JP6523842B2 - Rhabdomyolysis treatment - Google Patents

Description

  The present invention relates to novel rhabdomyolysis which is useful for the treatment of acute kidney injury etc. induced by rhabdomyolysis.

Rhabdomyolysis refers to a condition or condition in which rhabdomyolysis causes components in the myocytes to flow out into the bloodstream, and when it becomes severe, it causes acute kidney injury (AKI) and leads to death. Rhabdomyolysis induced AKI is a pathological condition proposed by Bywaters and Bearl at the time of World War II (Non-patent Document 1). , 8-15% of AKI patients in the United States (non-patent document 2). In addition, there is also a report that the incidence rate rises to 13-50% when the potential ones are included (Non-patent Documents 3 and 4). There are nontraumatic factors such as dehydration, drug side effects and glycolytic gene defects, and traumatic factors such as accidents and injuries, among which about 50% of these factors are traumatic factors. (Non-patent documents 5 and 6). Treatment for rhabdomyolysis AKI is symptomatic treatment, although infusion for fluid and blood replacement, administration of bicarbonate for alkalization of urine, or hemodialysis is performed (Non-Patent Document 7), it is still There is no effective treatment. In addition, in Rhabdomyolysis, blood transfusion (RBC) is performed because anemia occurs when the condition becomes severe. In particular, when traumatic hemorrhage is involved, the frequency of blood transfusion is increased.

  In a broad sense, crash syndrome is also included in traumatic rhabdomyolysis AKI. Crash syndrome is an underlay of a collapsing material that occurs during disasters, etc., resulting in prolonged compression of the extremities, resulting in skeletal muscle damage that appears rapidly due to relief such as rescue and general symptoms caused thereby. It is a disease group (non-patent document 8). In the past, there have been many victims during disasters such as large earthquakes and house collapses. Furthermore, if there is a chance of compression, it may also occur at clinical sites such as comatose patients (Non-patent Document 9), long-term bed rest of intraoperative patients (Non-patent document 10), cast fixation (Non-patent document 11).

Bywaters EG, Beall D. Crush injuries with impairment of renal function, 1941 J Am Soc Nephrol. 1998; 9 (2): 322-332 Moore KP, Holt SG, Patel RP, et al. A causative role for redox cycling of myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysis-induced renal failure. J Biol Chem. 1998; 273 (48): 31731- 31737 Rocuts F, Ma Y, Zhang X, ey al. Carbon monoxide suppressors membrane expression of TLR4 via myeloid differentiation factor-2 in beta TC3 cells. J Immunol. 2010; 185 (4): 2134-2139 Melli G, Chaudhry V, Cornblath DR. Rhabdomyolysis: An evaluation of 475 hospitalized patients. Medicine. 2005; 84 (6): 376-385 Factors that are necessary for hemodynamics ED patients with rhabdomyolysis. Am J Emerg Med. 2005; 23 (1): 1-7 Allison RC, Bedsole DL. The other medical causes of rhabdomyolysis. Am J Med Sci. 2003; 326 (2): 79-88 Sever MS, Erek E, Vanholder R, et al. Serum potassium in the crush syndrome victims of the Marmara disaster. Clin Nephrol. 2003; 59 (5): 326-333 Smith J, Greaves I. Crush injury and crush syndrome: a review. J Trauma. 2003; 54 (5): S226-S230 Show AD, Sjolin SU, McQueen MM. Crush syndrome following unconsciousness: need for urgent orthopaedic referral. BMJ. 1994; 309 (6958): 857-859 Godbaut B, Burchard KW, Slothman GJ, et al. Crush syndrome with death following pneumatic antishock garment application. J Trauma. 1984; 24 (12): 1052-1056 Development of the protracted compression syndrome after shoulder dislocation and application of an immobilizing bandage. Sud Med Ekspert 1977; 20 (4): 56 Murata I, Nozaki R, Ooi K, et al. Nitrite reduce ischemia / reperfusion-induced muscle damage and improve survival rates in rat crush Injury model. J Trauma. 2011; 72 (6): 1453-1463 J Trauma Acute Care Surg. 2012; 70 (6): 1548-1554 Murata I, Ooi K, Sakai H, et al. Characterization of systematic and histology Injury after crush syndrome and intervals of reperfusion in a small animals. I J J Ishig K, Wakabayashi H, Maeba T, et al. Lidocaine-metabolizing activity after warm ischemia and reperfusion of the rat liver in vivo. World J Surg. 2000; 24 (1): 49-53 Karuzina II, Archakov AI. The oxidative inactivation of cytochrome P450 in monooxygenase reaction as. Free Radic Biol Med. 1994; 16 (1): 73-97 3. Micropuncture study of the effects of mannitol and isotonic saline on individual nephron function. Nephron. 1967; 4 (6): 337- 355 Baliga R, Ueda N, Walker PD, et al. Oxidant mechanism in toxic acute renal failure. Am J Kidney Dis. 1997; 29 (3): 465-477 Boutaud O, Moore KP, Reeder BJ, et al. Acetoaminopheninhibits hemoprotein-catalyzed lipid peroxide and attenuates rhabdomyolysis-induced renal failure. Proc Natl Acad Sci U S A. 2010; 107 (6): 2699-2704: Drug metab Dispos. 2013; 41 (1): 141-8: Ogaki S, Taguchi K, Watanabe H, et al. Carbon monoxide-bound red blood cells protect red blood cell transfusion-induced hepatic cytochrome P450 impairment in hemorrhagic-shock rats. . Ogaki S, Taguchi K, Watanabe H, et al. Carbon monoxide-bound red blood cell resuscitation ameliorates therapeutic injuries induced by massive hemorrhage and red blood cell resuscitation via hepatic cytochrome P450 protection in hemorrhagic shock rats J 7): 2199-206. Billings FT, Ball SK, Roberts LJ, et al. Postoperative acute kidney incident is associated with hemoglobininemia an enhancement oxidative stress response. Free Radic Biol Med. 2011; 50 (11): 1480-1487 Pinheiro CH, Vitzel KF, Curi R. Effect of N-acetylcisteine on markers of skeletal injury after fatiguing contractile activity. Scand J Med Sci Sports. 2012; 22 (1): 24-33 Jastrow KM, Gonzalez EA, McGuire MF, et al. Early cytokine production risk risks traumatics trauma patients for multiple organ failure. J Am Coll Surg. 2009; 209 (3): 320-331 Yassin MM, Harkin DW, Barros D'Sa AA, et al. Lower limb ischemia-reperfusion Injury triggers a systematic inflammatory response and multiple organ dysfunction. World J Surg. 2002; 26 (1): 115-121 Sonoi H, Matsumoto N, Ogura H, et al. The effect of antithrombin on pulmonary endothelial damage induced crush injury. Shock. 2009; 32 (6): 593-600 Gonzalez D. Crush syndrome. Crit Care Med. 2009; 33 (1): S34-S41 Omura T, Sato R. The carbon monoxide-binding pigment of liver microsomes. J Biol Chem. 1964; 239 (7): 2370-2378 Sakai H, Masada Y, Horinouchi H, Yamamoto M, Ikeda E, Takeoka S, Kobayashi K, Tsuchida E ,. Hemoglobin-vesicles suspended in recombinant human serum albumin for resuscitation shock in anesthetized rats. Crit 31 Med. (2): 192-200 Taguchi K, Maruyama T, Iwao Y, et al. Pharmacokinetics of single and repeated injection of hemoglobin- vesicles on hemorrhagic shock rat model. J Control Release. 2009; 136 (3): 232-239

  The crash syndrome has a sudden change in symptoms upon compression rather than compression. That is, mediators such as potassium (K) and myoglobin (Mb) in the cell contents, and inflammatory cytokines triggered by injury are released from the injured muscle into the systemic circulation, accompanied by arrhythmia and low circulation. As a result of shocks, it is a major cause of death risk until hospital transport. Also, inhibition of renal excretion of potassium along with acute renal failure caused by Mb is also a factor that increases the risk of death due to arrhythmia. In other words, crash syndrome is a condition leading to death by a mechanism similar to rhabdomyolysis.

  The treatment for rhabdomyolysis acute kidney injury so far is symptomatic therapy, although administration of bicarbonate for fluid and blood replacement and alkalinization of urine or administration of hemodialysis is still effective. There is no such drug, and there is a strong demand for development of a drug that can be easily implemented at medical sites and even at disaster sites.

  The object of the present invention is to provide a novel therapeutic agent for rhabdomyolysis which is useful for the treatment of acute kidney injury (AKI) induced by rhabdomyolysis and the like.

In order to solve the above-mentioned problems, the inventor of the present invention has examined the usefulness of carbon monoxide-added erythrocytes (CO-RBC) against rhabdomyolysis-induced AKI in the following three pathological model, (1) dehydration / glycerol-induced Rhabdomyolysis comorbid AKI rat (nontraumatic model), (2) traumatic rhabdomyolysis AKI model rat complicated with hemorrhagic shock, and (3) Murata et al. (Non-patent documents 12 and 13) We examined using the crash syndrome model due to rubber band compression, which was established by As a result, it has been found that carbon monoxide-added erythrocytes (CO-RBC) show a therapeutic effect on rhabdomyolysis-induced AKI, and the present invention has been completed.

That is, according to the present invention, the following inventions are provided.
(1) A therapeutic agent for rhabdomyolysis comprising carbon monoxide-added erythrocytes as an active ingredient.
(2) The therapeutic agent for rhabdomyolysis according to (1), which is a therapeutic agent for rhabdomyolysis-induced acute kidney injury.
(3) The therapeutic agent for rhabdomyolysis according to (1) or (2), which is a therapeutic agent for crash syndrome.
(4) The therapeutic agent for rhabdomyolysis according to any one of (1) to (3), which is administered intravenously.

  The current treatment for rhabdomyolysis acute kidney injury is symptomatic treatment, and the ideal treatment is rapid hemodialysis, but complicated equipment and specialized technicians are required, and medical sites and disaster sites But it is practically difficult. The carbon monoxide-added erythrocytes used in the present invention can be easily and rapidly prepared simply by exposing carbon monoxide to the marketed concentrated red blood cell solution, and the practicality is very high. In addition, in Rhabdomyolysis, anemia is caused when the condition becomes severe, so the frequency of red blood cell transfusion is very high, and carbon monoxide-loaded red blood cells are used only as a treatment for rhabdomyolysis acute kidney injury. The role of blood transfusion for anemia and traumatic hemorrhage can be combined. Acute kidney injury by administering carbon monoxide-added red blood cells of the present invention to non-traumatic or traumatic rhabdomyolysis patients or to people (Crush syndrome preparatory group) who are laid down by collapse of the house It is a novel therapeutic agent for rhabdomyolysis acute kidney injury that suppresses the onset and improves the survival rate and the prognosis.

Figure 1 shows saline, red blood cells (RBC) or carbon monoxide red blood cells (CO-RBC) at 6 hours or 24 hours after control rat group (glycerol untreated) and rhabdomyolysis induction (glycerol intramuscular injection) 1) shows changes in the amount of A) plasma myoglobin (Mb), B) kidney myoglobin, C) plasma creatinine phosphor kinase (CPK) in rats treated with 2.). Each column shows the mean ± SD (n = 3-5). ** p <0.01 vs. control rat group FIG. 2 shows treated with saline, red blood cells (RBC) or carbon monoxide red blood cells (CO-RBC) at 24 hours after control rat group (glycerol untreated) and rhabdomyolysis induction (glycerol intramuscular injection) A) changes in plasma free heme, B) changes in the amount of kidney free heme in rats. Each column shows the mean ± SD (n = 5-6). ** p <0.01 vs. control rat group FIG. 3 shows saline, red blood cells (RBC) or carbon monoxide red blood cells (CO-RBC) treated with control rats (glycerol untreated) and rhabdomyolysis induced (glycerol intramuscular injection) 24 hours In the rat, A) total kidney CYP, B) total liver CYP content is shown. Each column shows the mean ± SD (n = 5). ** p <0.01 vs. control rat group FIG. 4 shows treated with saline, red blood cells (RBC) or carbon monoxide red blood cells (CO-RBC) at 24 hours after control rat group (glycerol untreated) and rhabdomyolysis induced (glycerol intramuscular injection) (A) blood urea nitrogen (BUN), (B) serum creatinine (SCr), (C) creatinine clearance (CCr) and D) the amount of N-acetyl-β-D-glucosaminidase (NAG) activity in urine in rats Indicates Each column shows the mean ± SD (n = 5-6). ** p <0.01 vs. control rat group FIG. 5 shows treated with saline, red blood cells (RBC) or carbon monoxide red blood cells (CO-RBC) at 24 hours after control rat group (glycerol untreated) and rhabdomyolysis induction (glycerol intramuscular injection) (A) Observation test of representative kidneys in rats, (B) Representative photomicrographs of PAS stained kidney sections. Magnification, B × 200 FIG. 6 shows treated with saline, red blood cells (RBC) or carbon monoxide red blood cells (CO-RBC) at 24 hours after control rat group (glycerol untreated) and rhabdomyolysis induction (glycerol intramuscular injection) A representative urine observation from rats is shown. FIG. 7 shows treated with saline, red blood cells (RBC) or carbon monoxide red blood cells (CO-RBC) at 24 hours after control rat group (glycerol untreated) and rhabdomyolysis induction (glycerol intramuscular injection) Figure 2 shows the concentration of urinary myoglobin (Mb) and hemoglobin (Hb) in rats. Each column shows the mean ± SD (n = 5-6). * p <0.05, ** p <0.01 vs. control rat group FIG. 8 shows changes in amounts of (A) red blood cells (RBC) and (B) hematocrit (Hct) in control rats and rhabdomyolysis-induced (glycerol intramuscular injection) rats. Each column shows the mean ± SD (n = 5-6). *: p <0.05, vs. control ^# : p <0.05 vs. saline FIG. 9 shows that treated with saline, red blood cells (RBC) or carbon monoxide red blood cells (CO-RBC) at 24 hours after control rat group (glycerol untreated) and rhabdomyolysis induction (glycerol intramuscular injection) Control (open circles) and saline (dotted circles), red blood cells (RBC) showing changes in the amount of plasma A) malondialdehyde (MDA), B) hydroperoxide in rats, 24 hours after glycerol injection The relationship between plasma free heme and MDA in rats treated with (closed circles) or carbon monoxide erythrocytes (CO-RBC) (grey circles) is shown. Each column shows the mean ± SD (n = 5-6). * p <0.05, ** p <0.01 vs. control rat group FIG. 10 shows treated with saline, red blood cells (RBC) or carbon monoxide red blood cells (CO-RBC) at 6 hours after control rat group (glycerol untreated) and rhabdomyolysis induced (glycerol intramuscular injection) Fig. 6 shows the absorption spectrum of oxidized myoglobin in rat kidney. Data are representative of 3 rats in each group with similar results. Figure 11. Survival of glycerol injection-induced rhabdomyolysis rats treated with saline (dotted circles), red blood cells (RBC) (black circles) or carbon monoxide red blood cells (CO-RBC) (grey circles) Indicates the rate. Survival rates were compared using Kaplan-Meier survival curves and log-rank test. **: p <0.01 vs. saline treated rats (n = 10). FIG. 12 shows hemorrhagic shock-induced glycerol-induced rhabdomyolysis model rat treated with saline (dotted circle), erythrocyte (RBC) (black circle) or carbon monoxide erythrocyte (CO-RBC) (gray circle) Shows the survival rate of Survival rates were compared using Kaplan-Meier survival curves and log-rank test. ***: p <0.001 vs. saline treated rats (n = 10). FIG. 13 shows the survival rates of crush syndrome model rats treated with saline (dotted circle), red blood cells (RBC) (black circles) or carbon monoxide red blood cells (CO-RBC) (grey circles). Survival rates were compared using Kaplan-Meier survival curves and log-rank test. ***: p <0.001 vs. saline treated rats (n = 10). FIG. 14 shows the progression of rhabdomyolysis-induced AKI by glycerol. FIG. 15 shows the suppression mechanism of non-traumatic rhabdomyolysis-induced acute kidney injury by carbon monoxide-added red blood cell (CO-RBC) treatment.

Hereinafter, the present invention will be described in more detail.
When rhabdomyolysis becomes severe, it causes AKI concomitantly, and the mortality rate rises sharply. Therefore, although appropriate management of rhabdomyolysis AKI is required, at the present time, only symptomatic treatment with fluid or hemodialysis is performed, and development of an effective therapeutic drug is desired. To summarize the previous reports, Mb in muscle and heme released from CYP (cytochrome P450) in the kidney play an important role in the pathogenesis and progression of Rhabdomyolysis AKI. From this, an antioxidant that suppresses Mb-induced oxidative stress disorder, a renal CYP protective agent, or a heme capture agent is considered as a candidate for an effective therapeutic agent for rhabdomyolysis AKI. So far, we have found that carbon monoxide-loaded erythrocytes (CO-RBC) show excellent protective effects against liver damage due to free heme and liver cytochrome P450 (CYP) (Non-patent document 19 and 20) CO-RBC is expected to exert therapeutic effects on rhabdomyolysis AKI. Therefore, non-traumatic rhabdomyolysis AKI, traumatic rhabdomyolysis AKI, and crush syndrome were created as the three types of pathological condition rats of rhabdomyolysis, and CO-RBC is useful for these. I examined the sex.

  In the glycerol-induced non-traumatic rhabdomyolysis AKI model, the following pathogenesis mechanism may be considered. First, glycerol treatment impairs the striated muscle, and Mb and CPK in muscle flow out into the blood and accumulate in plasma and kidney. Above all, Mb and its heme molecule induce oxidative stress, causing renal damage, and at the same time, the CYP of the kidney is destroyed and the heme molecule is released. The combination of these causes a rapid rise in free heme concentration in the kidney and further aggravates the renal function, resulting in a decrease in Mb excretion from the plasma and a more accumulation in the blood and kidney. It is a model that a vicious circle is formed and AKI is induced (Fig. 14).

  Saline, RBC or CO-RBC was administered 3 hours after glycerol treatment to this non-traumatic rhabdomyolysis AKI model rat, and the effect was evaluated 24 hours after glycerol treatment. 1) Release of muscle protein (CPK, Mb) into blood (Fig. 1), 2) Decreased renal function (Fig. 4), 3) Renal tubular disorder (nuclear shedding) (Fig. 5), 4) Metabolic acidosis・ The clinical findings specific to rhabdomyolysis AKI such as hyperkalemia (Table 1), 5) Hematuria, proteinuria (Table 2), 6) Increase in urine Mb and Hb (Figure 7) are not improved The Also, reflecting that, in the saline, RBC administration group, the pathological model rat died 80% and 60%, respectively (FIG. 11). On the other hand, CO-RBC administration suppressed the change of the above-mentioned clinical findings and improved the survival rate (100% survival).

  In addition to free heme, Mb is considered to be involved as an inducer of oxidative stress in this disease. The reason is that although Mb has less activity as an oxidant than free heme, when internal heme iron shifts to a divalent, trivalent, or tetravalent iron state, it acquires higher oxidizing power, which is a failure. The action is intensified (non-patent documents 16 and 21). In fact, one hour after glycerol treatment, Mb was accumulated not only in blood but also in renal pool, and also because an increase in Mb oxidant was observed in renal pool six hours after glycerol treatment. (FIG. 10) It is speculated that the Mb oxidant is involved in renal damage including degradation of renal CYP as a potent pro-oxidant. In addition, although CO-RBC administration group did not suppress the increase in Mb after 6 hours of glycerol treatment (Fig. 1), CO was released at an early stage of the pathological condition because it suppressed the promotion of formation to the Mb oxidant. It is conceivable that the amount of Mb is not suppressed but the quality, that is, the degree of oxidant, is suppressed.

  Furthermore, CO-RBC administration did not affect Mb and CPK, which are elevated in the early stage of the pathological condition, but conversely decreased at 24 hours after glycerol treatment when the pathological condition was formed. It was strongly suggested that the myopathy was secondarily suppressed by reducing the oxidative stress response mediated by the heme protein or free heme described above, rather than by suppressing the initial injury that causes the onset of the pathological condition. In fact, since skeletal muscle cells are impaired by lipid peroxidation (Non-patent Document 22), the indirect muscle protective effect by CO-RBC is supported. Based on these findings, FIG. 15 shows an outline of the pathological progression process of nontraumatic rhabdomyolysis-induced AKI assumed at present and the action point of CO-RBC.

  Furthermore, RBC administration did not show any efficacy against traumatic rhabdomyolysis AKI and crash syndrome model rats with hemorrhagic shock more lethal than the previous non-traumatic model. , CO-RBC exhibited excellent life-saving effect (100% survival rate) (Fig. 12, Fig. 13).

  Although the details of the mechanism of action of CO-RBC on traumatic rhabdomyolysis AKI or crash syndrome are unknown, the process after damage to muscle cells is similar to the non-traumatic model From these results, it is speculated that suppression of systemic ischemia-reperfusion injury by CO, stabilization of heme proteins, and organ protective effects against free heme may greatly contribute. In addition, rhabdomyolysis AKI and crash syndrome cause multiple organ failure associated with systemic inflammatory response syndrome and show severe pathological condition (non-patent documents 23 and 24), even symptomatic treatment, It is believed that early treatment after rescue is important (25 and 26). Ideally, on-site rapid hemodialysis, but currently not possible. Instead, if the rescue effect is exhibited by a single administration of CO-RBC, which can be intravenously administered, it can be used at disaster sites, and therefore, it is expected to be widely used. From the above results, CO-RBC is effective against a variety of rhabdomyolysis AKI (non-traumatic, traumatic rhabdomyolysis and crash syndrome) for which no effective therapeutic agent has been developed yet Was shown to be a good therapeutic agent.

The therapeutic agent for rhabdomyolysis of the present invention contains carbon monoxide-added erythrocytes as an active ingredient.
The red blood cells used in the present invention are mammalian red blood cells, preferably human red blood cells. It may be obtained from the patient being treated to be immunocompatible, may be blood donation from a volunteer, or may be marketed concentrated red blood cell fluid.

  The red blood cells used in the present invention are red blood cells to which carbon monoxide is added. Red blood cells to which carbon monoxide has been added can be produced by exposing carbon monoxide to red blood cell fluid. Specifically, CO is gently bubbled into the washed RBC (Hb concentration: 10 g / dL) for 5 minutes to produce CO-RBC. In addition, when addition of CO to RBC was evaluated by the amount of carbonyl Hb, HbCO content reaches 90% in 5 minutes of CO bubbling time.

Carbon monoxide-loaded erythrocytes can be used as a pharmaceutical composition used for the treatment of rhabdomyolysis.
Rhabdomyolysis is caused by extrinsic (crash syndrome), excessive muscle activity, muscle ischemia, metabolic disease, drugs, poisoning, infections, genetic diseases, heat stroke, etc. A disorder resulting from skeletal muscle damage with release to plasma, acute recurrent rhabdomyolysis, exertional rhabdomyolysis, familial paroxysmal rhabdomyolysis and idiopathic paroxysmal rhabdomyomas Lymphosis etc. are included. Clinical symptoms of rhabdomyolysis include limb weakness, numbness, muscle pain, muscle weakness, stiffness, swelling, reddish-brown urine and the like. Laboratory findings include myoglobin (Mb), creatinine phosphokinase (CPK), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), lactate dehydrogenase (LDH), muscle deprivation enzymes such as aldolase Rapid increase and muscle degeneration is observed.

  Treatment of rhabdomyolysis in the present invention includes, but is not limited to, treatment of rhabdomyolysis-induced acute kidney injury or treatment of crush syndrome. These diseases may be single or co-occurring or may be co-occurring with other diseases than those described above.

The administration method of the therapeutic agent of the present invention is preferably administered intravenously or arterially by injection or infusion, and more preferably intravenously.
The therapeutic agent of the present invention can be provided in the form of a pharmaceutical composition suitable for injection or infusion.
When large doses are administered, it is desirable to add a plasma expander, such as albumin, to maintain the glial osmotic pressure.

  Although the dosage of the therapeutic agent for rhabdomyolysis of the present invention varies depending on the patient's age, sex, symptoms, administration route, administration frequency, dosage form, as a specific example of dosage, 20 g of hemoglobin per day It is about 200 to 800 mL in a concentrated pack of / dl, and is administered once or a plurality of times. In the case of multiple doses, it may be divided into 3 to 4 times per day.

  According to the invention there is further provided carbon monoxide-loaded red blood cells for use in the treatment of rhabdomyolysis. Preferably, carbon monoxide-loaded red blood cells for use in the treatment of rhabdomyolysis-induced acute kidney injury, as well as carbon monoxide-loaded red blood cells for use in the treatment of crash syndrome are provided. The above carbon monoxide-loaded erythrocytes are preferably administered intravenously.

According to the present invention, there is further provided a method of treating rhabdomyolysis comprising administering a therapeutically effective amount of carbon monoxide-loaded red blood cells to a patient with rhabdomyolysis.
In the above-mentioned method for treating rhabdomyolysis, preferably, rhabdomyolysis-induced acute kidney injury can be treated or crush syndrome can be treated.
A therapeutically effective amount of carbon monoxide-loaded red blood cells can preferably be administered intravenously.
According to the invention there is further provided the use of carbon monoxide-loaded red blood cells for the production of rhabdomyolysis.

  The present invention will be described more specifically by the following examples, but the present invention is not limited by the examples.

[Method of experiment]
(1) Preparation of rhabdomyolysis model rats After 24 hours of model preparation, rats were treated with water-quenching, and then under ether anesthesia, rats were dosed with 50 mL of glycerol / saline at a dose of 8 mL / kg. Administered in the lower thigh muscle. Three hours after glycerol administration, saline, RBC (red blood cells) or CO-RBC (carbon monoxide-added red blood cells) were intravenously administered (1,400 mg Hb / kg, 22.4 mL / kg).

As RBC (red blood cells), blood was collected from rats and plasma was removed by centrifugation (1,200 rpm, 15 min, 4 ° C.). Washing and centrifugation with physiological saline (1,200 rpm, 15 min, 4 ° C.) were performed three times to prepare a washed RBC. The Hb concentration was measured by Hemoglobin B Test Wako (Wako Pure Chemical Industries, Ltd.) and diluted to a concentration of 10 g / dL with saline.
As CO-RBC (carbon monoxide-added erythrocytes), CO was gently bubbled into washed RBC for 5 minutes to produce CO-RBC.

(2) Measurement of renal function, myopathy, oxidative stress parameters Blood is collected at 1, 3, 6, 12 and 24 hours after administration of glycerol, and blood cell parameters (RBC, Hct) in whole blood are determined by hemocytometer ( Measured by Sysmec). In addition, after centrifuging the obtained blood (3,000 rpm, 10 min, 4 ° C.), using the supernatant as a measurement sample, the BUN, SCr, Mb, CPK, free heme and MDA measurement kits (BUN · SCr: It was measured according to the attached protocol using Wako Pure Chemical Industries, Mb: Life Diagnostics, CPK · free heme · MDA: Cayman Chemical).

(3) Preparation of Paraffin Section 24 hours after glycerol administration, the left kidney was excised, fixed with 4% paraformaldehyde (4 ° C., overnight), and then embedded in paraffin. Paraffin sections (2.5 μm) were prepared from paraffin-fixed kidneys.

(4) PAS Staining The prepared paraffin sections were deparaffinized and infiltrated with 1% periodate solution for 10 minutes. After washing with running water, the nuclei were dyed with hematoxylin, and an image was obtained with a microscope (BZ-8000, Keyence).

(5) Measurement of Urinary Parameters After glycerol administration, urine was collected for 24 hours, and the NAG activity in the resulting urine was measured using a measurement kit (Shionogi & Co., Ltd.) according to the attached protocol. In addition, with regard to urinary findings of urine obtained by collecting urine for 24 hours before and after administration of glycerol, urinary occult blood, protein and urine pH were scored and evaluated using a test paper (Pretest 5bII, Wako Pure Chemical Industries, Ltd.). For urinary occult blood, Hb and Mb concentrations of 0, 0.06, 0.15, and 0.75 mg / dL were each determined as a score of 0-4. For urine protein, the protein concentration (-), 30, 100, 300, and 1000 mg / dL were each determined as a score of 0-5.

(6) Measurement of blood biochemical parameters Before and after administration of glycerol, blood was collected at 6, 24 hours after administration, and pH, BE, lactic acid, K and Na values in the collected blood were measured using i-STAT 300F (Saiyaku Pharmaceutical Co., Ltd.) It measured using.

(7) Measurement of total CYP amount in liver and kidney microsomes The total CYP amount in liver and kidney microsomes was reported by Omura et al. Based on the absorption difference spectrum method. 1 mL of lysis buffer was added per 100 mg of liver or kidney, the homogenate prepared by the homogenizer was centrifuged at 10,000 g at 4 ° C. for 30 minutes, and the supernatant was collected. Furthermore, the supernatant was ultracentrifuged at 105,000 g at 4 ° C. for 60 minutes. The precipitate (microsome fraction) is dissolved in 10% glycerol containing 0.1 M potassium phosphate buffer (pH 7.4), and these are used as a microsome sample solution, to which 1 mg of sodium hydrosulfite as a reducing agent is added. The solution was bubbled with CO for 1 minute. The total CYP amount (nmol / mg protein) was calculated from the difference between the absorbance at around 450 nm and the absorbance at 490 nm with a spectrophotometer using a molecular absorption coefficient of 91 cm ⁻¹ mM ⁻¹ .

(8) Detection of Mb oxidant in kidney tissue
Based on the method of Moore et al. (Non-patent document 2), a 10 μm-cut rat kidney tissue piece was added with phosphate buffer (pH 7.4) containing 10 μM DTPA, and homogenized (Power Gen 125, Fisher Scientific) ) Was used for homogenization, and the supernatant obtained by centrifugation (6,000 rpm, 10 min, 4 ° C.) was measured for absorbance at 350-700 nm.

(9) Preparation of hemorrhagic shock-induced glycerol-induced rhabdomyolysis model rat After 24 hours of model preparation, rats were treated with water-quenching, and then under ether anesthesia, rats were dosed at 8 mL / kg with 50% glycerol / Saline was administered bilaterally into the lower thigh muscles. One hour later, blood was removed using a polyethylene tube (PE-50) inserted into the left femoral artery, and 40% of the total blood volume (22.4 mL / kg) (Non-patent Document 28) was removed and hemorrhagic A shock was induced (Non-patent Document 29). Each administration solution was administered 10 minutes after the end of the final blood removal.

(10) Creation of crash syndrome model rat The crash syndrome model applied a rubber band wound on a stainless steel pipe under a load of 2.0 kg to both rat hind legs according to the existing method (Non-patent documents 12 and 13) and compressed for 5 hours Later, the rubber band was released and used as a model.

[result]
(1) Therapeutic effect of CO-RBC on non-traumatic rhabdomyolysis AKI rats (1-1) Influence on deviation of muscle protein Plasma, one of the characteristic clinical findings of rhabdomyolysis The effects of CO-RBC on Mb in renal tissue and creatinine phosphokinase (CPK) in plasma were examined. Specifically, 3 hours after glycerol treatment, physiological saline (saline), RBC or CO-RBC is administered, and at 6 and 24 hours after glycerol treatment, Mb in plasma and kidney tissue, CPK in plasma Concentrations were measured and compared. As a result, in the saline and RBC administration groups, the concentrations of Mb and plasma CPK in plasma and kidney tissues were significantly higher than those in the control group (glycerol untreated group) (FIG. 1). On the other hand, in the case of CO-RBC administration, at 6 hours after glycerol treatment, although the improvement effect on the above-mentioned parameters was not observed, their increase was significantly suppressed after 24 hours. From this, it is considered that CO-RBC suppressed the renal and myopathies caused in the subsequent process of the pathophysiology rather than reducing the myopathies induced early in the pathophysiology.

(1-2) Effects on free heme and CYP concentration The free heme concentration in plasma and kidney tissue was measured 24 hours after glycerol treatment. Compared to the control group, the free heme concentration in plasma and kidney tissue was significantly increased in the saline and RBC administration groups (FIGS. 2A, B). On the other hand, in the CO-RBC administration group, this increase was suppressed.

  FIG. 3 shows the total amount of CYP in kidney and liver tissues 24 hours after glycerol treatment. As shown in the figure, the total amount of CYP in the kidney was significantly reduced in the saline and RBC administration groups as compared to the control group. On the other hand, the decrease was suppressed in the CO-RBC administration group (FIG. 3A). In contrast to the kidney, the total amount of CYP in the liver was unchanged in either group (FIG. 3B). This means that the degradation of CYP induced by rhabdomyolysis AKI mainly occurs in the kidney. From these results, the increase in free heme concentration in the kidney observed in the physiological saline and RBC administration groups largely contributes to the degradation of renal CYP, and CO-RBC inhibits it, thereby suppressing the kidneys. It was speculated that the increase in free heme concentration in storage was suppressed.

(1-3) Effects on blood biochemical parameters At the time of myocyte injury, various contents are released into the blood, so the balance of ion balance inside and outside the blood vessel is lost, hyperkalemia, metabolic acidosis Come to exhibit. Therefore, before glycerol treatment and at 6, 24 hours after treatment, lactic acid, K, and sodium (Na) levels in whole blood and blood pH and BE which are indicators of acidosis were measured. As a result, in the saline and RBC administration groups, a decrease in blood pH, base excess (BE) and a significant increase in blood lactic acid level were observed, and it was confirmed that metabolic acidosis was exhibited (Table 1) ). In addition, hyperkalemia has also occurred. On the other hand, acidosis and hyperkalemia were reduced in the CO-RBC administration group.

(1-4) Effects on Renal Function and Renal Histological Changes The renoprotective effect of CO-RBC against rhabdomyolysis AKI was evaluated from the viewpoint of renal function parameters. Specifically, physiological saline, RBC or CO-RBC was administered 3 hours after glycerol treatment, and renal injury parameters (urea nitrogen (BUN), serum creatinine (SCr)) were measured 24 hours after glycerol administration. In addition, urine was collected up to 24 hours after glycerol administration, and creatinine clearance (CCr) was calculated. In addition, urinary N-acetyl-β-D-glucosaminidase (NAG) activity is also measured, which is an enzyme deviating from the proximal tubule and appears early in the urine due to tubular disorder and is used as an indicator of AKI. did. As a result, significant increase in BUN, SCr and urinary NAG activity and significant decrease in CCr were observed in the saline administration group as compared to the control group (FIG. 4). Similar results were observed in the RBC administration group. In contrast, in the CO-RBC administration group, the reduction in renal function was significantly suppressed. This indicates that RBC administration does not improve the decrease in renal function due to rhabdomyolysis AKI, but that CO-RBC administration suppresses the decrease in renal function. From this, the utility of CO in the amelioration of this pathological condition was found.

  In addition, in rhabdomyolysis in humans, deposition of muscle-derived Mb in renal tissue causes significant tubular injury as a clinical finding (Non-patent Documents 14 to 16). Therefore, renal and renal tissue section images in the saline, RBC and CO-RBC administration groups were evaluated morphologically and histologically. FIG. 5A shows an image of the kidney. Unlike the deep-red, smooth-surfaced kidney in the control group, discoloration and atrophy were observed in the saline and RBC administration groups, and irregularities were observed on the organ surface. On the other hand, in the CO-RBC administration group, such a change in appearance was alleviated, and a form similar to the control was maintained. Therefore, next, kidney sections of each group were prepared, and histological observation was performed by periodic acid schiff (PAS) staining. As a result, in the saline and RBC administration groups, shedding of tubular nuclei was observed (FIG. 5B). On the other hand, in the CO-RBC administration group, no such remarkable tubular degeneration was observed, and the histology also was similar to the control group. These histological changes corresponded well to the changes in renal function parameters shown in FIG.

(1-5) Influence on Urine Findings One of the definitive diagnostic criteria for rhabdomyolysis is urinary findings. Specifically, since proteins such as Mb and hemoglobin (Hb) are excreted in the urine, occult blood is observed, exhibiting brown urine and acidifying the pH of the urine. Therefore, first, 24-hour urine accumulation after glycerol treatment was performed for the saline, RBC, and CO-RBC administration groups. As shown in FIG. 6, brown urine was observed in the saline and RBC administration groups. On the other hand, in the CO-RBC administration group, the degree of coloring was low. Next, analysis of urinary occult blood, urinary pH, and proteinuria revealed that in the saline and RBC administration groups, positive reactions of urinary occult blood and urinary protein were also observed, and a decrease in urinary pH was also observed. On the other hand, in the CO-RBC administration group, the fluctuation of the urinary findings was suppressed (Table 2). Furthermore, in order to examine the contents of occult blood, the Mb or Hb concentration present in urine storage (24 hours) was measured. As a result, Mb was significantly increased in the saline and RBC administration groups (FIG. 7). On the other hand, in the CO-RBC administration group, only about 1/3 of the accumulation was observed. Although Hb was not detected in the control group, the other three groups showed similar accumulation. However, when the accumulation amounts of Mb and Hb were compared, as in human, both groups were superior in Mb.

(1-6) Effects on Hemolysis In rhabdomyolysis, as a result of the increase of oxidative stress in blood, lipid peroxidation of red blood cells is enhanced, resulting in hemolytic symptoms. In addition, RBC transfusion will be performed as it becomes anemic if it gets severe. Therefore, in order to examine the influence of CO-RBC on hemolysis, RBC numbers and hematocrit (Hct) were measured over time for 24 hours after glycerol treatment. As a result, in the saline-administered group, the RBC numbers and Hct values were reduced 3 hours after glycerol treatment, as compared with the control group (FIGS. 8A, B). On the other hand, in the RBC or CO-RBC administration group, improvement in anemic symptoms was observed 3 hours after administration (6 hours after glycerol administration).

(1-7) Effects on oxidative stress in plasma and kidney The effect of CO-RBC on free heme-induced oxidative stress was examined. Here, after 24 hours of glycerol treatment, the lipid peroxide, malondialdehyde (MDA), which is a lipid peroxide, and the plasma hydroperoxide (oxidative metabolite), which is accumulated due to oxidative damage, were measured. As shown in FIGS. 9A and B, significant increases in plasma MDA and hydroperoxide concentrations were observed in the saline and RBC administration groups as compared to the control group. On the other hand, in the CO-RBC administration group, their increase was suppressed. In addition, when the relationship between free heme concentration and MDA was examined, a positive correlation tendency was observed between the two (FIG. 9C).

  When Mb in muscle leaks into blood and deposits in kidney tissue, intramolecular heme iron is oxidized from divalent to trivalent to generate Mb oxidant (Non-patent Documents 17 and 18). Since this oxidant causes more oxidative stress as a stronger oxidant, attention is focused on its relationship to the pathological condition. Since the Mb oxidant has an absorption maximum at around 410 nm, the presence or absence of the Mb oxidant in renal tissue in each group was measured using this as an index. FIG. 10 shows absorption spectra of renal homogenates in the physiological saline, RBC, and CO-RBC administration groups. In the physiological saline-administered group, the absorbance at around 410 nm increased compared to the control, and accumulation of Mb oxidant was observed. Similar spectral changes were also observed in the RBC administration group. On the other hand, the increase was suppressed in the CO-RBC administration group, and showed similar spectral characteristics to the control group. From the above results, it was shown that CO-RBC inhibits the accumulation of free heme and Mb oxidant in rhabdomyolysis AKI model rats, thereby suppressing the enhancement of oxidative stress in renal tissues.

(1-8) Effect on Survival Rate The rescue effect of CO-RBC on rhabdomyolysis AKI was examined. Saline, RBC or CO-RBC was post-administered 3 hours after glycerol treatment to glycerol-induced rhabdomyolysis AKI model rats, and the survival rate thereafter was assessed daily. As a result, in the saline and RBC treatment groups, death was confirmed from the second day of observation, and on the fifth day, 80% (8/10) in the saline treatment group and 60% in the RBC treatment group 10) died (Figure 11). On the other hand, no deaths were observed during the observation period in the CO-RBC-treated group (0/10). From the results, it was found that CO-RBC has an excellent life-saving effect on non-traumatic rhabdomyolysis AKI rats.

(2) Effects on survival rates of traumatic rhabdomyolysis AKI rats We investigated the therapeutic effect of CO-RBC on traumatic rhabdomyolysis, which accounts for the majority of rhabdomyolysis. Taking account of traumatic rhabdomyolysis AKI with hemorrhage, 40% blood removal is performed 1 hour after glycerol treatment, and resuscitation with saline, RBC, CO-RBC is performed simultaneously, and survival rate is temporally Evaluated. As a result, in the saline-administered group, death was observed 4 hours after the onset of hemorrhagic shock, and death of all rats was confirmed within 9 hours (0/10) (FIG. 12). In addition, even in the RBC administration group, death was confirmed 4 hours after hemorrhagic shock, and the survival rate after 24 hours was 10% (1/10). In contrast, all patients survived in the CO-RBC group (10/10). From this, it became clear that post-administration of CO-RBC exerts an excellent therapeutic effect also on traumatic rhabdomyolysis AKI.

(3) Influence on survival rate of crash syndrome model rat CO-RBC exerts excellent rescue effect on rhabdomyolysis, and therefore exerts therapeutic effect on crash syndrome The usefulness was evaluated by the survival rate in anticipation of not being. According to the existing method (Non-patent Documents 12 and 13), a rubber band wound around a stainless steel pipe was attached to a rat's both hind legs under a load of 2.0 kg, and a crash syndrome model was created by pressing for 5 hours. The rat's hind legs turned purple due to compression, and myopathy was suggested from the appearance. Five hours after wearing the rubber band, the pressure was released, and one hour later, saline, RBC or CO-RBC was administered, and the survival rate was monitored thereafter. As a result, in the saline-administered group, death was observed from 3 hours after pressure release, and all cases died within 9 hours (10/10) (FIG. 13). In addition, even in the RBC administration group, a fatal case appeared 6 hours after pressure release, and all cases died within 18 hours (10/10). In contrast, all cases survived in the CO-RBC administration group (0/10). From these results, it was found that CO-RBC also exerts a therapeutic effect on crash syndrome.

Claims (4)

A therapeutic agent for rhabdomyolysis comprising carbon monoxide-added erythrocytes as an active ingredient. The therapeutic agent for rhabdomyolysis according to claim 1, which is a therapeutic agent for rhabdomyolysis-induced acute kidney injury. The therapeutic agent for rhabdomyolysis according to claim 1 or 2, which is a therapeutic agent for crash syndrome. The therapeutic agent for rhabdomyolysis according to any one of claims 1 to 3, which is administered intravenously.