JP7493176B2 - Prediction of dermatitis due to radiation exposure - Google Patents

Description

The present invention relates to predicting the occurrence of dermatitis due to radiation exposure.

In recent years, radiation therapy and imaging-guided therapy (IVR) have become widely used, and side effects from radiation exposure have become a problem. Even when similar radiation therapy or IVR is performed, whether or not dermatitis, a side effect, occurs varies from case to case, making it difficult to predict whether dermatitis will occur. Furthermore, some skin reactions are immediate while others take days or weeks to develop. Even if dermatitis does occur, it may be a transient condition or a strong erythema may persist for 3 to 4 weeks, making it difficult to predict the severity of the dermatitis.

To date, methods have been proposed for diagnosing the risk of developing late breast effects after radiation therapy using biochemical markers related to T lymphocyte apoptosis and clinical parameters (Patent Document 1), a method for predicting the occurrence of dermatitis by identifying blood flow from images of the exposed area (Patent Document 2), and a method for predicting radiation sensitivity using cells collected from skin tissue (Patent Document 3).

JP 2019-532304 A International Publication No. 2015/146164 JP 2017-505630 A

However, the conventional methods have problems such as complicated steps leading to diagnosis, the need for special equipment, being highly invasive, and taking a long time to obtain results.
The problem to be solved by the present invention is to provide a method for collecting data to more simply predict the occurrence and severity of dermatitis after radiation exposure, and a kit related thereto.

In other words, after examining various methods, the inventors discovered that it is possible to predict the occurrence of dermatitis by measuring the glutathione concentration in red blood cells after exposure to radiation from radiation therapy or imaging-guided therapy, and thus completed the present invention.

The present invention, which solves the above problems, comprises the following:
(1) A biomarker for predicting the occurrence or non-occurrence of dermatitis after radiation exposure due to radiation therapy or radiotherapy, the biomarker being glutathione or its concentration.
(2) The biomarker according to (1), wherein the biomarker is reduced glutathione concentration/oxidized glutathione concentration in red blood cells.
(3) The biomarker according to (1) or (2), wherein the radiation exposure is due to exposure to X-rays, electron beams, gamma rays, proton beams, heavy particle beams, or neutron beams.
(4) The biomarker according to any one of (1) to (3), wherein the radiation exposure is due to radiation therapy for breast cancer, skin cancer, head and neck cancer, rectal cancer, esophageal cancer, brain tumor, metastatic brain tumor, liver cancer, lung cancer, or malignant lymphoma.
(5) A method for measuring a biomarker, comprising:
A method for measuring any one of the biomarkers according to (1) to (4) in a sample obtained from a subject after exposure to radiation through radiotherapy or radiotherapy.
(6) A kit comprising a means for measuring the biomarker described in (1) to (4).
(7) A method for collecting data for predicting whether or not dermatitis will occur after radiation exposure due to radiation therapy or imaging-guided therapy, the method comprising measuring a glutathione concentration in a sample obtained from a subject after radiation exposure, and predicting a high possibility of dermatitis occurring if the reduced glutathione concentration/oxidized glutathione concentration of the sample is below a first standard value.
(8) The method according to (1), in which it is predicted that when the reduced glutathione concentration/oxidized glutathione concentration is equal to or lower than a first standard value and higher than a second standard value, there is a high possibility that transient dermatitis will occur, and when the reduced glutathione concentration/oxidized glutathione concentration is equal to or lower than the second standard value, there is a high possibility that persistent dermatitis will occur.
(9) The method according to (7) or (8), wherein the radiation exposure is due to exposure to X-rays or electron beams.
(10) The method according to any one of (7) to (9), wherein the radiation exposure is due to radiation therapy for breast cancer, skin cancer, or head and neck cancer.
(11) A kit for predicting the occurrence or non-occurrence of dermatitis after radiation exposure due to radiation therapy or radiotherapy, comprising a means for measuring the concentrations of reduced glutathione and oxidized glutathione in a sample obtained from a subject.

The present invention also relates to the following inventions:
[1] A method for treating dermatitis occurring after radiation exposure due to radiation therapy or radiotherapy, comprising:
A method of treatment comprising: measuring a glutathione concentration in a sample obtained from a subject after radiation exposure; and initiating preventive treatment for dermatitis if it is predicted that the dermatitis is likely to occur.
[2] The treatment method described in [1], in which a high possibility of dermatitis occurring is predicted when the reduced glutathione concentration/oxidized glutathione concentration of the sample is below a first reference value.
[3] The method of treatment according to [1] or [2], wherein the radiation therapy is a treatment including irradiation with X-rays or electron beams.
[4] The therapeutic method according to any one of [1] to [3], wherein the radiation exposure is due to radiation therapy for breast cancer, skin cancer, head and neck cancer, rectal cancer, esophageal cancer, brain tumor, metastatic brain tumor, liver cancer, lung cancer, or malignant lymphoma.

The present invention provides a method for collecting data to more easily predict the occurrence and severity of dermatitis after radiation exposure, as well as related kits, markers, measurement methods, and treatment methods.

FIG. 1 is a diagram showing the degree of skin damage after radiation exposure. FIG. 1 shows glutathione concentrations in red blood cells after radiation exposure. * indicates significant differences between groups (P<0.05).

The following describes in detail the embodiments of the present invention. However, the following description of the components is a representative example of the embodiments of the present invention, and can be modified as appropriate without departing from the spirit of the present invention.

[First embodiment]
The biomarker according to the first embodiment of the present invention is glutathione or its concentration. The biomarker according to the present embodiment can be used to predict the occurrence of dermatitis after radiation exposure due to radiation therapy or radiotherapy.

Glutathione is a tripeptide consisting of glutamic acid, cysteine, and glycine, and is an antioxidant. There are reduced (GSH) and oxidized (GSSG) glutathione, and oxidized glutathione is formed by two molecules of reduced glutathione linked by a disulfide bond. Usually, 98% or more exists as reduced glutathione. Glutathione exists in cells at a relatively high concentration of 0.5 to 10 mM.
Glutathione includes total glutathione, reduced glutathione, oxidized glutathione, and combinations thereof, any of which can be used as a biomarker for predicting the occurrence or nonoccurrence of dermatitis after radiation exposure due to radiation therapy or imaging-guided therapy. However, taking into consideration the accuracy of prediction, it is preferable to use reduced glutathione concentration/oxidized glutathione concentration (the ratio of reduced glutathione concentration to oxidized glutathione concentration) as the biomarker.

In this embodiment, the biomarker may be any biomarker in a subject for which the onset of dermatitis needs to be predicted, and the subject may be not only a human, but also a mammal such as a cow, pig, monkey, guinea pig, rabbit, rat, mouse, or dog.

In this embodiment, glutathione or its concentration in a sample obtained from a subject is used as a biomarker, and the sample may be blood, urine, saliva, serum, etc., with blood being preferred.

In this embodiment, dermatitis refers to skin damage caused by radiation exposure. Symptoms of dermatitis include redness, erythema, swelling, desquamation, blisters, erosion, ulcers, necrosis, telangiectasia, and the like. Dermatitis can be classified into transient dermatitis, which tends to resolve after the onset of dermatitis symptoms, and persistent dermatitis, in which symptoms persist for one month or more.

Radiation generally means ionizing radiation, and is classified into electromagnetic radiation such as X-rays and gamma rays, and particle radiation such as alpha rays, beta rays, electron beams, proton beams, heavy particle beams, and neutron beams.
In one embodiment of the present invention, the radiation used in radiotherapy or imaging-guided therapy is preferably radiation selected from X-rays, electron beams, gamma rays, proton beams, heavy particle beams, and neutron beams.

Radiation therapy refers to the treatment of disease using radiation or radioactive substances. Radiation therapy aims to irradiate the lesion with radiation, but external irradiation is often used, in which radiation is irradiated from outside the body, and the radiation passes through the skin. A typical radiation therapy is administered at a range of 0.5 to 10 units (Grays) per day.

Diseases for which radiation therapy is performed include cancer. Cancers that are the subject of radiation therapy are not particularly limited by their characteristics, such as the organ of origin, tissue, stage, primary or metastatic status, and the subject's treatment history, but examples include breast cancer (mammary adenocarcinoma, breast squamous cell carcinoma, etc.), skin cancer (basal cell carcinoma, squamous cell carcinoma (squamous cell carcinoma), melanoma Kaposi's sarcoma, breast Paget's disease, extramammary Paget's disease, adnexal tumor, cutaneous T-cell lymphoma, etc.), head and neck cancer (oral cancer, pharyngeal cancer, oral cancer, nasal and paranasal sinus cancer, salivary gland cancer, thyroid cancer, etc.), lung cancer (small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, etc.), esophageal cancer, stomach cancer, liver cancer, kidney cancer, colon cancer, large intestine cancer, pancreatic cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, and vulvar cancer.

When the cancer to be treated has a lesion located shallow under the skin, radiation with relatively low energy, such as X-rays or electron beams, is used, which produces the maximum radiation dose at a shallow location under the skin. A characteristic of these types of radiation is that they have a strong effect on the skin close to the lesion, which can easily lead to the problem of dermatitis caused by radiation exposure after radiation therapy.

Therefore, in this embodiment, it is preferable that the radiation is radiation such as X-rays or electron beams that produces a maximum radiation dose at a shallow position beneath the skin, and that the radiation therapy is radiation therapy for cancers such as breast cancer, skin cancer, and head and neck cancer that have pathogens at a shallow position beneath the skin.

Interventional Radiology (IVR) is a treatment that uses radiological diagnostic technology, for example, treatment performed while viewing fluoroscopic images obtained by X-ray irradiation. IVR has the advantage of being able to treat lesions non-invasively, even in treatments that previously required open surgery, and is therefore rapidly becoming more widespread.
On the other hand, IVR is performed while irradiating the body with X-rays from outside, so the X-rays pass through the patient's skin.

Image-guided treatment is not particularly limited by the characteristics of the target disease, such as the organ where it originates, tissue classification, and progression, but examples include treatments involving the insertion of medical devices such as catheters and stents into blood vessels or ductal tissue, followed by embolization therapy, chemotherapy, drainage therapy, and stent placement therapy, as well as treatments involving the insertion of treatment needles into non-vascular lesion tissue, followed by pathological tissue sampling, wave ablation therapy, cryotherapy, and arterial embolization therapy.

The 60-120 kVp X-rays used in image-guided therapy have a maximum radiation dose at a shallow position under the skin, so the occurrence of dermatitis due to radiation exposure after treatment is likely to be a problem. Therefore, in this embodiment, image-guided therapy is preferably associated with treatment or examination of heart disease, treatment or examination of head and neck disease, or treatment or examination of liver disease.

[Second embodiment]
A method according to a second embodiment of the present invention is a method comprising measuring any of the biomarkers according to the first embodiment in a sample obtained from a subject after exposure to radiation by radiation therapy or imaging-guided therapy.

The method of this embodiment can obtain the concentration of glutathione, which is a biomarker. Here, glutathione includes total glutathione, reduced glutathione, oxidized glutathione, and combinations thereof. The method of this embodiment includes measuring the concentration of any of these glutathiones, but considering the accuracy of predicting the occurrence or non-occurrence of dermatitis after radiation exposure due to radiation therapy or imaging-guided therapy, it is preferable that the method includes measuring the reduced glutathione concentration/oxidized glutathione concentration (the ratio of the reduced glutathione concentration to the oxidized glutathione concentration). In this embodiment, the glutathione concentration may be an absolute concentration, or a value that correlates with the absolute concentration and allows comparison of the absolute concentration in each subject, or it may be a relative concentration, weight per volume, etc.

In one method according to this embodiment, the glutathione concentration is measured by centrifuging blood collected from a subject, collecting the red blood cells, which are the lower layer, and hemolyzing them, and quantifying the total glutathione concentration and oxidized glutathione concentration in the sample using a measurement kit such as a GSSG/GSH Quantification kit (Dojindo Laboratories), and the reduced glutathione concentration is calculated from the difference between the total glutathione concentration and the oxidized glutathione concentration. The measurement kit includes, for example, an enzyme, a coenzyme, a buffer, a substrate, an internal standard, etc.

Biomarkers may also be measured by other known methods using samples collected from subjects. For example, biomarkers may be measured by known methods such as direct competitive method, indirect competitive method, sandwich method, or other enzyme-linked immunosorbent assay (ELISA) method, RIA (radioimmunoassay) method, flow cytometry method, immunochromatography, etc., using antibodies specific to the biomarkers.

In this embodiment, the date of blood collection from the subject is not limited to a range in which glutathione concentration can be measured at a level that can predict the occurrence of dermatitis after radiation exposure, and can be determined by a person skilled in the art taking into consideration various conditions such as the animal species and condition of the subject, and the intensity and duration of radiotherapy. However, taking into consideration accuracy and simplicity, it is preferable to collect blood 1 to 14 days after exposure, more preferably 2 to 10 days, and even more preferably 2 to 6 days.

In this embodiment, the subject for which the biomarkers are measured may be any subject for which the onset of dermatitis needs to be predicted, and may be any mammal other than humans, such as cows, pigs, monkeys, guinea pigs, rabbits, rats, mice, and dogs.

[Third embodiment]
The concentration of the biomarker according to the first embodiment can be measured by the measurement method according to the second embodiment, and data for predicting the occurrence or non-occurrence of dermatitis after radiation exposure due to radiation therapy or radiotherapy can be collected.

That is, the method according to the third embodiment of the present invention is a method for collecting data for predicting the occurrence or non-occurrence of dermatitis after radiation exposure due to radiation therapy or imaging-guided therapy, which includes measuring the glutathione concentration of a sample obtained from a subject after radiation exposure, and predicts the high possibility of dermatitis occurring when the reduced glutathione concentration/oxidized glutathione concentration of the sample is equal to or lower than a first reference value.

In one such method of the present embodiment, the glutathione concentration in the red blood cells of a subject, in particular the reduced glutathione concentration/oxidized glutathione concentration in the red blood cells (the ratio of reduced glutathione concentration to oxidized glutathione concentration) is used as an indicator to predict the occurrence of dermatitis. Such an indicator can be measured by taking blood from the subject and using a measurement kit, for example, as described in the second embodiment. Therefore, the method of the present embodiment has the following characteristics: (1) the process is simple, (2) no special equipment is required, (3) it is less invasive to the subject, and (4) it takes a short time to obtain results.

For example, a group of subjects who have undergone a certain radiation therapy or imaging-guided therapy is measured for biomarkers after exposure. Then, the subjects can be classified based on the occurrence and severity of dermatitis after radiation exposure, and a first reference value can be determined. For example, the first reference value is the median or average of the reduced glutathione concentration/oxidized glutathione concentration in red blood cells in a group that has developed mild dermatitis or transient dermatitis after radiation exposure. Alternatively, the first reference value may be a value that can distinguish between a group that has developed dermatitis after radiation exposure and a group that has not developed dermatitis with a desired accuracy based on biomarkers in these groups.
In another embodiment, the first reference value may be 90%, 85%, 80%, 75% or 70% of the normal value of reduced glutathione concentration/oxidized glutathione concentration in red blood cells, and a value that allows distinction between these populations with the desired accuracy can be appropriately determined by a person skilled in the art.

In addition, in one method according to the present embodiment, it is predicted that when the reduced glutathione concentration/oxidized glutathione concentration in red blood cells is equal to or lower than a first reference value and higher than a second reference value, there is a high possibility that transient dermatitis will occur, and when it is equal to or lower than the second reference value, there is a high possibility that persistent dermatitis will occur.

For example, a biomarker in red blood cells is measured after exposure for a group of subjects who have undergone a specific radiation therapy or imaging therapy. The occurrence and severity of dermatitis after radiation exposure can then be examined, and the group can be classified into a group in which dermatitis did not occur, a group in which transient dermatitis occurred, and a group in which persistent dermatitis occurred, and the first and second reference values can be determined. For example, the second reference value is the median or average of the reduced glutathione concentration/oxidized glutathione concentration in red blood cells in a group in which persistent dermatitis occurred after radiation exposure. Alternatively, the first and second reference values may be appropriately determined by a person skilled in the art as values that can distinguish, with a desired accuracy, a group in which dermatitis did not occur, a group in which transient dermatitis occurred, and a group in which persistent dermatitis occurred, based on the biomarker concentrations in the groups in which transient dermatitis occurred and those in which persistent dermatitis did not occur after radiation exposure, and the biomarker concentrations in the groups in which persistent dermatitis occurred and those in which persistent dermatitis did not occur.

In another embodiment, the second reference value may be 65%, 60%, 55% or 50% of the normal value of the reduced glutathione concentration/oxidized glutathione concentration in red blood cells, and a value that allows distinction between these populations with the desired accuracy can be appropriately determined by a person skilled in the art.

Here, the normal value refers to the value before the subject is exposed to radiation. Alternatively, the normal value refers to the median or average value of the values in a population that has not been exposed to radiation (a population of subjects before undergoing radiation therapy or imaging therapy, or a population of healthy subjects).

Here, the skin condition that serves as the criterion for the occurrence or nonoccurrence of dermatitis can be appropriately set according to the level of dermatitis that a person skilled in the art wishes to predict.

The date of blood collection for measuring biomarkers in red blood cells after exposure is not particularly limited as long as it is within a range that allows measurement of glutathione concentrations at a level that can predict the occurrence of dermatitis after radiation exposure, and can be determined by a person skilled in the art taking into consideration various conditions such as the animal species and condition of the subject, and the intensity and duration of radiotherapy. However, taking into consideration accuracy and simplicity, it is preferable to collect blood 1 to 14 days after exposure, more preferably 2 to 10 days, and even more preferably 2 to 6 days.

In this embodiment, taking into consideration the accuracy of predicting the occurrence or nonoccurrence of dermatitis after radiation exposure, it is preferable that the predetermined radiation therapy or imaging-guided treatment for the subject group used when determining the first or second reference value is radiation therapy or imaging-guided treatment under the same conditions as the treatment that the subject to be predicted will receive.

Glutathione is present in all mammalian cells, and it is known that the glutathione concentrations in mouse and human red blood cells are similar (The Journal of Toxicological Sciences, Vol. 28, No. 5, 455-469, 2003 and Free Radic Biol Med. 2013 Dec;65:742-749). It is also known that many of the genes involved in glutathione synthesis and metabolism are common between mice and humans (https://www.genome.jp/dbget-bin/www_bget?hsa00480 and https://www.genome.jp/dbget-bin/www_bget?mmu00480).

[Fourth embodiment]
The kit according to the fourth embodiment of the present invention is a kit including a means for measuring a biomarker according to one embodiment of the present invention.

By using the kit of this embodiment, the concentration of glutathione can be measured. In addition, the kit of this embodiment can be used to collect data for predicting the occurrence or non-occurrence of dermatitis after radiation exposure, for example, due to radiation therapy or imaging-guided therapy according to the third embodiment of the present application, and to predict the occurrence or non-occurrence of dermatitis after radiation exposure based on the collected data.

Another kit according to this embodiment is a kit for predicting the occurrence of dermatitis after radiation exposure due to radiation therapy or imaging-guided therapy, which includes a means for measuring the concentrations of reduced glutathione and oxidized glutathione in red blood cells.

By using the kit of this embodiment, the concentrations of reduced glutathione and oxidized glutathione in the red blood cells of a subject can be measured, and it is possible to predict whether or not a subject exposed to radiation through radiation therapy or imaging-guided therapy will develop dermatitis.

Means for measuring the concentrations of reduced glutathione and oxidized glutathione in red blood cells include, for example, a measurement kit containing an enzyme, a coenzyme, a buffer, a substrate, an internal standard substance, etc., with enzymes, coenzymes, and an internal standard substance being particularly preferred.

Alternatively, the means may be an antibody specific to the biomarker and a visualization reagent thereof, and may include reagents for performing well-known methods such as direct competitive method, indirect competitive method, sandwich method, ELISA (enzyme-linked immunosorbent assay) method, RIA (radioimmunoassay) method, flow cytometry method, immunochromatography, etc.

The kit in this embodiment may further include instructions stating that it is predicted that there is a high possibility of dermatitis occurring when the reduced glutathione concentration/oxidized glutathione concentration in red blood cells is equal to or lower than a first reference value, or instructions stating that it is predicted that there is a high possibility of transient dermatitis occurring when the reduced glutathione concentration/oxidized glutathione concentration in red blood cells is equal to or lower than the first reference value and higher than a second reference value, and that there is a high possibility of persistent dermatitis occurring when the reduced glutathione concentration/oxidized glutathione concentration in red blood cells is equal to or lower than the second reference value.

[Fifth embodiment]
A treatment method according to a fifth embodiment of the present invention is a treatment method for dermatitis occurring after radiation exposure due to radiation therapy or imaging-guided therapy, comprising measuring a glutathione concentration in a sample obtained from a subject after radiation exposure, and initiating preventive treatment for dermatitis when it is predicted that dermatitis will occur.

According to the treatment method of this embodiment, when dermatitis is predicted to occur after radiation exposure using the biomarkers, methods for measuring biomarker concentrations, methods for collecting data, and/or kits of the first to fourth embodiments of the present application, preventive treatment can be started before dermatitis occurs, thereby reducing dermatitis.

In this embodiment, the animal that is predicted to develop dermatitis after radiation exposure and is the subject of treatment may be any subject that requires preventive treatment for dermatitis, and may be not only humans, but also mammals such as cows, pigs, monkeys, guinea pigs, rabbits, rats, mice, and dogs.

Preventive treatments include heparinoid creams, moisturizing creams, antioxidants, and anti-inflammatory drugs.

The present invention will be explained in more detail below with reference to examples, but the interpretation of the present invention is not limited to these examples.

[Example 1] Confirmation of the occurrence and degree of dermatitis after radiation exposure 1. X-ray irradiation of mice Six-week-old male C57BL/6J mice were placed in a simple inhalation anesthesia device for small animal experiments (NARCOBIT-E; Natsume Seisakusho) and anesthetized with sevoflurane (Fujifilm Wako Pure Chemicals). Gas concentration was 5% for induction and 3% for maintenance. The sleeping mice were placed in a container for irradiation, and X-rays were irradiated only to the right leg of the mouse using an X-ray irradiation device (MBR-1520R-3; Hitachi Power Solutions) under the following conditions: dose rate 0.69 Gy/min, focal point-table surface distance 550 mm, tube voltage 150 kV, current 20 mA, filter: 0.2 mm Cu and 0.5 mm Al, so that the doses were 5, 20, and 30 Gy, respectively.

2. Observation of Dermatitis The condition of the irradiated skin of the mice was observed with the naked eye for 80 days after X-ray irradiation. The skin condition was evaluated according to the following criteria, and the average of three scores was taken as the skin reaction score.
・Skin reaction evaluation criteria (score) (degree of skin reaction)
0 No change 0.5 50/50 difference from normal, suspicious 0.75 Definite but slight abnormality 1 Definite abnormality with redness 1.25 Severe redness and/or white scales and/or swelling 1.5 Scaly or crusty appearance in one very small area 1.75 Small area of moist desquamation (more distinct than 1.5)
2. Widespread damage (wet)
2.5 Damage to large areas of skin with clear moist exudate3 Damage to large areas of skin with moist exudate3.5 Complete moist decomposition of limbs - often attached to body

3. Results The results are shown in Figure 1.
As can be seen from FIG. 1, no erythema was observed in the mice irradiated with 5 Gy of X-rays, whereas erythema was observed 14 days after irradiation in the mice irradiated with 20 and 30 Gy of X-rays.
In mice that received 20 Gy of X-rays, symptoms began to subside after 20 days from irradiation, whereas in mice that received 30 Gy of X-rays, severe erythema or slight edema and crusts were observed 24 days after irradiation, and symptoms persisted up to 80 days after irradiation.
From the above, it was revealed that transient dermatitis occurred when exposed to 20 Gy of X-rays, and persistent dermatitis occurred when exposed to 30 Gy of X-rays.

[Example 2] Measurement of glutathione concentration after radiation exposure 1. X-ray irradiation of mice Mice were irradiated with X-rays in the same manner as in Example 1. Mice were irradiated with 20 Gy and 30 Gy of X-rays as treatment groups, and mice that were not irradiated with X-rays were prepared as a control group.

2. Measurement of glutathione concentration Each group of mice was placed in a simple inhalation anesthesia device for small animal experiments and anesthetized with sevoflurane. The gas concentration was 5% for induction and 3% for maintenance. On the 2nd and 6th days after X-ray irradiation, the right cheek of the sleeping mice was punctured with an animal lancet and blood was collected into a 1.5 mL microtube. The required blood volume was 50 μL. 20 μL of 2000 U of heparin sodium was used as an anticoagulant, and the blood was stored on ice after collection. The collected whole blood was centrifuged at 4°C for 1000 μL in a refrigerated centrifuge that had been cooled to 4°C in advance.
The mixture was centrifuged at 100 G for 10 minutes. After the centrifugation was completed and the mixture was cooled, the upper layer of plasma was aspirated with a micropipette and discarded.
200 μL of c Acid Dihydrate (SSA; Tokyo Chemical Industry Co., Ltd.) was added, and 50 μL of the lower layer of red blood cells was added and immediately stirred with a vortex. After confirming that the red blood cells had been hemolyzed, the cells were centrifuged at 4°C and 3000G for 10 minutes in a refrigerated centrifuge. After cooling after centrifugation, 100 μL of the supernatant was aspirated with a micropipette and dispensed into a new 1.5 mL microtube.
100 μL of MilliQ was added thereto and stirred, and the mixture was stored at −80 ° C. as a sample.
Next, GSSG/GSH Quantification kit (Dojindo Laboratories)
The total glutathione and GSSG (oxidized glutathione) in the sample were quantified using the method described above, and the amount of GSH (reduced glutathione) was calculated from the difference. The specific procedure is as follows: The thawed preserved sample was diluted 10-fold with milliQ and plated on a 96-well plate (FALCON) in 4 wells.
One well was for total glutathione, and the other was for GSSG. 1 μL of Masking solution (included in the kit) was added to the well for GSSG only.
After adding Buffer solution (included in the kit) to all wells,
Then, 60 μL of the solution was added to each well and incubated at 37° C. for 1 hour.
Add 60 μL of standard working solution (included in the kit), followed by enzyme/coenzyme working solution (included in the kit).
After leaving the mixture at room temperature for 10 minutes, the plate was read using a microplate reader (V
The absorbance of each was measured using a 412 nm filter with an Arioskan LUX (Thermo Fisher). After the measurement, the absorbance was converted to a concentration using a standard curve, and the amount of GSH was calculated using the following formula.
GSH amount = total glutathione amount - (GSSG amount x 2)

In addition, to compare the usefulness as an index, blood antioxidant capacity was measured according to the following method.
(Step 1 i-STrap (reaction))
20 μL of Solution A (tert-butyl hydroperoxide (tBuOOH); kit included) that had been brought to room temperature was dispensed into each reaction tube (kit included), 100 μL of physiological saline was added, and the mixture was stirred with a vortex. Then, 100 μL of the sample (whole blood) was added and further stirred with a vortex. 20 μL of Solution B (2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide (DPhPMPO); kit included) that had been brought to room temperature was added to each tube at 10 second intervals, and the mixture was stirred with a vortex. After adding to all the tubes, the mixture was further stirred with a vortex. After adding Solution B to the first tube, the tube was left to stand at room temperature for 30 minutes.

(Step 2 i-Strap (extraction))
After standing for 30 minutes, chloroform/methanol (2:1) was added to the reaction tube.
) solution was added to each tube at 10 second intervals (since it is a volatile solvent, the air in the micropipette was sufficiently replaced before adding it by reverse pipetting).
After adding the chloroform/methanol solution to the first tube, the tube was shaken with a vortex mixer for 10 minutes. After shaking for 10 minutes, the tube was placed in a refrigerated centrifuge (KUBOTA) that had been cooled to 4°C.
The reaction tube was placed in a dehydration tube (included in the kit, containing 0.3 g of sodium sulfate as a desiccant) and pre-cooled for 5 minutes so that the temperature inside the tube was 4°C. The tube was then centrifuged at 4°C and 3000 G for 10 minutes. After centrifugation and cooling, the upper layer (aqueous layer) was aspirated with a micropipette and discarded. The lower layer (chloroform/methanol layer) was then taken with a micropipette and placed in a dehydration tube (included in the kit, containing 0.3 g of sodium sulfate as a desiccant), gently shaken, and then cooled in ice for 1 hour.
After cooling for 5 minutes, the mixture was stored at -80 °C.

(Step 3 i-STrap (ESR measurement))
160 μL of each sample, which had been thawed and returned to room temperature, was placed in a quartz flat cell (RST-LC09F; Flashpoint) and measured by X-band ESR spectroscopy (JES-TE200; JEOL). The ESR conditions were as follows: microwave frequency: 9.423719000 GHz, microwave power: 2.00000 mW, field center: 332.000 mT, sweep width: 0.3000 mT, sweep time: 4.0 minutes, time constant: 0.3 seconds. The signal of the DPhPMPO spin adduct intensity was corrected by the intensity of the manganese marker (Mn2+) second from the left.

3. Results The ratios of the total glutathione concentration, GSSG concentration, GSH concentration, and GSSG concentration/GSH concentration in each treatment group on days 2 and 6 after X-ray irradiation to the values in the control group were calculated. The results are shown in FIG.
In addition, ROC analysis was performed on the total glutathione concentration, GSSG concentration, GSH concentration, GSSG concentration/GSH concentration, and blood antioxidant capacity 2 and 6 days after X-ray irradiation in each treatment group. The results are shown in Table 1.

From Table 1, it was shown that the AUC value of the method using GSSG concentration/GSH concentration was the highest and the prediction ability was high. In addition, in particular in the comparison between 20 Gy and 30 Gy, the AUC value when using glutathione concentration was higher than the AUC value when using blood antioxidant capacity, indicating that the method using glutathione concentration is superior to the method using blood antioxidant capacity in predicting the severity of dermatitis.

Claims (5)

A method for collecting data for predicting the occurrence or non-occurrence of dermatitis after radiation exposure due to radiation therapy or radiotherapy, the method comprising: measuring a glutathione concentration in red blood cells obtained from a subject after radiation exposure; a reduced glutathione concentration/oxidized glutathione concentration (ratio of reduced glutathione concentration to oxidized glutathione concentration) in the red blood cells being equal to or lower than a first reference value indicates a high possibility of the occurrence of dermatitis;
Here, the first standard value is a value that is determined based on the reduced glutathione concentration/oxidized glutathione concentration (the ratio of reduced glutathione concentration to oxidized glutathione concentration) in red blood cells in a population that develops dermatitis after radiation exposure and a population that does not, and that is capable of distinguishing between a population that develops dermatitis after radiation exposure and a population that does not . a reduced glutathione concentration/oxidized glutathione concentration (ratio of reduced glutathione concentration to oxidized glutathione concentration) being equal to or lower than the first reference value and higher than the second reference value indicates a high possibility of occurrence of transient dermatitis, and a ratio being equal to or lower than the second reference value indicates a high possibility of occurrence of persistent dermatitis,
The method according to claim 1, wherein the second standard value is a value capable of distinguishing between a population that will develop persistent dermatitis after radiation exposure and a population that will not develop persistent dermatitis after radiation exposure, the value being determined based on the reduced glutathione concentration/oxidized glutathione concentration (the ratio of reduced glutathione concentration to oxidized glutathione concentration) in red blood cells in a population that will develop persistent dermatitis after radiation exposure and a population that will not develop persistent dermatitis after radiation exposure. The method according to claim 1 or 2, wherein the radiation exposure is due to irradiation with X-rays, electron beams, gamma rays, proton beams, heavy particle beams, or neutron beams. The method according to any one of claims 1 to 3, wherein the radiation exposure is due to radiation therapy for breast cancer, skin cancer, head and neck cancer, rectal cancer, esophageal cancer, brain tumor, metastatic brain tumor, liver cancer, lung cancer, or malignant lymphoma. 5. A kit for use in the method of any one of claims 1 to 4 , comprising means for measuring the concentrations of reduced glutathione and oxidized glutathione in red blood cells obtained from a subject.

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