JP5084481B2 - Method for screening and evaluating blood pressure regulator - Google Patents

Description

  The present invention relates to a screening method and an evaluation method for a blood pressure regulator having a blood pressure lowering action or a blood pressure raising action.

  Nitric oxide produced from vascular endothelium (hereinafter referred to as “NO”) was found as an endothelium-derived relaxing factor (EDRF) in the early 1980s, and regulates vascular tonus and blood pressure. As known as important. In addition, the decrease in the activity of NO synthase (hereinafter referred to as “NOS”) has been closely related to various diseases (cardiovascular diseases, gastrointestinal diseases, renal / urological diseases, neurological diseases, etc.). It is known that Actually, those with high blood pressure values, which are indicators of cardiovascular disorders, etc., compared to those with low blood pressure values, the total nitric oxide in the blood (hereinafter referred to as “NOx”), which is a metabolite of NO produced by NOS NO), which is important as a factor that regulates vascular tonus and blood pressure, considering that the amount is reduced (Non-Patent Document 1) and that the decrease in NOS activity is associated with hypertension (Non-Patent Documents 2 to 4). And NOS is also considered important for blood pressure values.

  Therefore, it is considered to be very important to measure the blood pressure value and the amount of NOx in the blood in estimating the cardiovascular disorder and the like, but there are various problems. First, the events listed as problems of blood pressure measurement are compared with blood pressure at home (home blood pressure (hereinafter referred to as “HBP”)) and blood pressure under ambulatory blood pressure (hereinafter referred to as “ABP”). However, white blood pressure hypertension in which blood pressure (casual blood pressure (hereinafter referred to as “CBP”)) at the time of outpatient medical treatment at a medical institution, etc., and masked hypertension, etc., in which CBP is low compared to high values of HBP and ABP are known ( Non-patent document 5). As described above, it is difficult to accurately know the blood pressure that can be an index of circulatory disorders reflecting the internal environment.

  Thus, in order to know the blood pressure value accurately, a blood marker that assists the blood pressure value can be useful, but the amount of blood NOx that can be cited as a candidate has a problem in measurement. As an event raised as a problem of blood NOx measurement, NOx is often contained in food (Non-patent Document 6), and the amount of NOx in blood is influenced by the food ingested (Non-patent Document). 7) and contamination of the measurement sample for the amount of blood NOx from laboratory instruments (Non-patent Document 8). As described above, in order to know the blood pressure value accurately, it is desired to search for a blood marker other than NOx sufficient to assist the blood pressure value from the viewpoint of NOS.

  On the other hand, NO mentioned above is produced by NOS when arginine is metabolized to citrulline. As a factor responsible for NOS function regulation, binding sites of calmodulin (CaM), flavin (FAD, FMN), NADPH, heme protein and tetrahydrobiopterin (hereinafter referred to as “BH4”) have been analyzed so far. Has been found to exist in NOS. In fact, BH4 has been reported to be involved in NOS function control. In recent years, BH2 contained in biopterin (hereinafter referred to as “BP”) (BH4, dihydrobiopterin (hereinafter referred to as “BH2”) and BP) similar to BH4 has also been reported to be involved in NOS function control. , BH4, and BH2 are also attracting attention as to how to control the function of NOS (Non-Patent Documents 9 to 15).

  In the study of BPs involved in the regulation of NOS function, it has been known from laboratory reports that the amount of BH4 in vivo and the ratio of BPs (BH4 / BH2) decrease in vascular dysfunction at the onset of hypertension and diabetes in experimental animals. Is reported to be involved (Non-Patent Documents 16 to 21), and clinical trials of BH4 administration have been reported to improve vascular dysfunction (Non-Patent Documents 16 to 18 and 20). ). In addition, in humans, it has been reported that insulin resistance patients and renal disease carriers are associated with a decrease in the abundance ratio (BH4 / BH2) of BPs (Non-patent Documents 22 and 23). Furthermore, clinical trials of BH4 administration have been made for various diseases such as arteriosclerosis, coronary artery disease and hypertension, and it has been reported that vascular dysfunction is improved (Non-Patent Documents 24-37).

  Based on the above, BPs are important for various diseases such as prevention and treatment of arteriosclerosis, hypertension, hypercholesterolemia, diabetic vascular disorder related to vascular dysfunction in laboratory animals and humans It is believed that.

Node K et al. Hypertension (1997) 30: 405-408 Huang PL et al. Nature (1995) 377: 239-242 Forte P et al. Lancet (1997) 349: 837-842 Taddei S et al. Circulation (1996) 94: 1298-1303 Taku Ohara et al., Journal of Blood Pressure (2004) 11 (8) 19-23 Mariko Himeno J. Kanazawa Med. Univ (2001) 26: 170-180 Mariko Himeno et al. Journal of the Japan Society for Internal Medicine (2001) 90: 265 Takaharu Ishibashi et al. Nitric oxide (2000) 4 (5) 516-525 Gross SS et al. Nitric Oxide Biology and Pathobiology (2000) 167-187 Knowles RG et al. Biochem J (1994) 298: 249-258 Vasquez-Vivar J et al. Biochem J. (2002) 362 (Pt 3): 733-9 Blau N et al. Arterioscler Thromb Vasc Biol. (2003) 23 (5): 913-4 Vasquez-Vivar J et al. Proc Natl Acad Sci USA (1998) 95: 9220-9225 Vasquez-Vivar J et al. Free Radic Res (2003) 37 (2) 121-7 Kuzkaya N et al. J Biol Chem (2003) 278 (25) 22546-54 Katusic ZS et al. Am J Physiol Heart Circ Physiol (2001) 281 (3) H981-6 Landmesser U et al. J Clin Invest (2003) 111 (8) 1201-9 Shinozaki K et al. J Pharmacol Sci (2003) 91 (3) 187-91 Cosentino F et al Circulation (1995) 91: 139-144 Hong HJ et al. Hypertension (2001) 38: 1044-1048 Kerr S et al. Hypertension (1999) 33 (6): 1353-8 Shinozaki K et al. J Am Coll Cardiol (2001) (7) 1821-8 Yokoyama K et al. Nephrol Dial Transplant (2002) (6) 1032-6 Stroes E et al. J Clin Invest (1997) 99: 41-46 Fukuda Y et al. Heart (2002) 87: 264-269 Wyss CA et al. Eur J Nucl Med Mol Imaging (2005) 32 (1): 84-91 Tiefenbacher CP et al Circulation (2000) 102: 2172-2179 Verma S et al. J Thorac Cardiovasc Surg (2000) 120: 668-671 Maier W et al. J Cardiovasc Pharmacol (2000) 35: 173-178 Higashi Y et al. Am J Hypertens (2002) 15: 326-332 Fukuda Y et al. Circ J (2002) 66: 58-62 Setoguchi S et al. J Cardiovasc Pharmacol (2002) 39: 363-368 Ueda S et al. J Am Coll Cardiol (2000) 35: 71-75 Heitzer T et al. Circ Res (2000) 86: E36-E41 Heitzer T et al. Diabetologia (2000) 43: 1435-1438 Guzik TJ et al Circulation (2002) 105: 1656-1662 Ihlemann N et al. Am J Physiol Heart Circ Physiol (2003) 285: H875-H882

  As described above, a blood marker other than NOx, which is sufficient to assist the blood pressure value in order to know the blood pressure value accurately, is desired. As such blood markers, BPs are considered to be applicable, but the relationship between blood pressure values and BPs has not been clarified. Therefore, an object of the present invention is to clarify the relationship between the blood pressure value and the amount of BPs in blood, and to provide a screening method and an evaluation method for blood pressure regulators that focus on the concentration of BPs in blood.

  As a result of intensive studies by the present inventors in order to achieve the above-described object, the present inventors have completed the present invention by finding new knowledge that blood BH2 concentration correlates with blood pressure value among BPs. .

  That is, the method for screening a blood pressure regulator according to the present invention comprises a step of administering a candidate substance to a model animal other than a human and a step of measuring a dihydrobiopterin concentration in the blood of the model animal, When the dihydrobiopterin concentration is increased, the candidate substance is determined to have a blood pressure increasing action, and when the dihydrobiopterin concentration is decreased by the candidate substance, the candidate substance is determined to have a blood pressure lowering action. .

  In the method for screening a blood pressure regulator according to the present invention, for example, a hypertensive model animal is used as the model animal, and the antihypertensive agent is identified by measuring a decrease in dihydrobiopterin concentration in the blood of the model animal. can do.

  In the method for screening a blood pressure regulator according to the present invention, the method for measuring the concentration of dihydrobiopterin in blood is not particularly limited, and a conventionally known method can be used as appropriate. The blood concentration of dihydrobiopterin may be, for example, a value measured as a combined value of blood dihydrobiopterin and biopterin.

  The method for evaluating a blood pressure regulator according to the present invention includes a step of administering a substance to be evaluated to a model animal other than a human and a step of measuring a dihydrobiopterin concentration in the blood of the model animal. In particular, in the step of measuring the dihydrobiopterin concentration, it is preferable to evaluate the degree of variation of the dihydrobiopterin concentration and the variation timing. In the step of measuring the dihydrobiopterin concentration, it is preferable to evaluate the variation of the dihydrobiopterin concentration due to other factors such as other substances and various factors such as stress.

  According to the present invention, a substance that can be a blood pressure regulator can be screened out of various candidate substances based on a novel blood marker that assists blood pressure. In particular, according to the screening method for a blood pressure regulator according to the present invention, a change in blood pressure value can be measured with high accuracy according to the novel marker, so that screening with excellent reliability can be performed.

  Further, according to the present invention, it is possible to evaluate the blood pressure lowering action of the evaluation target substance based on the blood pressure marker. In particular, according to the method for evaluating a blood pressure adjusting agent according to the present invention, a change in blood pressure value can be measured with high accuracy according to the novel marker, and therefore, an evaluation with excellent reliability can be performed.

  Hereinafter, the present invention will be described in detail with reference to the drawings.

  The blood pressure regulator screening method and blood pressure regulator evaluation method according to the present invention are based on the novel finding that the dihydrobiopterin concentration in blood correlates with the blood pressure value. Here, the blood pressure regulator means to include both a blood pressure raising agent and a blood pressure lowering agent. That is, in the method for screening a blood pressure preparation agent according to the present invention, the blood pressure increasing action or blood pressure lowering action of the tested candidate substance is determined based on the fluctuation of the blood dihydrobiopterin concentration. That is, according to the screening method for a blood pressure regulator according to the present invention, a substance having an unknown or uncertain blood pressure regulating action is used as a candidate substance, and the blood pressure regulating action of the candidate substance is determined.

  In the method for evaluating a blood pressure regulator according to the present invention, the blood pressure increasing action or the blood pressure lowering action of the evaluation target substance is evaluated based on the fluctuation of the blood dihydrobiopterin concentration. That is, the method for evaluating a blood pressure adjusting agent according to the present invention determines the blood pressure adjusting action of the substance to be evaluated using a substance known or suggested to have a blood pressure adjusting action as the substance to be evaluated.

  Specifically, when the concentration of dihydrobiopterin in blood increases when a candidate substance or a substance to be evaluated is administered to a model animal, it is determined that these substances have a blood pressure increasing action. On the other hand, if the concentration of dihydrobiopterin in the blood decreases when a candidate substance or a substance to be evaluated is administered to a model animal, it is determined that these substances have a blood pressure lowering effect.

  In the present invention, a conventionally known method can be appropriately used as a method for measuring the concentration of dihydrobiopterin in blood. Here, the blood dihydrobiopterin concentration may be a value obtained by directly measuring BH2 in the blood, or the blood BH2 was oxidized under alkaline conditions to obtain biopterin. It may be a value (converted value). That is, the hydroxybiopterin concentration may be a value measured by converting BH2 contained in blood into biopterin.

  The blood contains BH4 (tetrahydroxybiopterin) in addition to BH2 as a reduced form of biopterin. According to a document disclosing the measurement method of BPs (T. Fukushima et al. Anal Biochem (1980) 102; 176-188), BH2 becomes biopterin when oxidized under both alkaline and acidic conditions. BH4 becomes pterin when oxidized under alkaline conditions and becomes biopterin under acidic conditions. Therefore, when oxidized biopterin is oxidized under alkaline conditions and the concentration of the generated biopterin is measured, it is measured as the combined value of biopterin originally present in the blood and biopterin derived from BH2. It will be. In this way, when the blood hydroxybiopterin concentration is measured by converting it into the biopterin concentration, BH2 can be measured with high accuracy without measuring BH4.

  Further, the candidate substance in the screening method according to the present invention and the evaluation target substance in the blood pressure regulator evaluation method according to the present invention are not particularly limited and may be any substance. These substances may be single substances or a mixture composed of a plurality of components. As these substances, for example, an unidentified substance such as an extract from a plant may be included, or a known composition may be included at a predetermined composition ratio. These substances may be any of proteins, nucleic acids, lipids, polysaccharides, organic compounds and inorganic compounds.

  When these substances are administered to a model animal, the dosage and administration interval are not particularly limited, and can be set as appropriate. Moreover, it does not specifically limit as a model animal, Mammals except humans, such as a mouse | mouth, a rat, and a monkey, can be mentioned. In particular, it is preferable to use a hypertensive model animal such as a hypertensive model rat.

  In the screening method and the evaluation method according to the present invention, blood is collected from the model animal after these substances are administered to the model animal, but the blood collection timing, the blood sampling interval, etc. are not particularly limited and can be appropriately set. .

  As described above, according to the method for screening a blood pressure regulator according to the present invention, a substance having a blood pressure increasing action or a substance having a blood pressure lowering action can be identified with high accuracy from the tested candidate substances. it can.

  For example, by comparing a known substance (for example, a known vasopressor or antihypertensive agent) having a blood pressure regulating action with a change in hydroxybiopterin concentration when administered to a model animal, the blood pressure regulating action by the known substance and a candidate It is also possible to characterize the similarity to a substance. Known antihypertensive agents and antihypertensive agents are classified into various types according to the timing at which their efficacy is exhibited, the degree of blood pressure adjustment, and the like. Therefore, a known blood pressure regulator is administered to a model animal, and the degree of variation and timing of the hydroxybiopterin concentration in the model animal are monitored in advance, and the degree of variation and timing of the comparison are compared, so that the known substances are known among the candidate substances. Substances similar to vasopressors and antihypertensives can be screened.

  The substance having a blood pressure increasing action identified by the method for screening a blood pressure preparation agent according to the present invention can be a candidate for a therapeutic or prophylactic drug for diseases caused by hypotension. In addition, the substance having a blood pressure lowering action identified by the screening method of the blood pressure preparation agent according to the present invention is a hypertension or a disease caused by a hypertension state, such as stroke, cerebral infarction, cerebral hemorrhage, cardiac hypertrophy, heart failure, narrowing. It can be a candidate for a therapeutic or prophylactic agent for heart disease, myocardial infarction, nephrosclerosis, renal failure and the like. In particular, it can also be used to prevent the onset of hypertension, stroke, etc. from a hypertensive state.

  In addition, according to the method for evaluating a blood pressure regulator according to the present invention, the blood pressure regulating action of the substance to be evaluated can be evaluated. In the method for evaluating a blood pressure regulator according to the present invention, a known substance having a blood pressure regulating action can also be used as a substance to be evaluated. Here, the evaluation means evaluation of the degree of the effect relating to the blood pressure adjustment effect, evaluation of the time when the effect relating to the blood pressure adjustment effect appears, evaluation of the influence of various factors on the blood pressure adjustment effect, and the like. That is, after the evaluation target substance is administered to the model animal, the degree of the effect on the blood pressure regulating action by the candidate substance and the onset time of the effect can be evaluated by monitoring the degree and timing of the fluctuation of the hydroxybiopterin concentration. . Furthermore, by further administering other substances or applying stress to the model animal to which the candidate substance has been administered, it is possible to evaluate the effect on blood pressure regulating action by various factors such as the other substances and stress. .

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.

[Example 1]
In this example, the relationship between the amount of blood BPs and nitrogen oxides in blood and the blood pressure value was examined.

<Meal control in the subject>
In this study, 16 healthy men who were not taking antihypertensive drugs were subject to testing. The blood sampling time in this study was between 9:00 am and 9:30 pm, taking into account the effect of daily fluctuations on the measurement items of this study. In addition, in order to avoid the influence on the measurement results due to fluctuations in the content of meal intake, all subjects took alcohol refusal from 3 days before and the same dinner on the previous day. Furthermore, on the day of blood collection, all subjects refrained from ingesting other than water and refrained from eating and drinking 1 hour before blood collection. All subjects finished eating 12 hours before blood collection.

<Blood pressure measurement>
The subject to be tested was rested for 10 minutes before measurement, and the blood pressure was measured twice using a fully automatic sphygmomanometer (TM-2655VP: manufactured by A & D CO., LTD). The average value of the obtained blood pressure was used as a measurement result.

<Measurement of blood total nitrogen oxide (NOx)>
A plasma sample for NOx measurement was prepared from a blood sample collected from the test subject. Plasma samples for NOx measurement were mixed with 100% methanol and 1: 1, and centrifuged at 4 ° C. and 10,000 × g for 20 minutes for deproteinization. Thereafter, the total nitrogen oxide amount contained in the supernatant was measured using an HPLC system (ENO-20; manufactured by Eicom) using Griess reagent reaction, and the amount of nitrogen oxide in plasma was calculated.

<Measurement of blood BP levels>
BPs contained in the blood were measured using a blood sample collected from the subject. This measurement is based on the method described in the literature (T. Fukushima et al. Anal Biochem (1980) 102; 176-188) disclosing the method for measuring BPs in all subjects. I went. According to this document, the amount of reduced BPs (ie, BH4) is determined based on the total amount of BPs (sum of the amount of reduced BPs and the amount of oxidized BPs) determined from the iodine oxidation reaction under acidic conditions and the alkaline conditions. It calculated from the amount of oxidized BPs obtained from the iodine oxidation reaction below. The abundance ratio of BPs was expressed as the amount of reduced BPs (BH4) / oxidized BPs (BH2).

<Iodine oxidation reaction under acidic conditions>
40 μL 1N HCl and 100 μL iodine oxidation solution (1% I ₂ , 2% KI) were added to and mixed with 200 μL of a plasma sample for measuring BPs. After standing at room temperature in a dark place for 1 hour, 20 μL 5% ascorbic acid and 40 μL distilled water were added to stop the reaction. After the reaction, the sample was subjected to ultrafiltration (Microcon 10NMWL: Millipore Corp., Bedford, MA, USA), and the filtrate containing iodine oxide was used as an HPLC sample.

<Iodine oxidation reaction under alkaline conditions>
A plasma sample for measuring BPs was prepared from a blood sample collected from a test subject. Then, 40 μL of 1N NaOH and 100 μL iodine oxidation solution (1% I ₂ , 2% KI) were added to and mixed with 200 μL of the plasma sample for measuring BPs. After standing at room temperature for 1 hour in the dark, 20 μL 5% ascorbic acid and 40 μL 2N HCl were added to stop the reaction. After the reaction, the sample was ultrafiltered, and a filtrate containing iodine oxide was used as an HPLC sample.

<Detection by HPLC>
The obtained HPLC sample (50 μL) was analyzed using an HPLC system (manufactured by Shimadzu), and the amount of BPs and BH4 / BH2 contained in plasma were calculated. The outline of the HPLC system is described below. 50 μL of the sample was injected into an inertsil ODS-3 column (4.6 x 250 mm, manufactured by GL Science). At a column temperature of 35 ° C, two eluents 50 mmol / L acetic acid / distilled water (pH 3.0) Elution was performed using L acetic acid / methanol under a linear gradient at a flow rate of 0.5 mL / min. Detection was performed at an excitation wavelength of 350 nm and a fluorescence wavelength of 450 nm.
(HPLC system overview)
System controller: SCL-10Avp
Column oven: CTO-10ACvp
Pump: LC10ADvp
Auto sampler: SIL-10ADvp
Detector: RF-10AxL

<Analysis of results>
Table 1 shows the results of measurement of the test subject's age, blood pressure, heart rate, BP amount, and NOx amount. The results shown in Table 1 were expressed as an average value ± standard deviation (Average (Ave) ± standard deviation (SD)). In Table 1, Age indicates age, SBP indicates systolic blood pressure, DBP indicates diastolic blood pressure, MBP indicates average blood pressure, HR indicates heart rate, and BP's indicates total BP amount. BH4 indicates the amount of BH4, BH2 indicates the amount of BH2, BH4 / BH2 indicates the abundance ratio of BPs, and NOx indicates the total amount of nitric oxide.

また、相関の検定には、PEARSONの検定を用い、有意差検定はStatView^TM（SAS Institute社製）を用い、FisherのγのZ変換を施行した。本検定においては、p値が0.05未満のものを統計学的有意差があるとした。PEARSONの検定結果を表２に示し、FisherのγのZ変換の結果（P値）を表３に示す。

In addition, PEARSON test was used for correlation test, and StatView ^™ (manufactured by SAS Institute) was used for significance test, and Fisher's γ Z conversion was performed. In this test, a p-value of less than 0.05 was considered to have statistical significance. PEARSON test results are shown in Table 2, and Fisher's γ Z conversion results (P values) are shown in Table 3.

表２に示した結果において、下線を記した数値は、絶対値が大きいため相関が高いと判断することができる。表３に示した結果において、P<0.05であるものに下線を記したが、これらP<0.05であるものは相関の度合いが統計的に有意であると判断することができる。

In the results shown in Table 2, it can be determined that the underlined numerical values have a high correlation because the absolute values are large. In the results shown in Table 3, those with P <0.05 are underlined, but those with P <0.05 can be judged to have a statistically significant degree of correlation.

  From the results in Table 3, it was revealed that blood pressure values (particularly systolic blood pressure and mean blood pressure) and BH2 amount (synonymous with BH2 concentration in blood) were statistically significantly correlated. Further, from the results of Table 2, it was revealed that there is a significant tendency in the correlation between the blood pressure value (particularly systolic blood pressure) and the BH4 / BH2 value.

[Example 2]
In this example, the change in blood pressure when BH2 was administered to a model animal was verified.

  In this example, Wister rats (spontaneously hypertensive rats (SHR): 10 weeks old, rabbit; purchased from Japan SLC) were used. Rats were preliminarily kept under a room temperature of 23 ± 2 ° C., humidity of 55 ± 10%, and a 12 hour light / dark cycle (light period; AM 7:00 to PM 7:00). During the breeding period, all rats were allowed to eat CE-2 solid food freely and tap water freely. During the test, SHR (♂; 18-26 weeks of age (N = 8)) was anesthetized by intraperitoneal administration (0.1 mL / 100 g body weight) of 1.5% α-chloralose + 7% urethane solution and dissolved in physiological saline. BH2 (12.5 μM / kg body weight) was administered intravenously from the right femoral vein. Saline was administered as a control. After administration, the amount of blood pressure fluctuation was measured over time by a pressure transducer (Polygraph System, manufactured by Nippon Denkou Co., Ltd.) that was cannulated in the left femoral artery.

The blood pressure of the BH2-administered group and the control group was measured every 10 seconds at an elapsed time of 0 to 40 seconds after administration, and the mean value ± standard deviation was calculated for the blood pressure values of each group. And, significant difference test using the StatView ^TM (manufactured by SAS Institute, Inc.), underwent Repeated Measures ANOVA. In this test, a p-value of less than 0.05 was considered to have statistical significance. The results are shown in FIG.

  From the results shown in FIG. 1, it was revealed that the blood pressure value significantly increased in the model animals administered with BH2. When considered together with the results of Example 1, it became clear that the BH2 concentration in blood is positively correlated with the blood pressure value, and that the BH2 concentration in blood can be used as a marker sufficient for monitoring the blood pressure value. .

Example 3
In this example, in order to demonstrate that blood pressure regulators can be screened from candidate substances based on fluctuations in BH2 concentration in blood, fluctuations in BH2 concentration when a known antihypertensive agent was administered to hypertensive model rats were examined. Verified.

  In this example, Wister rats (SHR: 10 weeks old, rabbit; purchased from Japan SLC) were used. Rats were preliminarily raised to 13 weeks of age under a light / dark cycle (light period; AM 7:00 to PM 7:00) of room temperature 23 ± 2 ° C., humidity 55 ± 10%, and 12 hours. During the breeding period, all rats were allowed to eat CE-2 solid food freely and tap water freely.

  After measuring the systolic blood pressure (tail-cuff method) during the pre-breeding period and confirming hypertension, chlorogenic acid (CQA: Cayman) (300 mg / kg) -containing physiological saline was forcibly forced by an oral sonde Administered. CQA is a substance having a blood pressure lowering action as shown in a reference experimental example described later. After a certain period of time after CQA administration, pentobarbital injection solution (40 mg / kg) was intraperitoneally administered to the rat, and after deep anesthesia was confirmed, whole blood was collected from the abdominal aorta. Whole blood is collected in a blood collection tube containing an anticoagulant (final concentration 2 mM EDTA) and a reducing agent (0.1% DTE), and the supernatant obtained by centrifugation at 2000 xg for 20 minutes at 4 ° C is used as plasma for BP measurement A sample was used. Plasma was stored at -80 ° C until use.

To the collected 200 μL of plasma, 40 μL of 1N NaOH and 100 μL of iodine oxidation solution (1% I ₂ , 2% KI) were added and mixed. After standing at room temperature in a dark place for 1 hour, 20 μL 5% ascorbic acid and 40 μL of 2N HCl were added to stop the reaction. After the reaction, the sample was subjected to ultrafiltration (using Microcon 10NMWL Millipore), and the filtrate containing iodine oxide was used as the HPLC sample. The outline of the HPLC system is described below. The sample was injected into a Hypersil ODS (C18) column (4.6 x 250 mm) in a volume of 50 μL, and the column temperature was 35 ° C. Elution was 15 mmol / L potassium phosphate buffer (pH 6.0): methanol (80%: 20%) Detection was performed at an excitation wavelength of 350 nm and a fluorescence wavelength of 450 nm.
(HPLC system overview)
System controller: SCL-10Avp
Column oven: CTO-10ACvp
Pump: LC10ADvp
Auto sampler: SIL-10ADvp
Detector: RF-10AxL

  The results of measuring the BH2 concentration after CQA administration are shown in FIG. As can be seen from FIG. 2, when 6 hours have passed since CQA known as an antihypertensive agent was administered, it was revealed that the BH2 concentration was significantly decreased from the time of CQA administration. As shown in a reference experimental example described later, when CQA was administered to a hypertensive rat under the same conditions as in this example, the blood pressure significantly decreased in about 6 hours after the administration. Considering the results of the reference experiment together, it became clear that the blood pressure lowering effect by CQA can be monitored with high accuracy by measuring the BH2 concentration in the blood.

  Therefore, when a candidate substance is administered to a model animal, the blood BH2 concentration fluctuation (BH2 concentration increase or decrease) is measured, and the blood pressure adjustment action (blood pressure increase action or blood pressure lowering action) of the candidate substance is measured. It became clear that it can be judged based on It was also clarified that the degree and timing of the blood pressure adjusting action of the candidate substance can be determined from the amount of fluctuation of the BH2 concentration and the fluctuation timing.

[Reference experiment example]
In this experimental example, the blood pressure lowering effect by CQA used in Example 3 was examined. In this experimental example, Wister rats (SHR: 10 weeks old, rabbit; purchased from Japan SLC) were used. Rats were preliminarily raised to 13 weeks of age under a light / dark cycle (light period; AM 7:00 to PM 7:00) of room temperature 23 ± 2 ° C., humidity 55 ± 10%, and 12 hours. During the breeding period, all rats were allowed to eat CE-2 solid food freely and tap water freely. The systolic blood pressure (tail-cuff method) was measured during the pre-breeding period, and after confirming hypertension, the saline containing CQA (Cayman) (200 mg / kg) used in Example 3 was used as an oral sonde. Forcibly. After a certain time after administration, systolic blood pressure (tail-cuff method) was measured, and the fluctuation rate (%) of blood pressure was calculated according to the following formula.

CQA投与後、血圧を測定し、血圧の変動率を算出した結果を図３に示す。図３から解るように、CQAを投与してから徐々に血圧が低下し、６時間を経過すると血圧（収縮期血圧）の変動率が最も低下していることが明らかとなった。

FIG. 3 shows the results of measuring blood pressure after CQA administration and calculating the blood pressure fluctuation rate. As can be seen from FIG. 3, it was revealed that the blood pressure gradually decreased after CQA administration, and that the fluctuation rate of blood pressure (systolic blood pressure) decreased most after 6 hours.

It is a characteristic view which shows the relationship between the elapsed time after BH2 administration, and a blood pressure change amount. FIG. 6 is a characteristic diagram showing the relationship between the elapsed time after CQA administration and the amount of BH2. It is a characteristic view which shows the relationship between the elapsed time after CQA administration, and the change rate of systolic blood pressure.

Claims (3)

Administering a substance to be evaluated to a model animal other than a human;
A method of evaluating a blood pressure regulator, comprising measuring a dihydrobiopterin concentration in the blood of the model animal and evaluating a variation degree and timing of the variation of the dihydrobiopterin concentration . The method for evaluating a blood pressure regulator according to claim 1 , wherein in the step of measuring the dihydrobiopterin concentration, a change in the dihydrobiopterin concentration due to other factors is evaluated. 3. The method for evaluating a blood pressure regulator according to claim 1, wherein the dihydrobiopterin concentration is measured as a sum of the dihydrobiopterin concentration and the biopterin concentration in blood.

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