JP4724810B2 - Ribosyl adenosine and ribosyl inosine as biomarkers of poly (ADP-ribose) metabolism - Google Patents

Description

Background of the Invention

FIELD OF THE INVENTION The present invention relates to a method for detecting morbidity of a disease associated with DNA damage or cell death and measuring the effect of anticancer drug treatment using ribosyl adenosine and ribosyl inosine as so-called biomarkers.

Background Art A poly-ADP-ribosylation reaction is one of post-translational modifications performed by poly (ADP-ribose) polymerase (Parp), and repairs DNA damage, transcription, regulation of chromatin conformation, induction of cell death, cell It is thought to be involved in the differentiation and canceration. Currently, 18 types of family proteins including Parp-1 have been identified in Parp. Parp-1 is present in the nucleus and centrosome and is activated by binding to the cleaved end after DNA damage, and using aspartic acid nicotinamide adenine dinucleotide as a substrate, aspartic acid of proteins such as self and histone, ADP-ribose is bound to glutamic acid and lysine residues. Further, with ADP-ribose as a unit, it is polymerized by (1 "α → 2 ' O ) glycosidic bond, and the protein is poly-ADP-ribosylated. The length is several to 200 residues or more. It has a linear or branched structure.

On the other hand, DNA damage is caused by oxidative stress in ischemic diseases, inflammation, and the like, and it is known that a large amount of poly (ADP-ribose) is synthesized by Parp and cell death is induced (Non-patent Document 1: Poly (ADP-ribose) polymerase gene disruption renders mice resistant to cerebral ischemia. Nature Med. 3: 1089-1095 (1997), Non-Patent Document 2: Poly (ADP-ribose) synthetase activation mediates mitochondrial injury during oxidant-induced cell death. J. Immunol. 161: 3753-3759, Non-Patent Document 3: Protection against peroxynitrite-induced fibroblast injury and arthritis development by inhibition of poly (ADP-ribose) synthetase. Proc. Natl. Acad. Sci. USA, 95: 3867- 3872.). At this time, cell destruction accompanying cell death occurs, and poly (ADP-ribose) synthesized in the cell may leak into the blood. Therefore, it is considered that poly (ADP-ribose) or its degradation product in blood can be a biomarker for diseases associated with DNA damage and cell death. At present, there is no report that clarifies what kind of metabolite is produced from poly (ADP-ribose) leaked out of the cell.
Nature Med. 3: 1089-1095 (1997) J. Immunol. 161: 3753-3759 Proc. Natl. Acad. Sci. USA, 95: 3867-3872

Summary of the Invention

  The present inventors have now succeeded in clarifying the metabolite of poly (ADP-ribose). Specifically, poly (ADP-ribose) degradation and metabolite identification by serum in vitro and poly (ADP-ribose) degradation and metabolite analysis in the mouse were performed. It was found that the major metabolites of poly (ADP-ribose) were ribosyl adenosine and ribosyl inosine. Therefore, the present inventors have found that these major metabolites can be useful markers for detecting diseases with DNA damage or cell death and measuring the effects of anticancer drug treatment. The present invention is based on these findings.

  Therefore, according to the present invention, there is provided a method for detecting morbidity of a disease associated with DNA damage or cell death and measuring the effect of anticancer drug treatment using ribosyl adenosine and ribosyl inosine as so-called biomarkers.

  Specifically, according to the first aspect of the present invention, there is provided a method for detecting morbidity in a disease associated with DNA damage or cell death in a test animal, the method comprising a biological obtained from a test animal. It is characterized by measuring the concentration of ribosyl adenosine or ribosyl inosine in a sample.

  According to a second aspect of the present invention, there is also provided a method for monitoring the effect of anticancer drug treatment, the method comprising continuing a biological sample from a patient receiving an anticancer drug. Collected and measured the concentration of ribosyl adenosine or ribosyl inosine in a biological sample. If the concentration of ribosyl adenosine or ribosyl inosine increases, the anticancer drug treatment is judged to be effective.

  Furthermore, according to a third aspect of the present invention, there is provided a method for determining the extent of DNA damage or cell death in an animal, the method taking a biological sample from the animal and ribosyladenosine in the biological sample. Alternatively, the concentration of ribosyl inosine is measured.

  Still further, according to a fourth aspect of the present invention, there is provided a method for screening an animal with a potential disease with DNA damage or cell death, wherein the method comprises collecting a biological sample from the animal, Characterized by measuring the concentration of ribosyl adenosine or ribosyl inosine in a biological sample.

Detailed description of the invention

  The disease detectable by the method according to the present invention is a disease accompanied by DNA damage or cell death, and specifically, ischemic disease, inflammation, which is considered to be a disease accompanied by cell destruction or enhanced poly ADP-ribosylation reaction. Or an autoimmune disease.

  When suffering from these diseases, poly (ADP-ribose) may leak into the blood, and this poly (ADP-ribose) is metabolized to ribosyl adenosine and ribosyl inosine by an experimental example described later. It was. Therefore, by using ribosyl adenosine and ribosyl inosine as indicators, it is possible to detect a disease with a DNA damage or cell death in a test animal. It is also possible to qualitatively or quantify DNA damage or cell death in animals. In addition, it is possible to screen for animals that may have a disease with DNA damage or cell death.

  DNA damage or cell death is also caused by administration of anticancer agents. Therefore, poly (ADP-ribose) leaks into the blood and metabolized into ribosyl adenosine and ribosyl inosine even with anti-cancer drug treatment. Therefore, if ribosyl adenosine and ribosyl inosine are used as indicators, anti-cancer drug treatment The effect can be determined.

  Therefore, according to the first aspect of the present invention, there is provided a method for detecting morbidity in a disease associated with DNA damage or cell death in a test animal, wherein in the method, in a biological sample obtained from a subject animal. Measuring the concentration of ribosyl adenosine or ribosyl inosine, preferably comprising comparing the concentration of ribosyl adenosine or ribosyl inosine to a normal value. When the concentration is larger than the normal value, it is determined that there is a possibility of suffering from a disease accompanied by DNA damage or cell death. According to another preferred embodiment, a threshold is set in advance with reference to the concentration of ribosyl adenosine or ribosyl inosine in a biological sample of an animal suffering from a disease involving DNA damage or cell death, and ribosyl When the concentration of adenosine or ribosylinosine is high, it can be determined that the patient may have suffered from a disease involving DNA damage or cell death.

  Moreover, according to the 2nd aspect of this invention, the method of monitoring the effect of anticancer agent treatment is provided. In this method, a biological sample is continuously collected from a patient receiving an anticancer drug, the concentration of ribosyl adenosine or ribosyl inosine in the biological sample is measured, and ribosyl adenosine or ribosyl inosine is measured. If the concentration of the drug increases, anticancer drug treatment is judged to be effective.

  Furthermore, according to a third aspect of the present invention, there is provided a method for knowing the degree of DNA damage or cell death in an animal, ie qualitatively or quantifying it. In this method, a biological sample is collected from an animal, the concentration of ribosyl adenosine or ribosyl inosine in the biological sample is measured, and preferably the concentration of ribosyl adenosine or ribosyl inosine is compared with a normal value. Comprising that. From the difference from the normal value, DNA damage or cell death can be qualitatively or quantitatively known. In another embodiment, the concentration of ribosyl adenosine or ribosyl inosine is compared to the concentration of ribosyl adenosine or ribosyl inosine in a biological sample of an animal that has undergone DNA damage or cell death. Thus, DNA damage or cell death can be qualitatively or quantitatively known.

  In addition, according to the fourth aspect of the present invention, there is provided a method for screening an animal having a possibility of a disease associated with DNA damage or cell death. In this method, a biological sample is collected from an animal, the concentration of ribosyl adenosine or ribosyl inosine in the biological sample is measured, and preferably the concentration of ribosyl adenosine or ribosyl inosine is compared with a normal value. Comprising that. Animals at concentrations significantly separated from normal values are determined to be likely to have a disease with DNA damage or cell death. According to another preferred embodiment, a threshold is set in advance with reference to the concentration of ribosyl adenosine or ribosyl inosine in a biological sample of an animal suffering from a disease involving DNA damage or cell death, and ribosyl If the concentration of adenosine or ribosylinosine is high, it is determined that the animal may have suffered from a disease with DNA damage or cell death.

  Furthermore, according to another aspect of the present invention, the use of ribosyl adenosine or ribosyl inosine as a marker for the degree of DNA damage or cell death, and ribosyl adenosine as a marker for the degree of effectiveness of anticancer drug treatment. Alternatively, the use of ribosylinosine is provided.

  In any of the above embodiments, the biological sample may be appropriately selected, but is preferably blood or urine.

  From this biological sample, the method for measuring the concentration of ribosyl adenosine or ribosyl inosine may be appropriately selected as long as the concentration can be measured. For example, TLC, HPLC, liquid chromatography-mass spectrometry (LC- MS) and the like. Moreover, in order to avoid the influence of the hybrid in a sample, it may be applied to these measurement techniques after preliminary purification.

  Details of the knowledge obtained from the experimental examples described later by the present inventors are as follows. First, poly (ADP-ribose) in serum was decomposed into phosphoribosyl-adenosine monophosphate (PR-AMP) and AMP. In blood, poly (ADP-ribose) was first decomposed into poly (ADP-ribose) by phosphodiesterase (PDE). The phosphodiester bond of (ADP-ribose) is cleaved. In cells, poly (ADP-ribose) glycohydrase (Parg) was thought to be the main degrading enzyme of poly (ADP-ribose), but PDE seems to work predominantly in blood. It is. Since PDE is localized in the cytoplasm in the cell and poly (ADP-ribose) is mainly synthesized in the nucleus, it is considered that degradation is performed by Parg localized in the nucleus in the cell. The presence of Parg in the blood is currently unknown, but no Parg activity was detected in fetal bovine serum and mouse blood. Although the ribose-ribose bond of phosphoribosyl-AMP and ribosyladenosine was degraded by Parg, it was considered possible to generate ribose phosphate, etc., but glutathione S-transferase fusion recombinant rat Parg does not degrade PR-AMP or ribosyladenosine. It was confirmed. Ribosyl adenosine and ribosyl inosine were also detected in urine, suggesting that the ribose-ribose bond is stable in blood.

Moreover, according to the experiment described later, the distribution of poly (ADP-ribose) in the living body has already moved out of the blood in about 5 minutes after intravenous injection, and is mainly distributed in the liver and kidney. Most of each organ after 5 minutes existed in a degraded state. Degradation products were detected in urine after 5 minutes, and approximately 5% of radioactivity was excreted after 1 hour, and poly (ADP-ribose) did not accumulate in specific organs. It has been reported by PM Mullen et al. (Strength of conjugate binding to plasmid DNA affects degradation rate and expression level) that plasmid DNA labeled with ³² P is also administered from the tail vein in about 10 minutes. in vivo. Biochim. Biophys. Acta. 2000 1523, 103-110). Thus, it was found that poly (ADP-ribose) is rapidly degraded in blood like DNA. Many distributions were found in the liver and kidneys, but it is not clear whether they are present in the blood of the organs or taken up into the cells. In the kidney, it decreases with time, so it is thought that it is excreted in the urine. The detected radioactivity was about 30% compared with the intravenous injection amount. It is considered that the radioactivity not detected is distributed to other organs, capillaries and the like.

Ribosyl adenosine and ribosyl inosine are also detected in urine and have a higher content than blood and are useful as biomarkers. Ribosyl adenosine can have ribose (1 ″ α → 2 ′ O ) ribose type and isomers having ribose (1 ″ β → 2 ′ O ) ribose type, but ribosyl adenosine is ribose ( The direct degradation product from poly (ADP-ribose) is O -α-ribosyl- (1 ″ → 2) -adenosine because it has only 1 ″ α → 2 ′ O ) ribose bond. it is conceivable that. Therefore, the ribosyl adenosine to be detected and measured in the present invention is preferably O -α-ribosyl- (1 ″ → 2) -adenosine.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples.

1. Preparation of reference material (1) ³² P-phosphate
T4 polynucleotide kinase (TAKARA) and γ- ³² P-ATP (Amersham) are added to the oligonucleotide DNA and reacted in T4 polynucleotide kinase buffer (TAKARA) at 16 ° C for 1 hour to phosphorylate the oligonucleotide DNA. Went. 50 μL of 1 M Tris-HCl (pH 8.0) was added to 40 μL of the reaction solution, and 1 μL of bacterial alkaline phosphatase (BAP, TOYOBO) was added and reacted at 60 ° C. for 1 hour to liberate ³² P-phosphate.

(2) Ribosyladenosine PDE is added to poly (ADP-ribose) and reacted overnight at 37 ° C. in PDE buffer [10 mM sodium-phosphate buffer (pH 7.0), 10 mM MgCl ₂ ]. And the peak of phosphoribosyl-AMP retention time of 17 minutes was separated by HPLC described later. BAP was added to phosphoribosyl-AMP in the presence of 0.5 M Tris-HCl (pH 8.0) and reacted at 60 ° C for 1 hour. Similarly, a peak with a retention time of 32 minutes was separated by HPLC, and the molecular weight was analyzed by LC-MS described later. Was 399 and its ultraviolet absorption spectrum was confirmed, and ribosyl adenosine was obtained.

(3) Ribosylinosine Ribosyl adenosine was reacted with adenosine deaminase (Sigma) 10 U / mL in 0.05 M pottasium-phosphate (pH 7.5) at 25 ° C. for 30 minutes. A peak with a retention time of 26 minutes was collected by HPLC, and its molecular weight was 400 and its ultraviolet absorption spectrum was confirmed by LC-MS.

2. TLC analysis (1) One-dimensional TLC
PEI (polyethyleneimine-impregnated) cellulose TLC plate (MACHERHY-NAGEL)
Developing solvent A: 0.1 M LiCl-3 M acetic acid-3 M urea
Developing solvent B: H ₂ O
(2) Two-dimensional TLC
Cellulose TLC plate (F1440, Schleicher & Schuell and Cellulose F, Merk))
Developing solvent 1: isobutyric acid / 25% NH ₄ OH / H ₂ O (capacity ratio is 50 / 1.1 / 28.9)
Developing solvent 2: 0.1 M sodium-phosphate (pH 6.3) / ammonium sulfate / n-propanol (100/60/2)
After deployment, radioactivity was analyzed using BAS2500 (Fuji Film).

3. Degradation of poly (ADP-ribose) by fetal bovine serum (1) Time course of poly (ADP-ribose) degradation in serum
To 100 ng ³² P-poly (ADP-ribose), add 20 μL of fetal bovine serum (PAA Laboratories, GmbH) to make a total volume of 50 μL, then 37 minutes, 0 minutes, 10 minutes, 30 minutes, 60 minutes, 3 hours, 5 hours The reaction was allowed for 24 hours. At each point, 10 μL of the reaction solution was collected, and SDS was added to 0.2% to stop the reaction. 2 μL was developed by one-dimensional TLC (developing solvent A).

(2) Identification of degradation products ¹⁴ C-poly (ADP-ribose) or non-radioactive label in reaction solution containing fetal bovine serum (Sanko Junyaku) 40%, 50 mM Tris-HCl (pH 7.5) 50 μg of poly (ADP-ribose) was reacted at 37 ° C. overnight. SDS was added to 1% to stop the reaction. ^{The 14} C-poly (ADP-ribose) reaction solution was analyzed by one-dimensional (developing solvent B) and two-dimensional TLC, and ribosyladenosine and Rf values were compared. For the non-radioactive labeled poly (ADP-ribose) reaction solution, the fraction of the degradation product was fractionated by HPLC and analyzed by LC-MS described later.

4). Measurement of organ distribution after intravenous injection of ³² P-poly (ADP-ribose)
Nembutal (Dainippon Pharmaceutical Co., Ltd.) 1.5 mg / 60 μL was intraperitoneally administered to ICR mice (11-13 weeks old, male), and then ³² P-poly (ADP-ribose) 10 μg / 4-8 from the tail vein. x 10 ⁴ cpm / 100 μL was intravenously injected, and the urinary tract was closed with an instantaneous adhesive for urine collection. At 5 minutes, 1 hour, and 3 hours after administration, the animals were dissected, blood was collected from the heart, and urine was collected from the bladder. The liver, kidney, spleen, and lung were removed and the weight of each organ was measured. Each organ was crushed in PBS using a homogenizer (Polytron). An equal volume of Solvable (Packard) was added to a portion of each organ, blood and urine and dissolved overnight at 50 ° C. 20% EDTA (pH 8.0) to each solution, added to the H ₂ O ₂ becomes 6%, was placed in room temperature for one day. Then, 10 to 15 mL of liquid scintillation count cocktail, Insta-gel plus (ParkinElmer) was added to the total amount and mixed until clear, and the radioactivity was measured with a liquid scintillation counter (Beckman).

5. Analysis of ³² P-poly (ADP-ribose) degradation in each organ A portion of the collected blood and crushed liver and kidney solution was treated with 0.5 N HClO ₄ for 20 minutes, and 3 N KOH / 0.7 M Gly- After neutralization with Gly, the mixture was centrifuged at 15,000 rpm for 10 minutes, and a part was divided into an acid-treated soluble fraction (supernatant) and an acid-treated insoluble fraction (precipitation). Poly (ADP-ribose) is recovered in the precipitate, and the degraded low molecular weight is recovered in the supernatant fraction. The precipitate was dissolved in 1 N NaOH, neutralized with 6 N HCl, SDS and proteinase K were added to a final concentration of 0.1% and 100 μg / mL, respectively, and allowed to react at 37 ° C. overnight. Extracted with phenol, and poly (ADP-ribose) was extracted by ethanol precipitation. It was washed with 70% ethanol, dried and dissolved in H ₂ O. In order to measure the degradation rate of poly (ADP-ribose), the supernatant after perchloric acid treatment and the sample after precipitation phenol extraction treatment were spotted on DE81 paper (Whatman), and the radioactivity was measured with BAS2500.

6). Analysis of degradation product of ³² P-poly (ADP-ribose) in mouse body To examine the degradation product of poly (ADP-ribose), two-dimensional TLC of the supernatant after perchloric acid treatment was performed. Urine was directly analyzed by two-dimensional TLC. In addition, poly (ADP-ribose) in blood dissolved in H ₂ O and poly (ADP-ribose) injected intravenously were run on a 20% polyacrylamide gel, and changes in chain length were examined (Panzeter et. Al., High resolution size analysis of ADP-ribose polymers using modified DNA sequencing gels. Nucleic Acids Res. 1990 Apr 25; 18 (8): 2194).

For analysis by LC-MS, 100 μL of 100 μg of non-radioactive labeled poly (ADP-ribose) physiological saline solution and 100 μL of physiological saline as a control were administered from the mouse tail vein, and blood and urine were collected 1 hour after administration. It was. The collected blood and urine were treated with HClO ₄ , and the supernatant was pretreated with a Sep-Pak cartridge as described below, and then a fraction with a retention time of 31 to 32 minutes during which ribosyladenosine was eluted by HPLC, and ribosylinosine Fractions with a retention time of 26 to 27 minutes were collected and analyzed by LC-MS.

7). LC-MS sample preparation
Sep-Pak cartridge light (Waters) was moistened with 5 mL of 50% CH ₃ CN, equilibrated with 5 mL of 50 mM triethylamine acetate (TEAA, Wako), and the supernatant after HClO ₄ treatment was adsorbed. Washed twice with 500 μL of H ₂ O and eluted with 1 mL of 20% CH ₃ CN. The eluate was concentrated, filtered through Ultra Free C3GV (MILLIPORE), and analyzed by HPLC to fractionate the degradation products. Each fraction was concentrated, filtered through Microcon YM3 (MILLIPORE), and analyzed by LC-MS as described below.

8). HPLC
Apparatus: HPLC analyzer (SCL-10A VP, Shimadzu Corporation)
Column: Develosil C30-UG-5 (f46 × 250 mm, Nomura Chemical)
Eluent: A solution 0.1 M CH ₃ COONH ₄ (Wako)
B solution 50 mM CH ₃ COONH ₄ -50% CH ₃ CN (Wako)
0 min, A: B = 98: 2 → 100 min, A: B = 0: 100 linear gradient flow rate: 0.5 mL / min
Detection: photo diode array detector (SPD-M10A VP, Shimadzu Corporation)

9. Liquid chromatography-mass spectrometry (LC-MS)
LC device (Series 1100, Agilent), column using Develosil C30-UG-5, eluent A solution 0.1 M CH ₃ COONH ₄ , solution B 50 mM CH ₃ COONH ₄ -50% CH ₃ CN, flow rate Analysis was performed with a linear gradient of 0.3 mL / min, from 2% to 50% of solution B for 50 minutes. Detection of UV 254 nm was performed at the same time. MS apparatus (Micromass ZQ, Waters) was ionized by electrospray ionization (ESI) and detected.

Test result 1: Identification of degradation products of poly (ADP-ribose) by fetal bovine serum (FBS) (1) Time course of degradation of ³² P-poly (ADP-ribose) and analysis of degradation products
The time course of degradation of ³² P-poly (ADP-ribose) by FBS was analyzed by one-dimensional TLC. Degradation products considered to be phosphoribosyl-AMP having an Rf value of 0.23 appeared, followed by spots corresponding to AMP having an Rf value of 0.7 and spots (degradation product 1) having an Rf value of 0.25. In order to examine the degradation products in more detail, the samples after the reaction at 37 ° C. for 3 hours and 24 hours were developed into two-dimensional TLC. AMP, phosphoribosyl-AMP, and degradation product 1 were detected in the reaction solution after 3 hours, and completely degraded to degradation product 1 after 24 hours. In order to identify the degradation product 1, the Rf value of one-dimensional and two-dimensional TLC of phosphoric acid, which is one of the predicted substances, was examined. When ³² P-phosphate was prepared from γ- ³² P-ATP and developed into one-dimensional and two-dimensional TLC, the Rf values almost coincided with the degradation product 1, respectively. This suggests that in serum, PDE that cleaves the phosphodiester bond is degraded to PR-AMP, and then the phosphate residue is released.

(2) Analysis of degradation products of ¹⁴ C-poly (ADP-ribose)
Analysis of degradation of ³² P-poly (ADP-ribose) suggested that phosphate residues were released after degradation into PR-AMP. Therefore, experiments using ³² P-poly (ADP-ribose) cannot identify major degradation products other than phosphate. Therefore, experiments were conducted using ¹⁴ C-poly (ADP-ribose) labeled with adenine and ribose residues.
^The reaction solution obtained by digesting ¹⁴ C-poly (ADP-ribose) with serum was analyzed by one-dimensional and two-dimensional TLC. As a result of one-dimensional TLC using a cellulose plate developed with H ₂ O, only one spot was detected, which showed an Rf value close to ribosyladenosine, unlike adenine or adenosine. The two-dimensional TLC showed the same Rf value as ribosyladenosine. In one-dimensional TLC, a reaction solution with a high FBS content is spotted on the TLC as it is, so it is considered that the mobility is slightly changed due to the influence of the serum components and SDS added to stop the reaction. From these results, it was suggested that the degradation product of poly (ADP-ribose) was ribosyladenosine. Because the blood is present adenosine deaminase (ADA), it was created ¹⁴ C-ribosyl inosine by treating the ¹⁴ C-ribosyl adenosine in ADA. ^In one-dimensional TLC of ¹⁴ C-poly (ADP-ribose), no ribosyl inosine was detected in the degradation product by serum.

(3) Identification of non-radioactive labeled poly (ADP-ribose) degradation product In order to further confirm that the degradation product was ribosyladenosine, identification was performed by LC-MS. First, 50 μg of non-radioactive labeled poly (ADP-ribose) was decomposed with FBS, analyzed by HPLC, and the peak pattern of HPLC was compared with the reaction solution without poly (ADP-ribose). A peak having a UV absorption corresponding to about 100% of poly (ADP-ribose) used in the reaction was detected around a retention time of 33 minutes, and this retention time was consistent with ribosyladenosine. The fraction in which this peak was detected was collected and analyzed by LC-MS, and at a retention time of 38 minutes, m / z = 268 (ES +) and m / z = 266 corresponding to the molecular weight of ribosyladenosine. Molecular ion peaks of (ES-) were detected, and molecular ion peaks corresponding to the fragment ions adenosine and adenine were also detected at the same retention time. Ribosyl adenosine was not detected in serum without poly (ADP-ribose), and ribosyl adenosine was identified as the major degradation product of poly (ADP-ribose) in serum.

(4) Degradation of ribosyl adenosine and phosphoribosyl-AMP by poly (ADP-ribose) polymerase (Parp)
¹⁴ C-ribosyl adenosine and ¹⁴ C-phosphoribosyl-AMP were reacted with rat glutathione S-transferase fusion Parg, and then developed with TLC to examine the presence or absence of degradation into adenosine, AMP, and ribose-phosphate. Both ribosyladenosine and phosphoribosyl-AMP showed almost no degradation after overnight reaction at 25 ° C.

Test result 2. Degradation of poly (ADP-ribose) in vivo and analysis of metabolites
(1) ³² P- poly (ADP-ribose) biodistribution above ³² P- poly (ADP-ribose) after intravenous injection were analyzed for the test results of intravenously injected from the mouse tail vein. The administered radioactivity was taken as 100%, and the ratio of radioactivity in each organ was calculated. From 5 minutes to 3 hours after administration, the residual radioactivity in the blood was about 2.3%, and almost 5 minutes after administration, the blood had moved out of the blood. The main distribution was in the liver and kidneys, with almost no distribution in the spleen or lungs. After 1 hour, about 5% of radioactivity was excreted in urine.

(2) ³² P-poly (ADP-ribose) In each organ, the distribution of radioactivity in the blood and each tissue after administration of degraded poly (ADP-ribose) was examined. In order to examine whether the radioactivity exists in the state of poly (ADP-ribose) or degradation products, the degradation rate was measured. The blood, liver, and kidney, which had a large amount of distribution, were examined for the degradation rate 5 minutes after administration. After treatment with HClO ₄ , the acid-treated soluble fraction (supernatant) and the acid-treated insoluble fraction (precipitate) were separated, and the radioactivity was measured. The ratio of radioactivity in the supernatant was 64% blood, 84% liver, 91% kidney, and a significant proportion was degraded.

(3) Analysis of ³² P-poly (ADP-ribose) metabolites in each organ Samples at each time in urine were analyzed by two-dimensional TLC. Spots were observed at the same position at any elapsed time. The Rf value of this spot was consistent with the Rf value of phosphoric acid, and it was found that phosphoric acid was excreted as a degradation product 5 minutes after intravenous injection. No spots of ADP-ribose produced when decomposed by AMP or Parg were observed. As for blood, liver, and kidney, when the degradation product after 5 minutes was analyzed by two-dimensional TLC, spots showing the same Rf values as phosphoric acid were observed as in urine. Since phosphate was also detected when ³² P-poly (ADP-ribose) was decomposed in serum, it was decomposed into phosphate and ribosyladenosine in vivo after poly (ADP-ribose) was intravenously injected. The possibility was suggested.

  In addition, when poly (ADP-ribose) extracted from the acid-treated insoluble fraction of blood was examined for chain length by 20% PAGE, it showed a shorter chain length distribution compared to intravenously injected poly (ADP-ribose). In particular, it was confirmed that the chain was shortened by decomposition.

(4) Analysis of degradation products in blood and urine From the experiment of intravenous injection of ³² P-poly (ADP-ribose) into mice, it was confirmed that phosphate was produced as one of the degradation products. The possibility that it was decomposed into ribosyl adenosine was suggested. Thus, degradation products in blood and urine after 1 hour of intravenous injection of 100 μg of poly (ADP-ribose) were analyzed by LC-MS.

  After pretreatment of acid-treated soluble fractions of blood and urine with a Sep-pak cartridge, fractions near the retention time of ribosyladenosine (fraction 1) and ribosylinosine (fraction 2) were collected using HPLC. . In the analysis by HPLC, a comparison was made between the intravenous injection of physiological saline and the UV absorption peak pattern at 254 nm, but no difference was observed. On the other hand, the results of LC-MS analysis of blood fraction 1 were as shown in FIG. 1 and FIG. 2 (control). Ribosyl adenosine and adenosine were close in retention time, and fraction 1 contained adenosine. Under these LC-MS conditions, adenosine was m / z = 268 (ES +) at 36 to 37 minutes, m / z = 266 (ES-), ribosyladenosine was around 38 minutes at m / z = 400 (ES +), A molecular ion peak of m / z = 398 (ES-) is detected. In the case of intravenous injection of physiological saline, only the molecular ion peak of adenosine was detected, but in the case of intravenous injection of poly (ADP-ribose), the molecular ion peak of ribosyladenosine was detected together with adenosine. It became clear that it was decomposed.

  Adenosine deaminase (ADA), which is a purine metabolizing enzyme, is present in blood and adenosine is deaminated and converted into inosine. Since the degradation product ribosyl adenosine may be metabolized to ribosyl inosine by ADA, the fraction 2 which is the retention time of ribosyl inosine was also analyzed by LC-MS. The results were as shown in FIGS. 3 and 4 (control). In the ribosyl inosine standard prepared by treating ribosyl adenosine with ADA, molecular ion peaks of m / z = 401 (ES +) and m / z = 399 (ES-) were detected at 31.4 minutes. Although it was not detected in an intravenous injection of physiological saline, a peak of m / z = 399 (ES-) was detected in the blood of a mouse intravenously injected with poly (ADP-ribose). Although no peak was detected at m / z = 401 (ES +), this peak was small even in the standard substance, and it was considered that it was not detected because the amount in the sample was small. In addition, although the retention time is slightly different from that of the standard substance, it is considered that it corresponds to ribosylinosine from the UV peak pattern. From the above, it was found that poly (ADP-ribose) was decomposed into ribosyl adenosine in the blood, and a part of it was metabolized to ribosyl inosine.

Whether these degradation products were excreted in urine was similarly analyzed by LC-MS. Urine 1 hour after poly (ADP-ribose) was intravenously treated with HClO _{4 and} pretreated with a Sep-pak cartridge, and fraction 1 (ribosyl adenosine) and fraction 2 (ribosyl inosine) were fractionated by HPLC. . When LC-MS analysis of each of these was performed, molecular ion peaks corresponding to ribosyladenosine and ribosylinosine were detected. The results were as shown in FIGS. Further, the results of the control mice intravenously injected with physiological saline were as shown in FIG. 6 and FIG. In control mice intravenously injected with physiological saline, ribosyladenosine in fraction 1 was not detected, while ribosylinosine in fraction 2 was detected at m / z = 401 (ES +), but the amount of poly (ADP- Less than ribose).

  Poly (ADP-ribose) is degraded into ribosyl adenosine and phosphoric acid in the mouse in the same manner as serum, but a part of it is metabolized to ribosyl inosine in vivo. It was also confirmed that these degradation products were excreted in urine.

It is the analysis result of HPLC and LC-MS of the ribosyl adenosine in the blood of the mouse | mouth which inject | poured the ³² P-poly (ADP-ribose) intravenously. ^{It is} a HPLC and LC-MS analysis result of ribosyl adenosine in the blood of a control mouse intravenously injected with physiological saline instead of ³² P-poly (ADP-ribose). ³² is a P- poly (ADP-ribose) analysis of HPLC and LC-MS of the ribosyl inosine in the blood of mice intravenously. It is the analysis result of HPLC and LC-MS of the ribosyl inosine in the blood of the control mouse which injected physiological saline intravenously instead of ³² P-poly (ADP-ribose). ^{It is} a HPLC and LC-MS analysis result of ribosyl adenosine in the urine of a mouse intravenously injected with ³² P-poly (ADP-ribose). ^{It is} a HPLC and LC-MS analysis result of the ribosyl adenosine in the urine of the control mouse | mouth which intravenously injected the physiological saline instead of the 32P-poly (ADP-ribose). ^{It is} a HPLC and LC-MS analysis result of ribosyl inosine in urine of mice intravenously injected with ³² P-poly (ADP-ribose). It is the analysis result of HPLC and LC-MS of the ribosyl inosine in the blood of the control mouse which injected physiological saline intravenously instead of ³² P-poly (ADP-ribose).

Claims (15)

A measurement method for detecting a disease with a DNA damage or cell death in a test animal,
Comprises measuring the concentration of ribosyl adenosine or ribosyl inosine in a biological sample obtained from a test animal,
The method wherein the disease involving DNA damage or cell death is ischemic disease, inflammation, autoimmune disease or cancer .   The method of claim 1, wherein the biological sample is blood or urine.   3. The method of claim 1 or 2, comprising comparing the concentration of ribosyl adenosine or ribosyl inosine to a normal value. Comparing said concentration of ribosyl adenosine or ribosyl inosine, the DNA damage or threshold value set in advance with reference to the concentration of the ribosyl adenosine or ribosyl inosine in a biological sample of an animal suffering from a disease accompanied by cell death 4. A method according to any one of claims 1 to 3, comprising . A measurement method for monitoring the effect of anticancer drug treatment,
For anticancer therapy to determine whether valid or not, comprising measuring the concentration of ribosyl adenosine or ribosyl inosine in a biological sample taken from patients receiving anti-cancer agents, Method.   6. The method of claim 5, wherein the biological sample is blood or urine. A measurement method for determining the degree of DNA damage or cell death in an animal,
Comprising measuring the concentration of ribosyl adenosine or ribosyl inosine in a biological sample taken from said animal, the method.   8. The method of claim 7, wherein the biological sample is blood or urine.   9. The method of claim 7 or 8, comprising comparing the concentration of ribosyl adenosine or ribosyl inosine to a normal value. 9. The method according to claim 7 or 8, comprising comparing the concentration of ribosyl adenosine or ribosyl inosine to the concentration of ribosyl adenosine or ribosyl inosine in a biological sample of an animal that has undergone DNA damage or cell death. . A measurement method for screening an animal having a possibility of a disease with DNA damage or cell death,
Comprises measuring the concentration of ribosyl adenosine or ribosyl inosine in a biological sample taken from said animal,
The method wherein the disease involving DNA damage or cell death is ischemic disease, inflammation, autoimmune disease or cancer .   Use of ribosyl adenosine or ribosyl inosine as a marker for the degree of DNA damage or cell death.   Use of ribosyl adenosine or ribosyl inosine as a marker representing the degree of effectiveness of anticancer drug treatment. A measurement method for knowing the amount of poly (ADP-ribose) released in blood in an animal,
Measuring the concentration of ribosyl adenosine or ribosyl inosine in a biological sample taken from said animal.   15. A method according to claim 14, wherein the biological sample is blood or urine.

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