect of the present invention relates to the monitoring kit according to the following (1) to (3), comprising means for measuring the ADMA concentration in erythrocytes of a blood sample obtained from a mammal: (1) A kit for monitoring the onset of renal anemia in mammals; (2) a kit for monitoring low ESA reactivity in mammals treated with ESA; or (3) a kit for monitoring the onset of heart disease in mammals.

  The kit for monitoring onset of renal anemia in a mammal according to the present invention comprises a means for receiving a blood sample from a mammal, a means for measuring the ADMA concentration in erythrocytes contained in the blood sample, and the measured ADMA concentration as a reference value. Means for comparing and means for determining whether the mammal has developed renal anemia and / or the extent of renal anemia from comparison with a reference value may be included.

  An ESA hyporesponsiveness monitoring kit for mammals treated with ESA according to the present invention is included in a means for receiving a blood sample from a patient undergoing ESA treatment and a blood sample obtained from a patient undergoing ESA treatment. Means for monitoring the ADMA concentration in the red blood cells, means for comparing the measured ADMA concentration with a reference value, and means for determining whether the patient exhibits low ESA reactivity from a comparison with the reference value.

  The monitor kit for the onset of heart disease in mammals according to the present invention comprises means for receiving a blood sample from a mammal, means for measuring ADMA concentration in erythrocytes contained in the blood sample, and the measured ADMA concentration as a reference value. Means for comparing and means for determining whether the mammal has developed a heart disease from a comparison with a reference value may be included.

  A fourth aspect of the present invention relates to the monitoring method according to (1) to (3), comprising screening for an increase in ADMA concentration in erythrocytes of a blood sample obtained from a mammal: (1) A method for monitoring the onset of renal anemia in mammals; (2) a method for monitoring low ESA reactivity in mammals treated with ESA; or (3) a method for monitoring the onset of heart disease in mammals. Here, in the method for monitoring the onset of renal anemia, an increase in ADMA concentration correlates with a decrease in hemoglobin (Hb) concentration. In the method for monitoring the onset of renal anemia, an increase in ADMA concentration is also correlated with a decrease in iron availability for hematopoiesis. Furthermore, in the method for monitoring the onset of renal anemia, an increase in ADMA concentration is also correlated with a decrease in serum iron and / or transferrin saturation (TSAT). Preferably, the method for monitoring low ESA responsiveness in a mammal treated with ESA comprises discriminating resistance to ESA treatment. In the method for monitoring the onset of heart disease, an increase in ADMA concentration correlates with an increase in NT-proBNP concentration.

  A fifth aspect of the present invention relates to a method for diagnosing renal anemia or heart disease in a mammal comprising screening for an increase in the concentration of ADMA in the red blood cells of the mammal.

  A sixth aspect of the invention comprises screening for an increase in ADMA concentration in the red blood cells of a mammal and reducing the dosage of ESA to an appropriate value if the ADMA concentration in the red blood cells exceeds a reference value. The present invention relates to an ESA administration method for preventing overdosage of ESA in a mammal.

  The present invention also includes a method for simultaneous examination of renal anemia and heart disease, comprising measuring the concentration of ADMA in red blood cells of a blood sample obtained from a mammal.

  A further aspect of the present invention also includes a kit for simultaneously monitoring the onset of renal anemia and the onset of heart disease, comprising means for measuring the ADMA concentration in red blood cells contained in a blood sample obtained from a mammal.

  Furthermore, the present invention also relates to a method for discriminating an ESA-resistant patient in renal anemia, which comprises monitoring ADMA concentration in erythrocytes contained in a blood sample obtained from a patient undergoing ESA treatment. , Regarding the method.

  Since erythrocyte ADMA has a strong correlation with renal anemia independently of EPO, it can be a renal anemia biomarker other than a decrease in EPO production. In addition, erythrocyte ADMA significantly increases when ESA is excessively administered or when ESA is less reactive, so it is optimal as an indicator of proper use of ESA in renal anemia. As a result, ESA treatment resistant patients in renal anemia can be identified. Also, by monitoring red blood cell ADMA when using ESA, the amount of ESA is optimized, leading to a reduction in ESA treatment-resistant patients and medical costs. Furthermore, when developing a new therapeutic drug for renal anemia, an appropriate index other than Hb is required, and erythrocyte ADMA can contribute as a marker for the development of a new drug.

FIG. 1 is a schematic diagram showing the process of generating ADMA from arginine. FIG. 2 is a schematic diagram showing the metabolic pathway of ADMA and a decrease in NO production. FIG. 3 is a schematic diagram showing the ADMA metabolic pathway in erythrocytes. FIG. 4 is a diagram showing the relationship between red blood cell ADMA concentration and anemia. FIG. 5 is a diagram showing the correlation between the red blood cell ADMA concentration and the iron metabolism index. (A) shows the correlation between erythrocyte ADMA concentration and serum iron, and (B) shows the correlation between erythrocyte ADMA concentration and TSAT. FIG. 6 is a diagram showing a correlation between erythrocyte ADMA concentration and NT-proBNP which is a heart disease marker. FIG. 7 is a graph showing the relationship between erythrocyte ADMA concentration and ESA low reactivity after ESA treatment. FIG. 8 is a diagram showing a comparison of ADMA concentrations in serum and erythrocytes between healthy subjects and CKD patients. FIG. 9 is a diagram showing the expression of erythroblast-related genes in CKD model mice. FIG. 10 is a diagram showing erythropoietin receptor inhibition through erythropoietin receptor expression inhibition by erythropoietin signal and ADMA accumulation. FIG. 11 is a diagram showing comparison of measured values by LC-MS and ELISA.

  The present invention relates to the use of ADMA as a biomarker for renal anemia. Specifically, it relates to the use of ADMA as a biomarker to monitor the following (1) to (3): (1) development of renal anemia in mammals; (2) erythropoiesis stimulating agent formulation (ESA) ) Low ESA responsiveness in mammals undergoing treatment treatment; or (3) development of heart disease in mammals.

  Hereinafter, the present invention will be described in detail. The following embodiment is an example for explaining the present invention, and is not intended to limit the present invention to this embodiment alone. The present invention can be implemented in various forms without departing from the gist thereof.

A. Renal anemia In the present invention, renal anemia refers to anemia caused by a decrease in EPO production ability in the kidney due to renal injury. Causes of renal anemia include factors such as uremic toxins, endotoxin, shortening of erythrocyte life due to inflammation, decreased EPO reactivity of hematopoietic cells, nutritional disorders, and residual blood in the circuit in hemodialysis (HD) patients . The standard of decrease in glomerular filtration rate (GFR) in which the frequency of renal anemia increases rapidly is as follows: serum creatinine (Cr) value ≧ 2 mg / dL or creatinine clearance (Ccr) value <20 to 35 mL / min (CKD stage 4 ~ 5) (Hakim RM et al., Am J Kidney Dis 11: 238-247, 1988; Chandra M et al., J Pediatr 113: 1015-1021, 1988; Chandra M et al., J Pediatr).

  The physiological Hb value of a healthy person varies depending on age, sex, race, and the like. Therefore, it is necessary to set the diagnostic criteria for anemia in consideration of these factors. When diagnosing anemia, it is necessary to distinguish various diseases that cause anemia. At that time, classification by mean red blood cell volume (MCV) is useful. The main cause of renal anemia is a decrease in EPO production associated with renal disorder, and is diagnosed for the first time when no other anemia-causing disease is observed.

  The diagnostic criteria for anemia in Japanese are: Hb value <13.5 g / dL (hematocrit (Ht) value <40%) for adult men, Hb value <11.5 g / dL (Ht value <35%) for adult women It is.

  EPO-resistant anemia, on the other hand, is not strictly defined, but as a rule, even if hemodialysis patients receive rHuEPO maximum dose 9,000 IU / week and CAPD patients receive rHuEPO maximum dose 6,000 IU / week. Patients with severe anemia that cannot be maintained at a value ≧ 25%, or hemodialysis patients with a maximum dose of 9,000 IU / week and CAPD patients with a maximum dose of 6,000 IU / week, Ht value ≧ 6% It is assumed that there is no increase in [Hb value ≧ 2 g / dL].

Causes of decreased EPO responsiveness include: (1) decreased red blood cell production (iron deficiency (most common cause), chronic infection (IL-1, TNF), malignant tumor complications, myelofibrosis, advanced secondary Hyperparathyroidism, aluminum accumulation (involved in resistance in the early stage of treatment), insufficient dialysis, nutritional deficiency (especially folic acid, vitamin B ₆ , B ₁₂ deficiency), use of drugs with bone marrow function inhibitory action), And (2) loss of red blood cells (bleeding (apparent, latent), large amount of blood sampling, advanced hypersplenism (combined with cirrhosis, etc.), hyperhemolysis (artificial valve, etc.), residue when dialyzer is reused ).

B. Erythropoiesis stimulating factor preparation (ESA)
The first-line drug for renal anemia is ESA. Currently available recombinant human erythropoietin preparations are epoetin α and epoetin β. Later, second generations such as darbepoetin alpha also appeared in the recombinant human erythropoietin preparation, and accordingly, the erythropoiesis stimulating factor preparation (ESA) including the second generation instead of the name human erythropoietin preparation (EPO). The name is now used.

  The dose and the number of doses should be determined in consideration of the type of ESA, the Hb value at the start of administration, the target value for improving anemia, the predicted or target rate of improving anemia, and the like.

  The monitoring method of the present invention can be used for any ESA. In adjusting the dosage of each ESA described below, the erythrocyte ADMA concentration can be used in place of the Hb concentration according to the present invention.

1. Epoetin alpha and epoetin beta
Epoetin α and epoetin β are administered to renal anemia as an initial dose 3 times a week, 1500 units once. Thereafter, the dose and administration frequency are adjusted so that the Hb concentration is maintained at 10 to 11 g / dL (30 to 33% in terms of hematocrit value). Epoetin α and epoetin β are intravenously injected from the blood circuit at the end of dialysis in principle. The upper limit of the dose of epoetin α or epoetin β is usually 3000 units three times a week. Epoetin alfa (genetical recombination) epoetin alfa is commercially available as Espoo (Kyowa Hakko Kirin). Epoetin beta (genetical recombination) epoetin beta is marketed as Epogin (Chugai Pharmaceutical).

2. Darbepoetin alfa
Darbepoetin α is a recombinant human erythropoietin preparation in which 5 of 165 amino acid residues of epoetin α are replaced with another amino acid residue, and two sugar chains are further added. The half-life of darbepoetin α is 25.3 hours, which is three times the half-life of epoetin α (8.5 hours). Therefore, with darbepoetin α, the Hb concentration (red blood cell ADMA concentration according to the present invention) can be maintained in the target range with a lower administration frequency (once a week to once every 2 weeks) than epoetin α and epoetin β.

  Darbepoetin α was previously supposed to be used by switching from these preparations in patients who have already used epoetin α or epoetin β. However, from August 2010, darbepoetin α may be used from the beginning for renal anemia. Darbepoetin alfa starts at 20 μg once a week. The upper limit of the dose of darbepoetin α is often 60 μg once a week. Darbepoetin alfa is also slowly injected intravenously from the blood circuit at the end of dialysis. Darbepoetin alfa is commercially available as Nesp (Kyowa Hakko Kirin).

3. Epoetin beta pegol Epoetin nesp beta pegol is a recombinant human erythropoietin preparation in which one molecule of linear methoxypolyethylene glycol (PEG) molecule is chemically bound to epoetin β. By such chemical modification, the half-life of epoetin beta pegol is 168 to 217 hours, about 20 times the half-life of epoetin beta 9.4 hours, and the half-life of darbepoetin alfa 32 to 49 hours. It became about 4 times. Therefore, epoetin beta pegol can increase the Hb concentration (red blood cell ADMA concentration according to the present invention) to an optimal range by administration once every four weeks (initially once every two weeks). Epoetin beta pegol is commercially available as Mircera (Chugai Pharmaceutical).

a. Initial dose Epoetin beta pegol is initially administered as an intravenous dose of 50 μg once every two weeks. If the Hb concentration does not increase moderately (according to the present invention, the erythrocyte ADMA concentration does not decrease moderately), the dose of epoetin beta pegol is increased. Since the hematopoietic effect of epoetin beta pegol persists for a long time, the transition of Hb concentration (red blood cell ADMA concentration according to the present invention) was sufficiently observed after the start of administration of epoetin beta pegol, and the effect was insufficient. If the effect is greater than expected, consider increasing or decreasing the dose as soon as possible. In principle, when increasing the dose of epoetin beta pegol, the dose is increased step by step according to Table 1.

Table 1: Administration stages for increasing the dose of epoetin beta pegol

  Once the target Hb concentration is obtained and stabilized during the initial administration period, the epoetin beta pegol administration interval can be extended. In such a case, the dose per administration is doubled and the administration interval is changed from once every two weeks to once every four weeks. If the Hb concentration (red blood cell ADMA concentration according to the present invention) cannot be maintained within the target range at a dose interval of once every 4 weeks, the dose is halved and returned to the dose interval once every 2 weeks. .

b. Initial dose when switching from epoetin α or epoetin β to epoetin beta pegol When switching from epoetin α or epoetin β to epoetin beta pegol, the transition of Hb concentration (red blood cell ADMA concentration according to the present invention) is stable. 100 μg of epoetin beta pegol for patients with a weekly dose of epoetin alfa or epoetin β of less than 4,500 IU, 150 μg for patients with a dose of 4,500 IU or more once every 4 weeks Administer intravenously. There is no report on the initial dose when switching from darbepoetin alfa to epoetin beta pegol.

c. Maintenance dose When the target Hb concentration (red blood cell ADMA concentration according to the present invention) is obtained and the concentration is stable, the epoetin beta pegol dose at that time is taken as the maintenance dose. The upper limit of the maintenance dose is 250 μg at a time.

  The target for the treatment of renal anemia is that the target Hb value for renal anemia in chronic kidney disease during the conservative period is 11 g / dL or more, and the administration start criteria for ESA is the time when the Hb value is less than 11 g / dL in multiple tests. And Since excessive improvement of anemia may lead to a worsening of the prognosis of life, such as adverse effects caused by administration of high doses of ESA, the ESA weight loss / drug withdrawal standard is set at over 13 g / dL. The upper limit for patients who already have cardiovascular complications or who are considered medically necessary is limited to 12 g / dL. In the present invention, the erythrocyte ADMA concentration can be monitored instead of these Hb values.

C. ADMA
ADMA is a modified amino acid in which two methyl groups are added to arginine, which is a kind of amino acid. ADMA is an endogenous inhibitor of nitric oxide (NO) producing enzyme. ADMA is formed in the process of degrading methylated protein and then excreted in the kidney or metabolized by dimethylarginine dimethylaminohydrolase (DDAH). Many cell types such as human endothelial cells and tubular cells have the ability to synthesize and metabolize ADMA. Increased blood levels of ADMA correlate with a number of diseases associated with endothelial dysfunction. For example, elevated blood ADMA levels in dialysis patients correlate with arteriosclerosis and cardiovascular disease risk. Furthermore, an increase in ADMA concentration has been confirmed in patients with hypercholesterolemia, hypertension, arteriosclerosis, chronic renal failure, chronic heart failure and the like, and is considered to be related to the restriction of endothelial vasodilation. In recent years, ADMA has been shown to be an important new cardiovascular risk factor.

  In the present invention, the erythrocyte ADMA concentration is used as an index of the onset / degree of renal anemia. To date, it has not been known that erythrocyte ADMA concentration correlates with renal anemia.

D. Monitoring of Erythrocyte ADMA Concentration To measure erythrocyte ADMA concentration, an erythrocyte sample is first prepared and then ADMA concentration measurement is performed. The red blood cell sample is prepared as follows. Whole blood collected from heparin is centrifuged (3,000 rpm, 4 ° C., 10 minutes), and plasma is collected. Add 2 volumes of PBS to the blood cell fraction, mix gently by inversion, and centrifuge (3,000 rpm, 4 ° C., 10 minutes). Remove PBS and buffy coat to make red blood cell fraction. An equal amount of 0.1 M phosphate buffer is added to the erythrocyte fraction to cause hemolysis (stored at −80 ° C. in the case of storage). The hemolyzed sample is centrifuged (12,000 rpm, 4 ° C., 20 minutes), and the supernatant is used as an erythrocyte sample.

  The ADMA concentration measurement is performed as follows. A sample (10 uL) is taken into a tube. Add 0.1% FA-acetonitrile (70 uL) and IS (internal standard) (20 uL). Stir with a mixer (30 seconds), then stir on an ultrasonic water bath (5 minutes). After centrifugation (16,400 × g, 4 ° C., 10 minutes), the supernatant is passed through a filter (0.2 μm) and analyzed by LC / MS / MS. Moreover, it can replace with LC / MS / MS and can also measure by ELISA. When measuring by ELISA, it is performed as follows. Add an equal volume of 10% trichloroacetic acid to an erythrocyte sample (100 uL), mix, centrifuge (15,000 rpm, 4 ° C., 20 minutes), take the supernatant (80 uL) in a new tube, and add an appropriate amount of 1M disodium hydrogen phosphate. Neutralize with a solution (pH 12) to obtain a measurement sample. 0.1M phosphate buffer (50 uL) and 0.1MN-succinimidyl 3-maleimidobenzoate / DMSO (20 uL) were added to the measurement sample and ADMA solution for calibration curve (each 10 uL), stirred for 30 minutes, and then centrifuged (15 Using a supernatant obtained at 1,000 rpm (4 ° C., 30 minutes), measurement is performed by competitive inhibition ELISA.

  Once the erythrocyte ADMA concentration is measured, the onset and extent of renal anemia can be determined from the correlation with the Hb concentration, and the amount of ESA used can be made appropriate, thereby allowing ESA overdose and low ESA response. Long-term use in sex patients can be avoided. Specifically, for example, when treatment is performed with epoetin α or epoetin β, the dose and frequency of administration so that the Hb concentration is maintained at 10 to 11 g / dL (30 to 33% in terms of hematocrit value). This can be done by monitoring the red blood cell ADMA concentration. The same applies to darbepoetin alfa and epoetin beta pegol. In the case of epoetin beta pegol, the hematopoietic effect lasts for a long time, so after starting administration of epoetin beta pegol, the transition of erythrocyte ADMA concentration is sufficiently observed, the effect is insufficient, or the effect is more than expected If so, consider increasing or decreasing the dose as soon as possible. In principle, when increasing the dose of epoetin beta pegol, the dose is increased step by step according to Table 1.

  EPO-resistant anemia is not strictly defined, but as a rule it is Ht value ≧ 9 000 IU / week for rHuEPO in hemodialysis patients and 6,000 IU / week for rHuEPO in CAPD patients ≧ Those with severe anemia that cannot be maintained at 25%, or hemodialysis patients with a maximum dose of 9,000 IU / week and CAPD patients with a maximum dose of 6,000 IU / week, Ht value ≧ 6% [Hb Value ≧ 2 g / dL].

  According to the present invention, it was found that erythrocyte ADMA concentration in patients already undergoing ESA treatment showed a significant positive correlation with ESA low reactivity index. From these results, the erythrocyte ADMA concentration reflects ESA low reactivity, and a decrease in erythrocyte ADMA concentration is a useful index for improving ESA low reactivity. Here, the ESA resistant index is expressed by ESA dose / week / Hb / BW, and is a value obtained by correcting the amount of ESA used per week by Hb value and body weight.

  Renal anemia is not only a CKD progression but also a risk factor of heart disease, and it is important to reflect both the degree of anemia and the risk of heart disease in grasping the disease state and determining the therapeutic effect due to renal anemia. Erythrocyte ADMA concentration showed a strong positive correlation with NT-proBNP, which is known as a risk factor for heart disease. This suggests that the erythrocyte ADMA concentration can predict not only the degree of anemia but also the risk of heart disease complications, and is considered to be a useful marker for determination of anemia treatment with the effect of preventing heart disease complications.

E. Kit The present invention also provides a kit for monitoring the onset of renal anemia in mammals and a kit for monitoring the onset of heart disease in mammals.

  The kit for monitoring the onset of renal anemia and / or heart disease according to the present invention includes a means for measuring the ADMA concentration in erythrocytes contained in a blood sample obtained from a mammal. The kit for monitoring onset of renal anemia according to the present invention also includes means for receiving a blood sample from a mammal, means for comparing the measured ADMA concentration with a reference value, and comparison with the reference value to develop renal anemia. And / or means for determining the extent of renal anemia and / or developing heart disease.

  The ESA hyporesponsiveness monitoring kit for patients undergoing ESA treatment according to the present invention includes a means for monitoring the ADMA concentration in red blood cells contained in a blood sample obtained from a patient undergoing ESA treatment. An ESA hyporesponsiveness monitoring kit in patients undergoing ESA treatment according to the present invention also includes means for receiving a blood sample from a patient undergoing ESA treatment, means for comparing the measured ADMA concentration to a reference value, and Means may be included for determining whether the patient exhibits low ESA responsiveness from a comparison with a reference value.

As for the above-mentioned standard value, there is no clear judgment standard for ESA low responsiveness at present, and the Japanese Society for Dialysis Medicine, 2008 edition “Guideline for the Treatment of Renal Anemia in Chronic Kidney Disease” also has a clear definition of ESA low responsiveness. Does not exist ", but as a guideline, it is defined as follows.
“HD (hemodialysis) patients: rHuEPO is 3,000 units once a week (9,000 units per week) intravenously, and DA is 60 mg once a week, anemia can be improved. When the target Hb value cannot be achieved, the response to ESA therapy is low.
PD (peritoneal dialysis) patients: rHuEPO is 6,000 units once a week (6,000 units per week) subcutaneously, and DA is 60 mg once a week intravenously. When the target Hb value cannot be achieved, the response to ESA therapy is low.
ND (conservative chronic kidney disease) patients: ND patients do not improve anemia even if rHuEPO is used subcutaneously at 6,000 units once a week (6,000 units per week), and target Hb When the value cannot be achieved, rHuEPO therapy is hyporesponsive. "

In the present invention, ADMA in erythrocytes can be derivatized and measured by competitive ELISA. For example, a kit according to the present invention may include:
A 96-well plate coated with ADMA-SMB-BSA (composite of ADMA and bovine serum albumin (BSA) cross-linked with N-succinimidyl 3-maleimidobenzoate (SMB)) SMB-ADMA preparation for preparing a calibration curve -SMB preparation for derivatizing ADMA in the sample-Primary antibody; Mouse monoclonal antibody that recognizes ADMA-SMB-Secondary antibody: HRP-labeled anti-mouse IgG antibody-Color reagent-Centrifugal ultrafiltration if necessary Unit (for deproteinization of erythrocyte hemolyzed sample)

A calibration curve can be created by determining the inhibition rate of each concentration with an OD value of 0 nM as 100% for ADMA in a concentration range of 0 to 500 nM.
F. Diagnostic Method The present invention further comprises a method for diagnosing renal anemia in a mammal, comprising screening for an increase in ADMA concentration in mammalian red blood cells, and a screening for an increase in ADMA concentration in mammalian red blood cells. A method for diagnosing a heart disease in a mammal is also provided.

  In accordance with the present invention, erythrocyte ADMA concentration is negatively correlated with renal anemia, so the erythrocyte ADMA concentration in a blood sample from a subject is measured and renal anemia is determined based on the erythrocyte ADMA concentration exceeding a certain reference value. Diagnosis can be made.

  Blood concentrations of B-type natriuretic peptide (BNP) and N-terminal pro-BNP (NT-proBNP) increase from the early onset of acute coronary syndrome (ACS). BNP and NT-proBNP are biomarkers of cardiac function and heart failure Known as. According to the present invention, it was found that erythrocyte ADMA concentration has a strong positive correlation with NT-proBNP, which is known as a risk factor for heart disease. Therefore, the red blood cell ADMA concentration in the blood sample from the subject can be measured, and a heart disease can be diagnosed based on the red blood cell ADMA concentration exceeding a certain reference value. Here, examples of heart diseases that can be diagnosed according to the present invention include heart failure, acute coronary syndrome (ACS), acute myocardial infarction, and the like.

  In the present invention, “mammal” includes humans, horses, cows, cats, dogs, pigs, goats, sheep, rabbits and the like, with humans being preferred.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.

Correlation between erythrocyte ADMA concentration and anemia The correlation between erythrocyte ADMA concentration and anemia was examined in 61 conservative CKD patients (<eGFR 60 ml / min / 1.73 m ² ). Of these, 57 patients were investigated with the exclusion of patients with strong inflammation and severe liver damage and 4 patients who had been diagnosed with acute kidney injury at that time or most recently, after surgery.

  Blood and urine were collected at the time of morning blood collection. The ADMA concentration was measured by LC-MS / MS analysis at Tohoku University. The concentration in erythrocytes was corrected with the protein concentration measured by the BCA method, and expressed in mol per g protein.

1. Method for Measuring Red Blood Cell ADMA (1) Preparation of Red Blood Cell Sample Whole blood (10 ml) collected from heparin was centrifuged (3,000 rpm, 4 ° C., 10 minutes), and plasma was collected. Two times the amount of PBS was added to the blood cell fraction, mixed gently by inversion, and centrifuged (3,000 rpm, 4 ° C., 10 minutes). PBS and buffy coat were removed to obtain an erythrocyte fraction. An equal amount of 0.1 M phosphate buffer was added to the red blood cell fraction (0.2 ml) to cause hemolysis (stored at −80 ° C. in the case of storage). The hemolyzed sample (0.4 ml) was centrifuged (12,000 rpm, 4 ° C., 20 minutes), and the supernatant was used as an erythrocyte sample.

(2) ADMA concentration measurement A sample (10 uL) was collected in a tube. 0.1% FA-acetonitrile (70 uL) and IS (internal standard) (20 uL) were added. The mixture was stirred with a mixer (30 seconds) and then stirred on an ultrasonic water bath (5 minutes). After centrifugation (16,400 × g, 4 ° C., 10 minutes), the supernatant was passed through a filter (0.2 μm) and analyzed by LC / MS / MS.

(3) Relationship between erythrocyte ADMA concentration and anemia In order to see the relationship between erythrocyte ADMA concentration and anemia in patients with conservative CKD, the erythrocyte ADMA concentration is plotted on the horizontal axis and the Hb value is plotted on the vertical axis. It was. The result is shown in FIG. As is clear from FIG. 4, a significant negative correlation was found in which the Hb value decreased as the erythrocyte ADMA concentration increased. From this, the erythrocyte ADMA concentration was considered to be an indicator of anemia.

  At this time, since the erythrocyte ADMA concentration also showed a correlation with eGFR, the erythrocyte ADMA concentration was divided by the median, and binomial logistic regression analysis was performed. The results are shown in Table 2.

表２から明らかなように、赤血球ＡＤＭＡ濃度は年齢や性別、ＢＭＩ、トータルコレステロール、ｅＧＦＲよりも強くＨｂと関係することがわかった。 Table 2: Binomial logistic regression analysis (Low rADMA <7.09 vs High rADMA> 7.09)

As is clear from Table 2, the erythrocyte ADMA concentration was found to be more strongly related to Hb than age, sex, BMI, total cholesterol, and eGFR.

Correlation between erythrocyte ADMA and iron metabolism index In order to clarify the reason that the erythrocyte ADMA concentration reflects the Hb value, an association with iron metabolism, which is an anemia exacerbation factor, was confirmed. Specifically, the correlation between erythrocyte ADMA concentration and serum iron and transferrin saturation (TSAT) was examined.

(1) Measurement of serum iron Whole blood (10 ml) collected by plain was centrifuged (3,000 rpm, 4 ° C., 10 minutes), and serum was collected. Using an automatic analyzer LABOSPECT008 (Hitachi High Technology) and a reagent L-type Wako Fe (Wako Pure Chemical Industries, Ltd.), the measurement was performed using a serum sample (7.5 ul).

(2) Measurement of TSAT Whole blood (10 ml) collected by plain was centrifuged (3,000 rpm, 4 ° C., 10 minutes), and serum was collected. Using an automatic analyzer LABOSPECT008 (Hitachi High Technology) and reagent L-type Wako UIBC (Wako Pure Chemical Industries, Ltd.), using serum sample (7.5 ul), UIBC (unsaturated iron binding capacity) ) Was measured. TSAT was calculated by the formula of serum iron / (serum iron + UIBC).

(3) Correlation between erythrocyte ADMA concentration and serum iron and TSAT Correlation between erythrocyte ADMA concentration and serum iron and TSAT is shown in FIG. As is clear from the results shown in FIG. 5, the erythrocyte ADMA concentration showed a significant negative correlation with serum iron (panel (A)) and TSAT (panel (B)). This suggests that an increase in erythrocyte ADMA concentration is associated with a decrease in iron utilization for hematopoiesis and reflects an exacerbation of anemia.

Correlation between erythrocyte ADMA and NT-proBNP (cardiac disease marker) Renal anemia is not only CKD progression but also a risk factor for heart disease. The degree of anemia and heart disease in understanding the pathological condition and treatment effect due to renal anemia Reflecting both risks was considered important. Therefore, the correlation between erythrocyte ADMA concentration and NT-proBNP, which is known as a risk factor for heart disease, was examined.

(1) Measurement of NT-proBNP Whole blood (10 ml) collected by plain was centrifuged (3,000 rpm, 4 ° C., 10 minutes), and serum was collected. NT-proBNP was measured using a serum sample (40 ul) using an immune analyzer Spherelite Wako (Wako Pure Chemical Industries, Ltd.) and a dedicated reagent Spherelite proBNP (Sanyo Chemical Industries).

(2) Correlation between erythrocyte ADMA concentration and NT-proBNP The correlation between erythrocyte ADMA concentration and NT-proBNP was examined. The result is shown in FIG. As is clear from the results shown in FIG. 6, the erythrocyte ADMA concentration showed a strong positive correlation with NT-proBNP, which is known as a risk factor for heart disease. This suggests that the erythrocyte ADMA concentration can predict not only the degree of anemia but also the risk of complications of heart disease, and is considered to be a useful marker for anemia treatment determination having an effect of preventing complications of heart disease.

表３から明らかなように、腎機能の影響を除いてもＮＴ−ｐｒｏＢＮＰは赤血球ＡＤＭＡ濃度と強い相関関係が認められた。 Similarly to Hb, binomial logistic regression analysis was performed by dividing the erythrocyte ADMA concentration by the median. The results are shown in Table 3.
Table 3: binomial logistic regression analysis (Low rADMA <7.09 vs High rADMA> 7.09)

As is apparent from Table 3, even when the influence of renal function was excluded, NT-proBNP was strongly correlated with the erythrocyte ADMA concentration.

Correlation between erythrocyte ADMA concentration and ESA low responsiveness index in patients already undergoing ESA treatment Correlation between erythrocyte ADMA concentration and ESA resistant index in patients already undergoing ESA treatment I investigated. The ESA low reactivity index was defined as ESA dose / week / Hb / BW. That is, the ESA low reactivity index is a value obtained by correcting the amount of ESA used per week by the Hb value and the body weight. The result is shown in FIG.

  As is clear from the results shown in FIG. 7, it was found that the erythrocyte ADMA concentration in a patient who had already undergone ESA treatment showed a significant positive correlation with the ESA low reactivity index. From this, the erythrocyte ADMA concentration reflects ESA low reactivity, and a decrease in erythrocyte ADMA concentration is a useful index for improving ESA low reactivity.

31 healthy subjects (male / female; 24/7, mean age ± standard deviation; 39.7 ± 7.9 years old) who have no obvious disease comparing the ADMA concentration in erythrocytes between the serum of healthy subjects and CKD patients The plasma ADMA concentration and erythrocyte ADMA concentration of 54 CKD patients were compared. Preparation of erythrocyte sample and measurement of ADMA concentration were carried out in the same manner as described in “Measurement method of erythrocyte ADMA” in Example 1. Comparison between the two groups was performed by an independent t-test.

  The results are shown in FIG. 8 (A: ** P <0.01; B: * P <0.05). As is clear from the results of FIG. 8, the CKD patients increased not only in the plasma ADMA concentration but also in the red blood cell ADMA concentration as compared with the healthy subjects. Although it has been reported so far that plasma ADMA concentration is increased in CKD patients, it was first shown by the present invention that red blood cell ADMA concentration was also significantly increased in CKD patients.

Expression of erythroblast-related genes in CKD model mice ADMA-degrading enzyme DDAH1Tg mice and C57BL6 mice as wild type were used, and CKD models were prepared by 5/6 nephrectomy. When evaluated at 12 weeks after 5/6 nephrectomy, plasma and red blood cell ADMA concentrations were significantly increased in the WT-CKD group compared to the sham group, but both were significantly suppressed in DDAH1Tg mice. It had been. There were no significant differences in blood pressure and renal function in the CKD group between WT and Tg. On the other hand, interestingly, the decrease in Hb and Ht observed in WT-CKD mice was mild but significantly improved in Tg mice, and the proportion of reticulocytes was also significantly increased. Disability improved.

  In mice, the spleen is a hematopoietic tissue. Spleen mRNA was extracted and RT-PCR was performed. The result is shown in FIG. As is clear from the results of FIG. 9, the expression of Epo receptor (EpoR) was significantly decreased in WT-CKD compared to WT-sham, but EpoR expression was significantly increased in DDAH1-Tg-CKD. It was. Accordingly, expression of genes (TfR-1, TfR-2, erythroferon) that are downstream of the Epo / EpoR signal and involved in erythroblast iron utilization was significantly increased in DDAH-1Tg mice.

  From this result, ADMA accumulation in erythroblasts in CKD decreases EpoR expression, inhibits Epo / EpoR signal, and causes erythroblast maturation, resulting in the onset of anemia and ESA resistance. It was suggested to be involved (FIG. 10). Since the ADMA concentration in erythrocytes reflects the accumulation of ADMA in erythroblasts, it is considered that the ADMA concentration in erythrocytes was correlated with anemia and ESA resistance index.

Comparison of measured values by LC-MS and ELISA Correlation between the case where ADMA concentration measurement was performed by LC / MS / MS and the case where it was performed by ELISA was examined.
For erythrocyte specimens from healthy individuals (n = 16), the ADMA concentration in the erythrocyte hemolyzed sample was measured by both LC / MS / MS and ELISA, and the correlation between the obtained measured concentrations was examined.
The result is shown in FIG. The correlation coefficient of the obtained Pearson was 0.552, and a positive correlation was observed between LC / MS / MS and ELISA.

  The number of patients with chronic renal failure is steadily increasing, and the number of patients receiving ESA is also increasing. The demand for ESA is increasing all over the world, and it always occupies the top in the statistics of drug sales. For this reason, not only in Japan but also in many countries, the burden on the medical economy is large, and there is an urgent need to establish a biomarker that can properly use ESA and to create a monitoring method that can grasp the treatment of renal anemia. The present invention can be used as a biomarker or monitoring method for monitoring proper use of ESA in renal anemia. The present invention can further be used as a research tool or biomarker in searching for ADMA inhibitors in erythrocytes.

Claims (16)

Use of asymmetric dimethylarginine (hereinafter referred to as ADMA) as a biomarker for monitoring the following (1) to (3):
(1) development of renal anemia in mammals;
(2) ESA hyporeactivity in a mammal undergoing treatment with an erythropoiesis stimulating agent preparation (ESA); or (3) Onset of heart disease in the mammal.   The use of ADMA as a biomarker according to claim 1, wherein the ADMA is ADMA present in erythrocytes of a blood sample obtained from a mammal or said mammal undergoing ESA treatment. The method according to the following (1) to (3), comprising measuring or monitoring the ADMA concentration in erythrocytes of a blood sample obtained from a mammal:
(1) Test method for renal anemia in mammals;
(2) A method for monitoring ESA hyporesponsiveness in mammals treated with ESA; or (3) A method for examining heart disease in mammals. The monitoring kit according to any one of (1) to (3) below, comprising means for measuring the ADMA concentration in erythrocytes of a blood sample obtained from a mammal:
(1) a kit for monitoring renal anemia in mammals;
(2) A kit for monitoring low ESA reactivity in mammals treated with ESA; or (3) a kit for monitoring the onset of heart disease in mammals.   5. The kit for monitoring renal anemia in a mammal according to claim 4, wherein means for receiving a blood sample from the mammal, means for measuring the ADMA concentration in red blood cells contained in the blood sample, and the measured ADMA concentration as a reference The method according to claim 4, comprising means for comparing with a value and means for determining whether the mammal has developed renal anemia and / or the extent of renal anemia from comparison with a reference value. Anemia onset monitoring kit.   The kit for monitoring ESA hyporesponsiveness in a mammal treated with ESA according to claim 4 comprises means for receiving a blood sample from a patient undergoing ESA treatment, and a blood sample obtained from a patient undergoing ESA treatment. Means for monitoring the ADMA concentration in the contained red blood cells, means for comparing the measured ADMA concentration with a reference value, and means for determining whether the patient exhibits low ESA reactivity from a comparison with the reference value. Item 5. A kit for monitoring ESA hyporeactivity in mammals treated with ESA according to Item 4.   The monitor kit for developing heart disease in a mammal according to claim 4, wherein means for receiving a blood sample from the mammal, means for measuring the ADMA concentration in erythrocytes contained in the blood sample, and the measured ADMA concentration as a reference The kit for monitoring the onset of heart disease in a mammal according to claim 4, comprising means for comparing with a value and means for determining whether the mammal has developed a heart disease based on a comparison with a reference value. The monitoring method according to (1) to (3), comprising screening for an increase in ADMA concentration in erythrocytes of a blood sample obtained from a mammal:
(1) A method for monitoring the onset of renal anemia in mammals;
(2) A method for monitoring low ESA reactivity in mammals treated with ESA; or (3) a method for monitoring the onset of heart disease in mammals.   9. The method of monitoring the development of renal anemia according to claim 8, wherein an increase in ADMA concentration correlates with a decrease in hemoglobin (Hb) concentration.   9. The method for monitoring the onset of renal anemia according to claim 8, wherein an increase in ADMA concentration correlates with a decrease in iron availability for hematopoiesis.   9. The method of monitoring renal anemia development according to claim 8, wherein an increase in ADMA concentration correlates with a decrease in serum iron and / or transferrin saturation (TSAT).   9. The method of monitoring the onset of heart disease according to claim 8, wherein an increase in ADMA concentration correlates with an increase in NT-proBNP concentration.   A method for diagnosing renal anemia or heart disease in a mammal, comprising screening for an increase in the concentration of ADMA in the red blood cells of the mammal.   Preventing overdose of ESA in mammals, including screening for increased ADMA concentration in red blood cells of mammals and reducing ESA dosage to an appropriate value when ADMA concentration in red blood cells exceeds a reference value ESA administration method.   9. The method of monitoring ESA hyporesponsiveness of an ESA-treated mammal according to claim 8, wherein the method of monitoring ESA hyporesponsiveness of a mammal treated with ESA comprises determining ESA-resistant resistance. Method.   The use, method or kit according to any one of claims 1 to 8, or 13 to 15, wherein the mammal is a human.

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