JP5527535B2 - Test method of effect of taking steroid by autotaxin measurement - Google Patents

Description

  The present invention relates to a method for monitoring steroid use or effect by measuring autotaxin concentration in a human sample, a reagent using the method, and a method for reducing human autotaxin by taking steroids.

Steroid drugs are drugs that are used in a wide range of diseases because of their anti-inflammatory, antiallergic, and immunosuppressive effects. In particular,
(1) In internal medicine and pediatrics,
(1-1) Chronic adrenocortical dysfunction, acute adrenocortical dysfunction, adrenal genital syndrome, subacute thyroiditis, thyroid poisoning, malignant ocular bulge associated with thyroid disease, ACTH (adrenocorticotropic hormone) alone Deficiency,
(1-2) Rheumatoid arthritis in rheumatic diseases, juvenile rheumatoid arthritis, rheumatic fever, rheumatic polymyalgia, lupus erythematosus in collagen disease, systemic vasculitis, polymyositis, dermatomyositis, scleroderma,
(1-3) nephrotic and nephrotic syndrome in renal diseases,
(1-4) Congestive heart failure in heart disease,
(1-5) Bronchial asthma in allergic diseases, asthmatic bronchitis, allergies / addiction due to drugs or other chemicals, serum diseases, severe infections in severe infections,
(1-6) hemolytic anemia in blood diseases, leukemia, granulocytopenia, purpura, aplastic anemia, bleeding predisposition due to coagulation factor disorder,
(1-7) localized enteritis, ulcerative colitis in digestive organ disease,
(1-8) improvement of general condition in severe debilitating disease,
(1-9) Fulminant hepatitis in liver disease, cholestatic acute hepatitis, chronic hepatitis, cirrhosis,
(1-10) sarcoidosis in lung disease, diffuse interstitial pneumonia,
(1-11) pulmonary tuberculosis in tuberculous disease, tuberculous meningitis, tuberculous pleurisy, tuberculous peritonitis, tuberculous peritonitis,
(1-12) Encephalomyelitis, peripheral neuritis, myotonia, myasthenia gravis, multiple sclerosis, chorea, facial nerve palsy, spinal fistula retinitis in neurological diseases,
(1-13) malignant lymphoma in malignant tumors and similar diseases (related diseases), recurrent metastasis of breast cancer,
(1-14) idiopathic hypoglycemia, fever of unknown cause as other medical diseases,
(2) Adrenalectomy in the surgical field, organ / tissue transplantation, post-invasive pulmonary edema, surgical invasion for patients with adrenal cortical dysfunction, snake venom / insect poison,
(3) Ankylosing spondylitis in the orthopedic field,
(4) Prevention of adhesion after oviduct surgery in obstetrics and gynecology, ovulation disorder due to adrenal cortex dysfunction,
(5) Prostate cancer in the urological field, penile induration,
(6) Eczema / dermatitis group, prurigo group, psoriasis, palmoplantar pustulosis, erythematous pustulosis, lichen planus, adult edematous sclerosis Erythematosis, anaphylactoid purpura, Weber Christian disease, mucocutaneous ocular syndrome, Raynaud's disease, alopecia areata, pemphigus group, Duhring herpes zoster, congenital epidermolysis bullosa, herpes zoster, erythroderma Disseminated miliary lupus, allergic vasculitis, ulcerative chronic pyoderma, neonatal sclerema, eosinophilic fasciitis,
(7) Inflammatory diseases of the internal eye, optic nerve, orbit, and eye muscles in the ophthalmic field, inflammatory diseases of the external and anterior eyes, postoperative inflammation,
(8) Acute / chronic otitis media in the field of otolaryngology, exudative otitis media / ear canal stenosis, Meniel disease and Meniel syndrome, acute sensorineural hearing loss, vasomotor rhinitis, allergic rhinitis, hay fever, sinusitis / Nasal polyposis, progressive gangrenous rhinitis, laryngitis / laryngeal edema, esophageal inflammation and esophageal dilatation, postoperative treatment in otolaryngology area, refractory stomatitis and glossitis, olfactory disturbance, acute / chronic salivary glanditis,
It is used for the treatment.

  Although it is a steroid drug that has been used in a wide variety of ways as described above, side effects, particularly side effects caused by long-term use or large-scale use, are problematic. Side effects include elevated blood pressure, edema, impaired glucose tolerance, lipid metabolism disorder, osteoporosis, aseptic necrosis of the head, peptic ulcer, muscle weakness, moon face, psychiatric symptoms, insomnia, acne, skin striae, cataract, glaucoma, leukocytes Increases, hypokalemia, adrenocortical dysfunction, etc. are known.

  Therefore, when administering a steroid drug, it is preferable to stop the administration or reduce the dose as soon as the therapeutic effect is confirmed. However, in vivo concentration measurement of a steroid drug is not easy, and extraction from a blood sample, analysis with a large apparatus, and the like are necessary, and thus it cannot be widely used. In addition, steroid drugs are used in a large number of diseases as described above, and there are many cases in which compliance with medication is not observed depending on patients who have been prescribed steroid drugs, but it is difficult to confirm it. Prescriptions for steroid drugs are mostly adjusted by confirming the appearance of therapeutic effects and the occurrence of side effects, etc., and since there is no quantitative control such as in vivo concentration, side effects due to overdose are a concern. Yes. Therefore, a steroid drug monitoring method that can easily monitor the concentration of a steroid drug in a living body and avoids taking a steroid drug more than necessary is eagerly desired.

WO2008 / 016186

J. et al. Biol. Chem. 256, 2524-2529, 1992 Nat. Rev. Cancer 3, 582-591, 2003 Int. J. et al. Cancer 10, 109, 833-838, 2004 Blood, 106, 2138-2146, 2005 Clin. Chim. Acta, 388, 51-58, 2008

  The treatment of taking steroids is carried out for a wide variety of diseases, but it is preferable to avoid overdosing because of the large side effects. In the treatment of taking steroids, the current situation is to control the reduction or stop of the dose by confirming the cure of the disease, or to control the dose based on the confirmation of the onset of side effects. In addition, since steroid drugs are widely used, there are many cases in which compliance with medication is not observed, and a method capable of quantitatively monitoring the dose is desired. However, conventional methods for monitoring steroid drugs have not been able to be carried out simply because they require advanced analytical techniques and equipment.

  Therefore, an object of the present invention is to provide a method capable of easily monitoring a steroid drug and a reagent using the method.

  As a result of intensive studies by the inventor in view of the above problems, it has been found that by taking steroids in the treatment of skin diseases, the autotaxin concentration in serum decreases, and the phenomenon is caused by taking steroids regardless of the type of skin disease. The present invention has been completed by finding that it is a general phenomenon.

Specifically, this application includes the following inventions:
(1) A method for examining the effect of taking a steroid by measuring the autotaxin concentration in a human sample.
(2) The test method according to (1), wherein the autotaxin concentration in a human sample before taking a steroid is used as an index.
(3) The test method according to (1) or (2), wherein the steroid is any one of prednisolone, cortisone, hydrocortisone, methylprednisolone, dexamethasone, and betamethasone.
(4) The test method according to any one of (1) to (3), wherein the autotaxin concentration in a human specimen is measured by an immunochemical technique.
(5) The test method according to any one of (1) to (3), wherein the method for measuring autotaxin concentration in a human specimen is a method for measuring lysophospholipase D activity.
(6) A test reagent for the effect of taking a steroid using the test method according to any one of (1) to (5).
(7) A method for reducing human autotaxin by taking steroids.
(8) A method for reducing human autotaxin by taking a steroid and an autotaxin antagonist in combination.
(9) The reduction method according to (7) or (8), wherein the steroid is any one of prednisolone, cortisone, hydrocortisone, methylprednisolone, dexamethasone, and betamethasone.
(10) The reduction method according to any one of (7) to (9), which is intended to inhibit malignant tumor growth or metastasis.
(11) A method for reducing human autotaxin by administering a steroid for a human suffering from a disease requiring reduction of human autotaxin.
(12) The method according to (11), wherein the disease requiring reduction of human autotaxin is a disease selected from malignant tumors, pregnancy-induced hypertension syndrome and chronic liver disease.
(13) A pharmaceutical composition for reducing human autotaxin comprising a steroid.
(14) The pharmaceutical composition according to (13), further comprising an autotaxin antagonist.
(15) A method for examining the effect of taking a steroid by measuring the autotaxin concentration in a human specimen and comparing it with a reference.
(16) The steroid according to (15), based on an autotaxin concentration predicted value calculated from an autotaxin concentration in a human sample measured before taking a steroid, based on a calibration curve for a steroid dose and an autotaxin concentration Examination method for taking effect.

  According to the present invention, a steroid intake monitor can be quantitatively measured by measuring the autotaxin concentration in a human sample. Autotaxin can be measured by measuring lysophospholipase D activity, which is an enzyme activity of autotaxin. However, when it is measured using the immunoassay reagent described in Patent Document 1, the endogenous content contained in the sample is measured. The human autotaxin can be quantified in a short time without being affected by the measurement interfering factors and competitive enzymes. Furthermore, since human autotaxin is reduced by taking steroids, it is expected to reduce human autotaxin efficiently by taking treatments aimed at reducing human autotaxin, such as taking steroids for cancer growth and metastasis. it can.

The fluctuation | variation of the serum autotaxin density | concentration at the time of taking prednisolone in an eosinophilic fasciitis patient (A) and a dermatomyositis patient (B) is shown. The numerical value in the figure shows the prednisolone dose (mg / day) of the previous day. The fluctuation | variation of the serum autotaxin density | concentration at the time of taking prednisolone in two patients with systemic scleroderma (A) (B) is shown. The numerical value in the figure shows the prednisolone dose (mg / day) of the previous day. Fig. 3 shows changes in autotaxin concentration and lysophospholipase D activity when prednisolone is taken in systemic scleroderma. In the figure, black circles indicate autotaxin concentration, and white squares indicate lysophospholipase D activity. The numerical value in the figure shows the prednisolone dose (mg / day) of the previous day. Fig. 4 shows the correlation between prednisolone dose and autotaxin concentration (A) or lysophospholipase D activity (B).

Hereinafter, the present invention will be described in detail.
Human autotaxin was developed in 1992 by M.M. L. It is a glycoprotein having a molecular weight of about 125 KDa isolated by Stracke et al. As a substance that induces cell motility from A2058 human melanoma cell culture medium (Non-patent Document 1). Autotaxin produces lysophosphatidic acid (LPA) using lysophosphatidylcholine as a substrate due to its lysophospholipase D activity. Many researchers have shown that LPA is involved in cancer growth and metastasis in vivo (Non-Patent Documents 2 to 4), and the causal relationship between autotaxin, which is its production enzyme, and various diseases has been studied. Has been. Recently, a method for quantifying human autotaxin has been established (Patent Document 1, Non-Patent Document 5), and is expected as a diagnostic marker for various diseases.

Therefore, as a result of an intensive study by the inventor on the relationship between steroid use and autotaxin concentration,
It was found that the autotaxin concentration in human blood was lowered by taking steroids, and further, the autotaxin concentration in human blood was lowered in proportion to the dose of steroids. The autotaxin concentration in a human specimen can be accurately quantified by the method using the immunochemical technique described in Patent Document 1, for example, without requiring pretreatment of the specimen. Therefore, for example, by measuring the autotaxin concentration in a human sample with a reagent using the method described in Patent Document 1, quantitative monitoring of the effect of taking steroids, which has been difficult until now, becomes easy, and excessive administration of steroid drugs Can be controlled quantitatively. Further, according to the method of the present invention, it is possible to confirm whether or not the compliance is observed.

  When a steroid is taken, the autotaxin concentration decreases in a dose-dependent manner according to the steroid dose and the type of steroid (FIG. 4A). Therefore, the autotaxin concentration of a patient after taking a steroid is predicted using a calibration curve obtained by plotting the steroid dose and the autotaxin concentration (or reduction ratio) and performing linear regression as shown in FIG. 4A. be able to. A calibration curve must be established for each steroid.

  The physician can predict the patient's autotaxin concentration from the prescribed steroid dose based on a calibration curve for steroid dose and autotaxin concentration (or percent reduction), against which the autotaxin concentration in the patient When the measured value of is significantly high or low, it can be determined that the compliance is not observed. For example, when a doctor prescribes 30 mg of prednisolone per day, according to the calibration curve of FIG. 4A, it is predicted that the autotaxin concentration before taking will be reduced by about 30%, which is significantly higher than this predicted concentration. If it is high or low, medication compliance is not being observed. Specifically, when there is a divergence of at least 10%, preferably 20%, 30%, 40%, 50%, 70%, 80%, or 90% with respect to the predicted value obtained from the calibration curve, It can be determined that compliance is not observed.

  When monitoring the effect of taking steroids, it is desirable to keep the rate of decrease from the autotaxin concentration before taking a certain range determined according to the treatment stage. Steroids are generally known to have strong side effects and withdrawal symptoms. Therefore, in order to reduce the withdrawal symptoms in order to treat side effects and reduce withdrawal symptoms, a relatively high autotaxin concentration is reduced at the initial stage of treatment. It is desirable to control the amount of steroid so that the rate of reduction, for example, 40% to 70%, preferably 50% to 60%, is maintained and the rate of decrease in autotaxin concentration is gradually reduced as the treatment progresses. . For example, it is desirable to control the amount of steroid so as to maintain a reduction rate of 20 to 50%, preferably 30 to 40% in the middle stage of treatment, and 10 to 30%, preferably 10 to 20% in the late stage of treatment. .

  Due to the phenomenon of lowering autotaxin concentration in human specimens by taking steroids found this time, it is expected that taking steroids can be a technique to reduce human autotaxin. Therefore, the method of taking steroids to patients can be a method for controlling the growth and metastasis of cancers involving human autotaxin, particularly cancers involving LPA produced by lysophospholipase D activity possessed by human autotaxin. I can expect. In particular, steroid drugs are expected to improve the autotaxin antagonism of molecular target drugs by using them as adjuvants in the treatment with antagonists such as molecular target drugs for the purpose of reducing human autotaxin it can. Steroid drugs themselves have already been confirmed to be safe, and the mechanism of action differs from that of new drugs such as molecular target drugs. Is thought to be easy.

  The steroid used in the present invention is not particularly limited as long as it is a steroidal anti-inflammatory drug usually used in the treatment of diseases. Difurazone, difluprednate, thixortorol, dexamethasone, dexibudesonide, desoxycorticosterone, deflazacort, deprodon, triamcinolone, tolterodine, hydrocortisone, parameterzone, halcinonide, halopredone, halometasone, hydroxycortisone, hydrocortisone, budesonide, flucinone , Fluocinolone acetonide, fluorometholone, fluticasone, fludrocortisone, Lunisolid, fluprednisolone, flumethasone, flulandrenolide, predniscarbate, prednisolone, progesterone, beclomethasone, betamethasone, methylprednisolone, medorizone, meprednisone, mometasone, rimexolone, rofleponide, and pharmaceutically acceptable salts and solvates thereof Etc. are exemplified. Preferred steroids include prednisolone, cortisone, hydrocortisone, methylprednisolone, dexamethasone, betamethasone, beclomethasone, and fluticasone.

  Examples will be shown below to describe the present invention in more detail. However, these examples show examples of the present invention, and the present invention is not limited to the examples. The serum samples used in the examples were collected and measured at the University of Tokyo Hospital and the research ethics were approved by the Ethics Committee of the University of Tokyo Graduate School of Medicine. Further, the autotaxin concentration was measured using an automatic immunoassay device AIA series (manufactured by Tosoh Corporation) based on the method of Patent Document 1, and the lysophospholipase D activity was measured according to the conventional method.

Measurement of serum human autotaxin concentration A two-step two-antibody sandwich ELISA using human autotaxin monoclonal antibody R10.23 as a solid phase antibody and human autotaxin monoclonal antibody R10.21 as a secondary antibody Human autotaxin was measured. The solid phase antibody R10.23 was converted to F (ab) ₂ by pepsin digestion and used. R10.23 was added to a 96-well immunoplate (manufactured by NUNC) at a concentration of 2 μg / mL at 50 μL / well and allowed to bind to the plate at 4 ° C. overnight. After washing 3 times with TBS, TBS containing 3% BSA was added at 250 μL / well and blocking treatment was performed for 2 hours. After washing 3 times with TBS, human autotaxin standard (0, 0.34, 0.675, 1.35, 2.70, 5.40 μg / mL) and human serum of unknown concentration were washed with ELISA assay buffer. Diluted to 5/5 and added at 50 μL / well. After 2 hours of reaction at room temperature, the plate was washed 4 times with TBST, and ELISA assay buffer containing 0.8 μg / mL of biotin-labeled R10.21 was added at 50 μL / well. After standing at room temperature for 2 hours, the plate was washed 4 times with TBST, and an ELISA buffer containing HRP-labeled streptavidin (manufactured by Zymed) diluted 1000 times was added at 50 μL / well. After standing at room temperature for 1 hour, the plate was washed 6 times with TBST, and TMB substrate was added at 50 μL / well. After 30 minutes at room temperature, the reaction was stopped with 1N-phosphoric acid, and the absorbance at 450 nm was measured. Regression of the calibration curve with human autotaxin standard was performed by cubic regression. An increase in absorbance at 450 nm was confirmed depending on the human autotaxin concentration. Using this calibration curve, the concentration of human autotaxin in the specimen was calculated from the absorbance at 450 nm obtained with an unknown concentration of human serum.

Measurement of serum lysophospholipase D activity The lysophospholipase D activity was measured by slightly changing FEBS letters 571, 197-204, 2004. Specifically, 20 μL of a sample solution and 20 μL of a substrate solution containing 2 mM lysophosphatidylcholine (14: 0-lysophosphatidylcholine), 100 mM Tris-HCl, 500 mM NaCl, 5 mM MgCl2, 0.05% Triton X-100 (pH 9.0) And reacted at 37 ° C. for 6 hours to overnight. Subsequently, 0.5 mM TOOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline), 10 Unit / mL horseradish peroxidase, for quantifying choline produced by the enzyme reaction, After adding 150 μL of an R1 solution consisting of 0.01% Triton X-100 and 100 mM Tris-HCl (pH 8.0) and allowing to stand for 5 minutes, 1 mM 4-aminoantipyrine, 10 Unit / mL choline oxidase, 0.01% Triton X- 50 μL of an R2 solution consisting of 100, 100 mM Tris-HCl (pH 8.0) was added. After 30 minutes, the produced choline was measured at an absorbance of 550 nm, and the lysophospholipase D activity value was determined based on a known concentration of choline chloride.

Example 1: Autotaxin concentration monitor in patients with eosinophilic fasciitis and dermatomyositis When taking prednisolone, a steroid, for female eosinophilic fasciitis and male dermatomyositis patients, The autotaxin concentration in serum with the passage of time was measured. In patients with eosinophilic fasciitis, the autotaxin concentration when prednisolone was not taken was 1.187 mg / L, but the autotaxin concentration was lowered by taking prednisolone, and the prednisolone dose was up to 13.5 mg / day. It was shown that the low value was maintained (FIG. 1A). In patients with dermatomyositis, it was shown that the autotaxin concentration decreased from a low dose of 8 mg / day to a high dose of 75 mg / day, and the autotaxin concentration was low in the course of continuous 20 mg / day prednisolone. (FIG. 1B). In addition, in each of patients with eosinophilic fasciitis and dermatomyositis, an increase in autotaxin concentration was observed when the prednisolone dose was reduced (FIGS. 1A and B).

Example 2: Monitor of autotaxin concentration in patients with systemic scleroderma The concentration of autotaxin in serum with the lapse of time was measured when prednisolone was taken in two patients with systemic scleroderma. In any patient, the autotaxin concentration after taking prednisolone was lower than that before taking prednisolone (FIGS. 2A and B). Further, when the prednisolone dose was reduced, an increase in autotaxin concentration was observed (FIG. 2B).

Example 3: Monitoring autotaxin concentration and lysophospholipase D activity in patients with systemic scleroderma Serum autotaxin concentration and lysophospholipase D over time when prednisolone was taken to patients with systemic scleroderma Activity was measured. It was confirmed that both the serum autotaxin concentration and the lysophospholipase D activity can monitor the prednisolone dose equally (FIG. 3). In addition, when the relationship between prednisolone dose and autotaxin concentration or lysophospholipase D activity was plotted, the autotaxin concentration (correlation coefficient 0.906) (Fig. 4A) and lysophospholipase D activity with respect to prednisolone dose were very good. Correlation (correlation coefficient 0.978) (FIG. 4B). Thus, it can be seen that the prednisolone dose can be monitored by measuring autotaxin or lysophospholipase D activity.

Claims (6)

A method for measuring the autotaxin concentration in human specimens in order to test the effects of taking steroids . The measurement method according to claim 1, wherein the autotaxin concentration in a human sample before taking a steroid is used as an index. The measuring method according to claim 1 or 2, wherein the steroid is any one of prednisolone, cortisone, hydrocortisone, methylprednisolone, dexamethasone, and betamethasone. The measurement method according to claim 1, wherein the autotaxin concentration in a human sample is measured by an immunochemical technique. Measurement method according to any one of claims 1 to 3 the method of measuring the autotaxin concentration in human specimen is a method of measuring the lysophospholipase D activity. A test reagent for the effect of taking a steroid for use in the measurement method according to claim 1 ,
An immunological test reagent containing an anti-autotaxin antibody, or
A test reagent for measuring lysophospholipase D activity containing lysophosphatidylcholine as a substrate and choline oxidase and horseradish peroxidase as enzymes.

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