# Patent

## Title
Method for determining whether or not a mammal is affected with a lung cancer

## Abstract

The present invention relates to a method for determining whether or not a mammal is affected with a lung cancer. The method for determining whether or not a mammal is affected with a lung cancer of the present invention includes the steps of:

    
    
        (a) detecting whether or not anthranilic acid is contained in the urine excreted from the mammal; and
        (b) determining that the mammal is affected with a lung cancer when the anthranilic acid is detected as being contained in the urine.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for determining whether or not a mammal is affected with a lung cancer.
2. Description of the Related Art
Substances (a kind of proteins) that exist in a large amount only in cancer cells but either do not exist, or even though exist, do exist in just a slight amount in normal cells have been known. Such proteins are referred to as “tumor markers”. Tumor marker tests, in general, quantitatively determine a tumor marker in blood, and have been used for the purpose of aiding diagnosis of cancer, or confirming the degree of progression of cancer.
Conventional tumor markers are high molecular substances such as hormones, enzymes, isozymes or fragmented proteins. Since there are great differences among individuals in blood levels of these conventional tumor markers, usability in diagnosing cancer has been unsatisfactory due to occurrence of false negative and false positive.
SUMMARY OF THE INVENTION
The present inventors investigated utilization of a low molecular compound, to which attention has not been drawn hitherto, as a tumor marker, and consequently discovered that by quantitatively determining anthranilic acid in urine excreted from a mammal, determination is enabled as to whether or not the mammal is affected with a lung cancer. Accordingly, the present invention was accomplished.
The present invention relates to a method for determining whether or not a mammal is affected with a lung cancer, the method comprising the steps of:
(a) detecting whether or not anthranilic acid is contained in the urine excreted from the mammal; and
(b) determining that the mammal is affected with a lung cancer when the anthranilic acid is detected as being contained in the urine.
According to the present invention, a method of determination is provided which is useful for diagnosing lung cancer in mammals.
The above objects, other objects, features and advantages of the present invention will be apparent from the following detailed description of preferred embodiments with reference to attached drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1(a) shows an ion chromatogram of control mouse (normal mouse) urine subjected to an ultrafiltration treatment.
FIG. 1(b) shows an ion chromatogram of cancer-bearing mouse urine subjected to an ultrafiltration treatment.
FIG. 2(a) shows an extracted ion chromatogram of a specified component A in cancer-bearing mouse urine.
FIG. 2(b) shows an MS/MS spectrum of the specified component A shown in FIG. 2(a).
FIG. 2(c) shows an ion chromatogram of anthranilic acid.
FIG. 2(d) shows an MS/MS spectrum of anthranilic acid.



DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
1. Production Method of Cancer-bearing Mouse, and Urine Collection Method
1.1 Animals Used for Experiments
As immunocompromised mice, C.B-17/Icr-scid/scid Jcl (CLEA Japan, Inc.), 5 weeks-old male (50 animals) and female (15 animals) mice were used. The mice were fed separately in a glass metabolism cage for mouse (Metabolica, manufactured by Sugiyama-Gen Co., Ltd.). The mice were fed with CE-2 (CLEA Japan, Inc.) sterilized with radioactive ray as a chow, and given purified water which had been subjected to steam sterilization under pressure (121° C., 30 min) as drinking water.
1.2 Cells for Transplantation
A549 (DS Pharma Biomedical Co., Ltd.) that is a lung cancer cell line derived from human was used as cells for transplantation. Using a DMEM medium containing 10% fetal calf serum (FBS), A549 cells were proliferated by culturing at 37° C., in the presence of 5% CO2.
1.3 Production of Cancer-Bearing Mouse
With respect to all individuals of the mice, the body weight was measured using an electronic balance (BJ600, manufactured by Sartorius K.K.). Then, the mice were grouped such that the average body weight of each group became equalized. The cancer-bearing group included 29 male mice and 17 female mice, whereas the control group (normal group) included 9 male mice and 5 female mice. A549 cells proliferated by culture were recovered, and transplanted into the mice of the cancer-bearing group subcutaneously at the right abdomen in an amount of 5×107/0.1 ml/mouse.
1.4 Urine Collection Method
Collection of the urine from mice was carried out on both the cancer-bearing group and the control group. The day on which A549 cells were transplanted into the mice of the cancer-bearing group was defined as test day 0, and the urine was collected every day until test day 30. The mouse was retained, and the urine collected into a 0.5 ml tube was provided as fresh urine. The fresh urine was stored at −80° C.
2. Pretreatment of Urine Sample
2.1 Thawing of Urine Sample
The fresh urine from the male mouse which had been freeze preserved at −80° C. (urine collected on day 26 to day 30 from starting the test) was thawed on ice. The fresh urine in a plurality of 0.5 ml tubes was assembled in a single 1.5 ml tube.
2.2 Ultrafiltration Treatment
The thawed fresh urine was subjected to centrifugal separation using Millipore Ultrafree-MC filters (UFC3 LGC 00, MW: 10,000, manufactured by Millipore) at 7,000 G at 0° C. for 90 min to allow for ultrafiltration of the fresh urine. The urine left on the filter was set on a new filter, and a similar operation was repeated. The urine that passed through the filter was set on a Microcon centrifugation system filter unit YM-3 (MW: 3,000, manufactured by Millipore), and an operation similar to that described above was carried out. The filtrate obtained by this operation was dispensed 50 μl each into 0.5 ml tubes, and stored at −80° C. until mass spectrometry was carried out.
3. Mass Spectrometry
3.1 Reagents and Apparatuses
As reagents for use in the mass spectrometry, acetonitrile for HPLC (Wako Pure Chemical Industries, Ltd.) and formic acid for LC/MS (Wako Pure Chemical Industries, Ltd.) were used.
Upon the mass spectrometry, a high performance liquid chromatograph (manufactured by Shimadzu Corporation, Prominence series) and a mass spectrometer (manufactured by Shimadzu Corporation, LCMS-IT-TOF) were used.
3.2 Analysis Conditions
Experimental conditions of the mass spectrometry were as in the following.
Column: Develosil RPAQUEOUS-AR (internal diameter: 2.0 mm, length: 250 mm, particle size: 5 μm)
Column temperature: 40° C.
Flow rate: 0.2 ml/min
Mobile phase: solvent A: 0.1% aqueous formic acid solution, and solvent B: 0.08% formic acid/acetonitrile
Gradient condition:

    
    
        A:B=100:0 (0 min)→100:0 (5 min)→80:20 (45 min)→70:30 (55 min)→50:50 (70 min)→5:95 (71 min) 5:95 (75 min)
    
    


Amount of infusion: 5 μl
Ionization method: ESI+, ESI−
4. Identification and Quantitative Determination of Specified Component that Serves as a Cancer Marker
4.1 Confirmation of Specified Component A
A lung cancer cell culture fluid and a normal cell culture fluid were subjected to an ultrafiltration treatment in a similar manner to the fresh urine, and comparison of total ion chromatogram of the filtrate was made. A peak increased in the lung cancer cell culture fluid was confirmed by visual inspection, which was denoted as a specified component A. Comparison of the total ion chromatogram of the filtrate was made also with regard to the urine from the control mouse and the cancer-bearing mouse, and it was confirmed that the concentration of the specified component A increased in the cancer-bearing mouse urine (FIG. 1(b)) than in the control mouse urine (FIG. 1(a)). The m/z value of the specified component A was 138.051 [M+H]+ as in FIG. 1(a) and FIG. 1(b).
4.2 Searching of Candidate Compound
A compound to be the candidate for the specified component A was searched on a database. On the basis of the m/z value of the specified component A (138.051 [M+H]+) obtained by the mass spectrometry, a candidate compound was searched using a compound database Madison Metabolomics Consortium Database (http://mmcd.nmrfam.wisc.edu/). Furthermore, using MassBank.jp (http://www.massbank.jp/) which is an MS/MS spectral database, the MS/MS spectral pattern of the specified component A was confirmed as to whether or not it agrees with the MS/MS spectral pattern on the database. As a result, the m/z value of the specified component A obtained by the mass spectrometry, and the MS/MS spectral pattern agreed with the information of anthranilic acid (m/z: 138.055 [M+H] (measured)) retrieved from the database.
4.3 Preparation of Standard Sample and Identification of Specified Component A
For the purpose of identifying the specified component A, anthranilic acid (Wako Pure Chemical Industries, Ltd., guaranteed reagent) was purchased, and using an anthranilic acid solution as a standard solution, an LC-MS/MS analysis was carried out. The anthranilic acid was dissolved in water, which was provided as a primary standard solution. Furthermore, the primary standard solution was diluted in water to produce a 10 μM standard solution for analysis. With respect to this standard solution for analysis, the LC-MS/MS analysis was carried out. As a result, the retention time and the MS/MS spectral data of the specified component A (FIG. 2(a) and FIG. 2(b)) were confirmed to agree with the retention time and the MS/MS spectral data of anthranilic acid in the standard solution for analysis (FIG. 2(c) and FIG. 2(d)). Thus, the specified component A was identified to be anthranilic acid.
4.4 Production of Calibration Curve
For the purpose of measuring the concentration of anthranilic acid in the cancer-bearing mouse urine, standard solutions for producing a calibration curve were prepared by diluting the primary standard solution in water to give the concentrations of 10 μM, 5 μM, 2 μM and 1 μM. A calibration curve was produced by plotting the concentrations and the peak areas of these standard solutions for producing a calibration curve. The concentration of anthranilic acid in the urine of the cancer-bearing mouse and the control mouse was calculated on the calibration curve. As a result, the concentration of anthranilic acid in the urine was 0.062 μM in the cancer-bearing mouse, and the detection limit or less in the control mouse, as shown in Table 1.







TABLE 1







Peak Area in Urine and Concentration in Urine of Anthranilic Acid










Cancer-bearing mouse
Control mouse


Acid
(day 26-30)
(day 26-30)












First analysis peak area
29985
0


Second analysis peak area
30548
0


Third analysis peak area
0
0


Mean peak area *
30267
0


Concentration in urine 
0.062
detection limit or less


(μM)





* The Mean peak area is an average of the first analysis peak area and the second analysis peak area.






From the aforementioned experimental results, it was confirmed that anthranilic acid is detected in urine excreted from a mammal affected by a lung cancer. For almost all conventional tumor markers, it has been necessary to collect blood from a patient, and then measure the concentration of the tumor marker in plasma. However, according to the present invention, since the tumor marker which is a target of detection is a component in urine, an inspection sample can be noninvasively obtained without imposing a burden to the patient. According to the present invention, an efficient determination as to whether or not a mammal is affected with a lung cancer is enabled.

## Claims

1. A method for determining whether or not a mammal is affected with a lung cancer, said method comprising the steps of:
(a) measuring the concentration of anthranilic acid contained in urine excreted from the mammal; and
(b) determining that the mammal is affected with a lung cancer if the concentration of anthranilic acid contained in the urine is more than 0 M, and determining that the mammal is not affected with a lung cancer if the concentration of anthranilic acid contained in the urine is 0 M.