Patent Application
20100167322
The present invention relates to methods of detecting cerebral stroke or asymptomatic cerebral infarction using the contents of acrolein, and interleukin-6 and CRP which are inflammatory markers, polyamine oxidase activity or polyamine oxidase protein contents and subjects' ages as indications.
In Japan, the number of deaths caused by cerebrovascular diseases is the third largest after those by neoplasm malignants and cardiac diseases. Sequela following the affection of the cerebrovascular diseases not only pose serious problems for patients in the daily life of their own, e.g., involving paralysis or akinesia, but also contribute to increased psychic stress on caregivers. Cerebral stroke which is the majority of the cerebrovascular diseases is a disease difficult to detect and treat early. Although initiation of treatment of patients in the stages of cerebral infarctions without any subjective symptoms of cerebral infarctions, such as hemiplegia, hemiparesis, numbness, decreased sensation, hand-foot movement disorders, disturbance of consciousness and language disorders, (asymptomatic cerebral infarctions) is effective, the majority of cases with asymptomatic cerebral infarctions are accidentally found using diagnostic imaging and there is no diagnosis marker utilized in, e.g., blood examination or urinalysis under present circumstances. Therefore, development of simple and highly reliable diagnosis methods which do not require expensive devices, such as diagnostic imaging, is desired.
Acrolein and polyamine oxidases generated from polyamine have been known as biomarkers having correlations with cerebral infarction. Acrolein is detoxicated by aldehyde dehydrogenase in a cell but, when leaked extracellularly, demonstrates a strong toxicity. Therefore, acrolein is considered to have a high correlation with a cell injury score, and thus its correlations with symptoms such as renal disorders, cerebral stroke and asymptomatic cerebral infarction have been studied. See non-patent documents 1 to 3 and patent documents 1 and 2.
Non-Patent Document 1
Sakata, K. et al (2003) Biochem. Biophys. Res. Commun. 305, 143-149
Non-Patent Document 2
Tomitori, H. et al (2005) Stroke 36, 2609-2613
Non-Patent Document 3
Igarashi, K. et al (2006) Amino Acids 31, 477-483
Patent Document 1
Japanese Unexamined Patent Application Publication No. 2002-95942
Patent Document 2
Japanese Unexamined Patent Application Publication No. 2005-304476
However, for example, the accuracy of conventional diagnosis and screening methods for cerebral stroke and asymptomatic cerebral infarction based on acrolein or polyamine oxidase activity as described in the patent document 2 was still not sufficient, particularly in terms of practical use.
In contrast, interleukin-6, which was found as a B cell differentiation-inducing factor, is known to be a malignant cell growth factor for multiple myeloma and to be involved in various inflammatory diseases and autoimmune diseases. Therefore, studies of interleukin-6 as a biomarker for cerebral stroke have been conducted. See non-patent documents 4 and 5 and patent document 3.
CRP (C-reactive protein) is also found as a serum protein (β-globulin) which undergoes a precipitation reaction with C-polysaccharide extracted from a cell wall in Streptococcus pneumoniae, and blood CRP concentrations are known to be increased in many diseases such as infections (particularly bacterial infections), myocardial infarction and autoimmune diseases. In recent years, technological improvement in measuring devices has lead to development of methods for measuring high sensitivity CRP (hs-CRP), which allow sensing of even extremely mild inflammatory reactions without definite inflammatory diseases such as infections and malignant tumors (minimum detectable sensitivity: 0.01 mg/L). In a large clinical study by Ridker et al., CRP was reported to become an independent prediction marker for ischemic heart disease. Therefore, studies of CRP as a biomarker for cerebral stroke have been conducted. See non-patent documents 6, 7 and 8 and patent document 3.
Non-Patent Document 4
Smith, C. J. et al (2004) BMC Neurol. 15, 4:2
Non-Patent Document 5
Tzoulaki, I. et al (2006) Circulation. 115, 2119-2127
Non-Patent Document 6
Ridker PM. et al (2002) Engl J Med 347, 1557-1565
Non-Patent Document 7
Wakugawa, Y. et al (2006) Stroke 37, 27-32
Non-Patent Document 8
Tzoulaki, I. et al (2006) Circulation. 115, 2119-2127
Patent Document 3
Japanese Unexamined Patent Application Publication No. 2005-522669
However, there are no cases of using interleukin-6 or CRP individually as a biomarker for diagnosis of cerebral infarction, and a technique of distinguishing the stroke type of a patient after the onset of a cerebral stroke symptom in combination of interleukin-6 or CRP with B-type natriuretic peptide, matrix metalloprotease-9, S-100β, thrombin-antithrombin III complex and one or more types of von Willebrand factors is disclosed. Furthermore, neither suggestion nor teaching about the relationships of, in particular, samples from patients with asymptomatic cerebral infarction before the onset of stroke symptoms, with interleukin-6 and CRP are given.
The objective of the invention is to provide a method of detecting or screening cerebral stroke or asymptomatic cerebral infarction at an elevated accuracy, as being able to be practically used.
As a result of extensive research to achieve the above-mentioned objective, the present inventors found that the values which are obtained newly by mathematically statistically analyzing the contents of acrolein, and interleukin-6 and CRP which are inflammatory markers, in subject samples, polyamine oxidase activity and subjects' ages are significantly statistically correlated with the presence of asymptomatic cerebral infarction, and the invention was thus accomplished based on the results.
More specifically, the present invention includes each aspect as described below,
(1) A method of detecting cerebral stroke or asymptomatic cerebral infarction, comprising measuring the contents of a aldehyde compound formed from polyamine, interleukin-6 and C-reactive protein, polyamine oxidase activity or a polyamine oxidase protein content in a biological sample obtained from a subject and using the measurement data thus obtained and a subject's age as indications.
(2) The method according to a first aspect of the present invention, wherein the aldehyde compound formed from the polyamine is acrolein.
(3) The method according to a first or second aspect of the present invention, further comprising: measuring the contents of the aldehyde compound formed from the polyamine, the interleukin-6 and the C-reactive protein, the polyamine oxidase activity or the polyamine oxidase protein content in the biological sample; carrying out mathematically statistical analysis of the measurement data thus obtained and a subject's age, thereby obtaining a value which exhibits a statistically significant change; and detecting cerebral stroke or asymptomatic cerebral infarction based on the value.
(4) The method according to a third aspect of the present invention, further comprising carrying out the mathematically statistical analysis by a neural network technique.
(5) A kit for detecting cerebral stroke or asymptomatic cerebral infarction, comprising a reagent for measuring the contents of an aldehyde compound formed from polyamine, interleukin-6 and C-reactive protein, polyamine oxidase activity or a polyamine oxidase protein content in a biological sample.
(6) A system for detecting cerebral stroke or asymptomatic cerebral infarction, comprising: a reagent for measuring the contents of a aldehyde compound formed from polyamine, interleukin-6 and C-reactive protein, polyamine oxidase activity or a polyamine oxidase protein content in a biological sample; and an electronic treating device and software for carrying out mathematically statistical analysis.
The method according to an aspect of the present invention allows detection of a patient with cerebral stroke or an asymptomatic patient at a high accuracy of about 95%. Accordingly, a patient with cerebral stroke or asymptomatic cerebral infarction can be detected and diagnosed early before developing a symptom of cerebral infarction, or can be screened.
A most preferred embodiment of the present invention will be described in detail below, but many other different embodiments according to the present invention, which are apparent to those skilled in the art, are possible, and the present invention is not limited to the embodiment described below.
The biological samples from the subjects, used in the method according to an aspect of the present invention, are preferably plasma used in the example described below. However, other biological samples can also be used appropriately. Such other biological samples can include, for example, urine, saliva, cerebral spinal fluid and bone marrow fluid.
The term “polyamine” herein refers to a straight-chain aliphatic hydrocarbon having two or more primary amino groups. Known biogenic polyamines may include, but are not limited to, putrescine, cadaverine, spermidine, spermine, 1,3-diaminopropane, caldine, homospermidine, 3-aminopropylcadaverine, norspermine, thermospermine, caldopentamine. Preferred polyamines in accordance with an aspect of the present invention may be putrescine, spermidine and spermine.
The above polyamines are metabolized by oxidation, acetylation, transamination or carbamoylation, and a polyamine oxidase (acetylpolyamine oxidase (AcPAO) or spennine oxidase (SMO)) is an enzyme involved in the oxidation of polyamines. The term “polyamine oxidase” herein refers to an enzyme that oxidizes a diamine or a polyamine as a good substrate and generates hydrogen peroxide. Such a polyamine is oxidatively deaminated by a polyamine oxidase, resulting in the formation of aldehyde compounds such as acrolein. In accordance with an aspect of the present invention, such a preferred aldehyde compound may be acrolein, but is not limited thereto.
A plasma acrolein content can be determined by any procedure well-known to those skilled in the art, for example, measuring the content of FDP-lysine (N-formyl-piperidino-lysine), which is an amino acid adder with acrolein. An FDP-lysine content can be measured, for example, by using ACR-LYSINE ADDUCT ELISA SYSTEM (NOF CORPORATION), according to the attached manual. The acrolein content may also be measured in the form of derivatives other than FDP-lysine. Furthermore, it is also possible to measure the acrolein content directly, and such a procedure is described in a report, for example, by Alarcon et al. (Alarcon, R. A. (1968) Anal. Chem. 40, 1704-1708). However, there is such a problem that the reactivity of acrolein with other molecules is high so that the amount of free acrolein in the blood is very small. Thus, considering the measurement of acrolein in the form of FDP-lysine is simple and easy, it is a preferred aspect in the present invention to measure acrolein in the form of FDP-lysine.
Specifically, subject plasma and a standard solution are dispensed into a plate immobilized with antigen by 50 μl/well, and further the same amount of primary antibody solution is added. The fluid is removed after resting for 30 minutes at room temperature and washed with a washing solution, followed by dispensing 100 μl/well of secondary antibody solution. It is washed with the washing solution after resting for 1 hour at room temperature, then color is developed by adding a coloring reagent of 100 μl/well and leaving it at rest for 15 minutes at room temperature, and reaction stop solutions are dispensed by 50 μl/well, followed by determining the absorbance at 450 nm using a plate reader. A plasma acrolein content is displayed as an FDP-lysine content per milliliter of plasma (nmol/ml plasma).
A plasma interleukin-6 content can be measured by any procedure well-known to those skilled in the art, for example, by using Human IL-6 ELISA (ENDOGEN CORPORATION), according to the attached manual.
Specifically, primary antibody solutions are dispensed into a 98-well plate by 50 and further the same amounts of subject plasma and standard solution are added. The fluid is removed after resting for 2 hours at room temperature and washed with a washing solution, followed by dispensing 100 μl/well of secondary antibody solution. It is washed with the washing solution after resting for 30 minutes at room temperature, then color is developed by adding a coloring reagent of 100 μl/well and leaving it at rest for 30 minutes at room temperature, and reaction stop solutions are dispensed by 100 μl/well followed by determining the absorbance at 450 nm using a plate reader. A plasma interleukin-6 content is displayed as a plasma content per milliliter of plasma (pg/ml plasma).
A plasma CRP content can be measured by any procedure well-known to those skilled in the art, for example, by using Human CRP ELISA KIT (Alpha Diagnostics International, Inc.), according to the attached manual.
Specifically, each well is washed with a washing solution, followed by dispensing subject plasma and standard solution into a 98-well plate by 10 μl/well and further adding an abzyme labeling solution of 100 μl/well. The fluid is removed after resting for 30 minutes at room temperature and washed with a washing solution. A coloring reagent is added, color is developed while shaking the mixture for 10 minutes at room temperature, and reaction stop solutions are dispensed by 50 μl/well, followed by determining the absorbance at 450 nm using a plate reader. A plasma CRP content is displayed as a CRP content per milliliter of patient plasma (mg/dl plasma).
The measurement of the activities of polyamine oxidases (AcPAO and SMO) can be conducted by any procedure well-known to those skilled in the art, for example, by incubating 0.06 ml of reaction mixture containing 10 mM Tris-hydrochloric acid (pH 7.5), 0.2 mM substrate (acetyl spermine or spermine) and 0.05 ml of patient plasma for 48 hours at 37° C. Trichloroacetic acid (TCA) is added to 0.02 ml of the reaction mixture to a final concentration of 5%, and it is centrifuged. A part of obtained supernatant is used for polyamine assay. Polyamine oxidase activity can be displayed as the content of a spermidine generated by the decomposition of acetyl spermine or spermine per milliliter of patient plasma (nmol/ml plasma).
The procedures of measuring the enzyme activity of polyamine oxidase are described in various reports, including a report by Sharmin et al. (Sharmin et al., (2001) Biochem. Biophys. Res. Commun. 282, 228-235), a report by Sakata et al. (Sakata et al., (2003) Biochem. Biophys Res. Commun. 305, 143-149), and a report by Igarashi et al. (Igarashi et al., (1986) J. Bacteriol. 166, 128-134). Based on the descriptions of such reports, those skilled in the art can measure the enzymatic activity of polyamine oxidase by making appropriate modifications.
Furthermore, the protein content of polyamine oxidase can be measured by any procedure well-known to those skilled in the art, for example, by enzyme-linked immunosorbent assay (EILSA), western blotting analysis or immunoprecipitation method using a specific antibody for polyamine oxidase. Such procedures are heretofore known and commonly used, and therefore, those skilled in the art can measure the protein content of the enzyme using the above procedures by setting adequate conditions appropriately. An antibody against polyamine oxidase used for conducting these measurements may be a monoclonal antibody or a polyclonal antibody.
The polyclonal antibody against polyamine oxidase can be obtained, for example, by immunizing rabbits with the peptide fragment of polyamine oxidase by using a conventional technique for production of a peptide antibody. The production of a peptide antibody can be confirmed through assaying whether the antibody has reached to sufficient titer by collecting blood from rabbits administered with the peptide and measuring its antibody titer. The procedures for producing a peptide antibody are described in various experimental manuals and well known to those skilled in the art, so the antibody against polyamine oxidase can be obtained by making various modifications based on those descriptions.
In accordance with an aspect of the present invention, the contents of an aldehyde compound formed from polyamine, interleukin-6 and C-reactive protein, polyamine oxidase activity or a polyamine oxidase protein content in the biological sample are measured; mathematical statistical analysis of the measurement data thus obtained and a subject's age is carried out, thereby consequently obtaining a value which exhibits a statistically significant change; and cerebral stroke or asymptomatic cerebral infarction can be detected based on the value.
The mathematical statistical analysis may be carried out by using procedures well-known to those skilled in the art, preferably by using a neural network technique. The neural network technique can be conducted, for example, by using NEUROSIM/L (Fujitsu Ltd.), according to the-attached manual.
“Cutoff value” is a value set for the detection of a specific disease. Asymptomatic cerebral infarction can be detected using a value obtained as described above as “cutoff value.” Cerebral stroke or asymptomatic cerebral infarction can also be detected using the value obtained as described above, which statistically significantly varies from the “cutoff value.” For example, in a patient with asymptomatic cerebral infarction and a healthy subject, an ROC (Receiver Operating Characteristic) curve is developed by using commercially available statistical analysis software based on values obtained by measuring the contents of the above biomarkers, the optimal sensitivity and specificity are determined, and a cutoff value may be set depending on the purpose of detection, for example, so that a higher sensitivity is given priority in detection for a purpose such as primary screening and a specificity is higher in detection for the purpose of close investigation.
Based on the findings in accordance with an aspect of the present invention, a method of prophylaxis of cerebral stroke and prevention of progress of the symptom by suppressing the contents of acrolein, and interleukin-6 and CRP which are inflammatory markers, polyamine oxidase activity or these protein contents in a subject sample can also be provided.
Furthermore, there is also provided a method of screening (exploration) of a new drug which is effective for treatment of cerebral stroke by administering an experimental animal with a candidate compound which may be effective for treatment of cerebral stroke and assaying whether the compound has the activity of inhibiting the contents of acrolein, and interleukin-6 and CRP which are inflammatory markers, polyamine oxidase activity or these protein contents in the experimental animal.
In accordance with an aspect of the present invention, a kit for detecting cerebral stroke or asymptomatic cerebral infarction is further provided. The kit comprises a reagent for measuring the contents of an aldehyde compound formed from polyamine, interleukin-6 and C-reactive protein, polyamine oxidase activity or a polyamine oxidase protein content in a biological sample. Furthermore, e.g., any measuring instrument or apparatus, standard solution and buffer well-known to those skilled in the art may be optionally contained.
In accordance with an aspect of the present invention, there is also provided a system for detecting cerebral stroke or asymptomatic cerebral infarction, comprising: a reagent for measuring the contents of a aldehyde compound formed from polyamine, interleukin-6 and C-reactive protein, polyamine oxidase activity or a polyamine oxidase protein content in a biological sample; and an electronic treating device and software for carrying out mathematically statistical analysis. Such electronic treating devices for carrying out mathematically statistical analysis may include, e.g., an appropriate computer well-known to those skilled in the art; and such software for carrying out mathematically statistical analysis may include, e.g., one allowing the above neural network technique.
Hereinafter, the present invention will be further concretely described referring to an example concerning the construction of a cerebral infarction judgment model employing an artificial neural network, but the invention is not limited thereto.
The presence or absence of infarction was examined by taking head tomographic images by magnetic resonance imaging (MRI) with the permission and consent of subjects. An as a result, the subject group was divided into 44 patients with asymptomatic cerebral infarction and 53 healthy subjects. Blood was collected from the patients with asymptomatic cerebral infarction and the healthy subjects to compare the contents of acrolein, interleukin-6 (IL-6) and CRP and polyamine oxidase activity in plasma.
The plasma acrolein content was determined by measuring the content of FDP-lysine (N-formyl-piperidino-lysine), which is an amino acid adder with acrolein. The above procedure was conducted using ACR-LYSINE ADDUCT ELISA SYSTEM (NOF CORPORATION). The plasma interleukin-6 content was measured by the above procedure using Human IL-6 ELISA (ENDOGEN CORPORATION). The plasma CRP content was measured by the above procedure using Human CRP ELISA KIT (Alpha Diagnostics International, Inc.). The measurement of the activities of polyamine oxidases (AcPAO and SMO) was conducted by the above procedure.
To construct a neural network model, NEUROSIM/L version 4 (Fujitsu) was used as software, and learning was conducted by an error back-propagation method at learning constants of a learning rate (e)=5.0 and a momentum term (a)=0.4. In the neural network, three input layers were set at five of the plasma biomarkers (polyamine oxidase, acrolein, interleukin-6 and CRP) and a subject's age, and, based on empirical rules, intermediate and output layers were set at three and one, respectively. Values of “1” for the patients with asymptomatic cerebral infarction and “0” for the healthy subjects were input into teacher data in an output layer. Learning data were from 97 samples in total consisting of 44 samples from the patients with asymptomatic cerebral infarction and 53 samples from the healthy subjects.
To judge the presence or absence of asymptomatic cerebral infarction by the practical detection of the images and to examine the accuracy of the results of prediction in the neural network model, receiver operating characteristic (hereinafter, ROC) curve analyses and areas under the ROC curve (AUC) were calculated using the predicted values calculated by the above software analysis. GraphPad PRISM 4 (GraphPad Software Corporation) was used for the calculation of the ROC curve analyses and the AUCs. The accuracy of the cutoff values was evaluated with sensitivity, specificity, predictive values (positive and negative reaction predictive values) and likelihood ratios (positive and negative likelihood ratios).
Cerebral stroke or asymptomatic cerebral infarction can be detected and diagnosed early by using the detection method according to an aspect of the present invention. Furthermore, a possibility of paving a new way for prophylaxis or early treatment of a cerebral stroke and asymptomatic cerebral infarction, such as development of a drug for prophylaxis or treatment of cerebral stroke and asymptomatic cerebral infarction, is provided by exploring a compound that removes acrolein, polyamine oxidase, and interleukin-6 and CRP which are inflammatory markers by utilizing the findings of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2007-132239 | May 2007 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2008/052701 | 2/19/2008 | WO | 00 | 3/2/2010 |
