JP7633481B2 - Metabolic diagnostic biomarkers for drug-induced interstitial pneumonia - Google Patents

Description

The present invention relates to a method for assisting in diagnosing the onset and progression of drug-induced interstitial pneumonia.

Drug-induced interstitial pneumonia is a general term for inflammatory adverse reactions of the pulmonary interstitial space caused by medicines. Interstitial pneumonia can be divided into those caused by clear causes such as medicines, pneumoconiosis, and collagen disease, and idiopathic types with unknown causes, and are further classified into various disease types. Differentiation between these types is clinically important, as the treatment methods and prognosis differ for each type.

Drug-induced interstitial pneumonia is diagnosed based on its similarity to known interstitial pneumonias such as idiopathic (Non-Patent Document 1). Among the types of drug-induced interstitial pneumonia, the most severe type is diffuse alveolar damage (DAD). DAD is a typical type seen in acute respiratory distress syndrome and acute exacerbation of idiopathic pulmonary fibrosis (Non-Patent Document 2), and even in drug-induced cases, DAD has poor response to treatment and a poor prognosis (Non-Patent Documents 1 and 2). Therefore, early detection and diagnosis of DAD are particularly important. In addition, other types of DAD include organizing pneumonia (OP) and nonspecific interstitial pneumonia (NSIP), but there are cases that are difficult to classify using imaging tests or pathological tests, such as cases that do not fit any pattern or cases where multiple patterns are mixed, and image pattern classification has limitations (Non-Patent Document 3).

Diagnosing drug-induced interstitial pneumonia requires ruling out factors other than those caused by the drug, and it often takes time to confirm the diagnosis. Since imaging and pathological diagnosis alone impose a heavy financial and physical burden on patients, the use of biomarkers is considered useful.

Currently, sialylated glycan antigen KL-6 (KL-6), pulmonary surfactant protein A (SP-A), and pulmonary surfactant protein D (SP-D) are used as biomarkers for drug-induced interstitial pneumonia to aid in diagnosis, and it is known that KL-6 has a high positive rate for DAD (Non-Patent Documents 4, 5). These are indicators that reflect drug-induced interstitial pneumonia, but they can also increase in diseases other than smoking and interstitial pneumonia, and do not correlate with the severity of lung damage. In addition, although it is recommended to follow the progress of KL-6 based on the value before drug administration, it also increases in the exacerbation of existing interstitial pneumonia and opportunistic infections. In other words, these markers are not specific to drug-induced interstitial pneumonia, especially DAD-type drug-induced interstitial pneumonia, and are of low usefulness as markers for distinguishing disease types. In addition, KL-6, SP-D, and SP-A have the problem of remaining at high levels for a long period of time even after the condition has improved (Non-Patent Documents 4, 5), and therefore new biomarkers are needed. To date, proteins such as UBE2T, HK1, PMSE1, USO1, IFI16, and GLTP (Patent Document 1), as well as autoantibodies (Patent Document 2), have been proposed as marker candidates and patent applications have been filed.

Diagnosis and treatment of drug-induced lung injury Japanese Respiratory Society Acute interstitial pneumonia (AIP) Journal of the Japanese Respiratory Society 42(1), -23-27, 2004 Masahiko Kusumoto CT diagnosis of drug-induced lung injury Lung Cancer 55,807-809.2015 Ishikawa N, et al., Utility of KL-6/MUC1 in the clinical management of interstitial lung diseases, Respiratory Investig. 50, 3-13, 2012 Kawase S, et al., Change in serum KL-6 level from baseline is useful for predicting life-threatening EGFR-TKIs induced interstitial lung disease, Respiratory Research, 12:97, 2011

JP2015-90323A International Publication WO2014/148429 Brochure

The present invention aims to develop biomarkers for diagnosing and predicting the disease progression and type of drug-induced interstitial pneumonia.

We used metabolomics, a method for comprehensively analyzing metabolite molecules, including more than 1,000 types of amino acids, amino acid derivatives, and lipids, using high-performance liquid chromatography and mass spectrometry, to analyze blood samples from patients with drug-induced interstitial pneumonia. Focusing on DAD and OP, we searched for metabolite molecules that significantly changed in the acute phase of drug-induced interstitial pneumonia compared to the convalescent phase group, using statistical significance and effect size (Hedge's g value) as indicators, based on the ion intensity of each metabolite molecule. While there were many samples in which the KL-6 and SP-D values did not change, we found lysophosphatidylcholine (LPC) molecules from plasma as a lipid molecule group that decreased particularly in the acute phase of DAD-type drug-induced interstitial pneumonia, and kynurenine molecules (kynurenine and quinolinic acid, which are involved in the kynurenine pathway) and their parent compound tryptophan as metabolite molecules that increased particularly in the acute phase of DAD-type drug-induced interstitial pneumonia.

Receiver operating characteristic curve (ROC) analysis showed that the LPC molecules distinguished the acute phase of DAD-type drug-induced interstitial pneumonia from the recovery phase of all drug-induced interstitial pneumonia patients with high accuracy (area under the curve: AUC 0.903-0.938), and from non-disease cases taking similar drugs with high accuracy (area under the curve: AUC 0.929-0.971). They also distinguished the acute phase of DAD-type drug-induced interstitial pneumonia from other lung diseases (lung cancer cases, pulmonary nontuberculous mycobacterial disease cases, idiopathic interstitial pneumonia cases, collagen disease cases, COPD (chronic obstructive pulmonary disease) cases, and bronchial asthma cases) with high accuracy (area under the curve: AUC 0.892-0.978).
On the other hand, the kynurenine molecule group distinguished the acute phase of DAD-type drug-induced interstitial pneumonia from the recovery phase of all drug-induced interstitial pneumonia patients with high accuracy (area under the curve: AUC 0.915-0.947). In addition, the acute phase of DAD-type drug-induced interstitial pneumonia was also distinguished with high accuracy (area under the curve: AUC 0.882-0.957) from other lung diseases (lung cancer cases, pulmonary nontuberculous mycobacterial disease cases, idiopathic interstitial pneumonia cases, collagen disease lung cases, and COPD cases). It was shown that the LPC molecule group and the kynurenine molecule group could be useful biomarkers for drug-induced interstitial pneumonia, especially DAD-type drug-induced interstitial pneumonia.

The present invention has been completed based on these findings, and the gist of the present invention is as follows.
(1) A method for testing for drug-induced interstitial pneumonia, comprising measuring the levels in a sample from a subject of at least one molecule selected from a group of molecules included in the kynurenine pathway and/or at least one molecule selected from a group of lysophosphatidylcholine (LPC) molecules.
(2) The method described in (1), wherein the measured value assists in diagnosing the progression of drug-induced interstitial pneumonia.
(3) The method described in (1), wherein the measured value assists in the specific diagnosis of drug-induced interstitial pneumonia.
(4) The method according to any one of (1) to (3), wherein the drug-induced interstitial pneumonia is DAD-type drug-induced interstitial pneumonia.
(5) The method according to any one of (1) to (4), wherein the group of molecules involved in the kynurenine pathway includes kynurenine, quinolinic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, 2-amino-3-carboxymuconic acid semialdehyde, picolinic acid, and tryptophan.
(6) The method according to any one of (1) to (5), wherein the LPC molecule group comprises LPC(14:0), LPC(15:0), LPC(16:0), LPC(16:1), LPC(17:0), LPC(18:0), LPC(18:1), LPC(18:2), LPC(19:0), LPC(20:0), LPC(20:3), LPC(20:4), LPC(20:5) and LPC(22:6).
(7) A method for diagnosing drug-induced interstitial pneumonia, comprising:
a1. Obtaining a sample from a subject;
b1. Measuring the level of at least one molecule selected from the group of molecules involved in the kynurenine pathway and/or at least one molecule selected from the group of lysophosphatidylcholine (LPC) molecules in a sample from a subject; and
c1. The method includes determining whether or not drug-induced interstitial pneumonia has developed based on the measured value of b1 and/or the ratio calculated from the measured values of b1 of the two molecules.
(8) A method for diagnosing drug-induced interstitial pneumonia, comprising:
a1. Obtaining a sample from a subject;
b1. Measuring the level of at least one molecule selected from the group of molecules involved in the kynurenine pathway and/or at least one molecule selected from the group of lysophosphatidylcholine (LPC) molecules in a sample from a subject; and
c1. The method according to claim 1, further comprising determining the progression of drug-induced interstitial pneumonia based on the measured value of b1 and/or a ratio calculated from the measured values of b1 of the two molecules.
(9) A method for diagnosing and treating drug-induced interstitial pneumonia, comprising:
a1. Obtaining a sample from a subject;
b1. Measuring the level of at least one molecule selected from the group of molecules involved in the kynurenine pathway and/or at least one molecule selected from the group of lysophosphatidylcholine (LPC) molecules in a sample from a subject;
c1. Determining the presence or absence of drug-induced interstitial pneumonia based on the measured value of b1 and/or the ratio calculated from the measured values of b1 of the two molecules; and
d1. The method comprising administering treatment for drug-induced interstitial pneumonia to a subject determined to be highly likely to have developed drug-induced interstitial pneumonia.
(10) A method for diagnosing and treating drug-induced interstitial pneumonia, comprising:
a1. Obtaining a sample from a subject;
b1. Measuring the level of at least one molecule selected from the group of molecules involved in the kynurenine pathway and/or at least one molecule selected from the group of lysophosphatidylcholine (LPC) molecules in a sample from a subject;
c1. Determining the severity of drug-induced interstitial pneumonia based on the measured value of b1 and/or the ratio calculated from the measured values of b1 of the two molecules; and
d1. The method comprising administering treatment for drug-induced interstitial pneumonia to a subject determined to be likely to be in the acute phase of drug-induced interstitial pneumonia.
(11) A kit for testing for drug-induced interstitial pneumonia, comprising a reagent capable of measuring the level in a sample from a subject of at least one molecule selected from a group of molecules involved in the kynurenine pathway and/or at least one molecule selected from a group of lysophosphatidylcholine (LPC) molecules.

By measuring the blood or urinary concentrations of the metabolites discovered in this study, either alone or in combination, it is possible to diagnose with a high degree of accuracy whether a patient suspected of having drug-induced interstitial pneumonia has drug-induced interstitial pneumonia, particularly DAD, as well as the progression of the patient's disease and the activity of their drug-induced interstitial pneumonia.
This specification includes the contents described in the specification and/or drawings of Japanese patent application No. 2020-21306, which is the priority basis of this application.

Plasma concentrations of LPC molecules in drug-induced interstitial pneumonia (acute and recovery phase), cases without interstitial pneumonia, and cases of other lung diseases. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 for A-DAD, †, p < 0.05; ††, p < 0.01; †††, p < 0.001; ††††, p < 0.0001 for A. One-way ANOVA and post-hoc. A, All acute-phase drug-induced interstitial pneumonia cases, A-DAD, DAD-type acute-phase drug-induced interstitial pneumonia cases, B, All convalescent-phase drug-induced interstitial pneumonia cases, C, Non-disease cases of drug-induced interstitial pneumonia caused by the same drug, D, Lung cancer cases, E, Fungal pneumonia cases, F, Bacterial infection cases, G, Nontuberculous mycobacterial pulmonary disease cases, H, Idiopathic interstitial pneumonia cases, I, Connective tissue disease lung cases, J, COPD case, K, bronchial asthma case. Serum concentrations of kyrunine, tryptophan, and quinolinic acid, and blood kyrunine/tryptophan ratio in patients with drug-induced interstitial pneumonia (acute and recovery phase), patients without interstitial pneumonia, and patients with other lung diseases. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 for A-DAD, †, p < 0.05; ††, p < 0.01; †††, p < 0.001; ††††, p < 0.0001 for A. One-way ANOVA and post-hoc. A, All acute-phase drug-induced interstitial pneumonia cases, A-DAD, DAD-type acute-phase drug-induced interstitial pneumonia cases, B, All convalescent-phase drug-induced interstitial pneumonia cases, C, Non-disease cases of drug-induced interstitial pneumonia due to use of the same drug, D, Lung cancer cases, F, Bacterial infection cases, G, Nontuberculous mycobacterial pulmonary disease cases, H, Idiopathic interstitial pneumonia cases, I, Connective tissue disease cases, J, COPD cases, K, Case of bronchial asthma. Urinary concentration of quinolinic acid in drug-induced interstitial pneumonia (acute and recovery phase). **, p < 0.01 for A-DAD, ††, p < 0.01 for A. One-way ANOVA and post-hoc. A, All cases of acute drug-induced interstitial pneumonia, A-DAD, DAD-type acute drug-induced interstitial pneumonia, B, All cases of recovery phase drug-induced interstitial pneumonia.

Hereinafter, an embodiment of the present invention will be described in detail.
The present invention provides a method for testing for drug-induced interstitial pneumonia, comprising measuring the levels of at least one molecule selected from a lysophosphatidylcholine (LPC) molecule group and/or at least one molecule selected from a kynurenine pathway molecule group in a sample from a subject. The drug-induced interstitial pneumonia may be DAD-type drug-induced interstitial pneumonia.

Drug-induced interstitial pneumonia is defined as a drug-related respiratory disorder that occurs during drug administration and has inflammatory lesions in the interstitial area. The mechanism of its onset is largely unknown except for a few drugs, but it is generally believed to be caused by direct toxicity or immune reaction. There is no specific clinical type of drug-induced interstitial pneumonia, and it is characterized by clinical findings, imaging findings, and histopathological patterns, and is basically classified based on the similarity to interstitial pneumonia caused by other causes. In general, acute onset mainly shows the clinical features of diffuse alveolar damage (DAD) type and hypersensitivity pneumonia (HP) type, while subacute to chronic onset often shows the clinical features of nonspecific interstitial pneumonia (NSIP) type and organizing pneumonia (OP) type. Of these, DAD type is poorly responsive to treatment and has a poor prognosis, and even if it recovers, fibrosis remains, so its diagnosis has great clinical significance.

Lysophosphatidylcholine (LPC) molecules are phospholipids with one fatty acid chain, and are the most abundant and widely distributed in the body. They are produced by hydrolysis of phosphatidylcholine (PC), a phospholipid with two fatty acid chains, by phospholipase A. LPC itself is a component of cell membranes and extracellular vesicles, and acts as a ligand for G protein-coupled receptors. In addition, active phospholipase A, which is responsible for the production of LPC, and phospholipases C and D, which are responsible for the degradation of LPC, are activated by stimuli such as inflammation and immune responses, and their blood concentrations fluctuate in various diseases such as arteriosclerosis, diabetes, and cancer.

The LPC molecule group may include LPC(14:0), LPC(15:0), LPC(16:0), LPC(16:1), LPC(17:0), LPC(18:0), LPC(18:1), LPC(18:2), LPC(19:0), LPC(20:0), LPC(20:3), LPC(20:4), LPC(20:5) and LPC(22:6). The numbers in parentheses following LPC indicate the ratio of the number of carbons in the acyl group to the number of double bonds.

The kynurenine pathway is the main metabolic pathway for tryptophan, in which kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, 2-amino-3-carboxymuconic acid semialdehyde, quinolinic acid, and picolinic acid are synthesized in that order from the substrate tryptophan.

The enzymes involved in the kynurenine pathway are distributed in a wide range of tissues, and some are produced in response to stimulation of the immune system or nervous system. It has also been reported that molecules in the produced kynurenine pathway act on the immune system and nervous system, and are associated with imbalances in the activity of these enzymes and intermediate metabolites and products.

Therefore, the molecules involved in the kynurenine pathway may include tryptophan, kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, 2-amino-3-carboxymuconic acid semialdehyde, quinolinic acid and picolinic acid.

In the present invention, the subject is a mammal suspected of developing drug-induced interstitial pneumonia, but may be any mammal that is considered to be at risk of developing drug-induced interstitial pneumonia. Typically, the subject is a human. Examples of samples derived from the subject include cells, tissues, and body fluids obtained from the subject, specifically, blood (e.g., whole blood, serum, plasma, plasma exchange fluid, etc.), urine, and bronchoalveolar lavage fluid of the subject. Whole blood, serum, or plasma obtained in a normal blood test (clinical test) may be used as the blood sample. When measuring molecules of the LPC molecule group, the sample derived from the subject may be plasma, and when measuring molecules of the molecule group involved in the kynurenine pathway, the sample derived from the subject may be serum or urine.

In the method of the present invention, the levels of at least one molecule selected from the lysophosphatidylcholine (LPC) molecule group and/or at least one molecule selected from the molecule group included in the kynurenine pathway are measured in a sample derived from a subject. Furthermore, the ratio of the measured values of the two molecules may be calculated. For example, for the molecule group included in the kynurenine pathway excluding tryptophan, the ratio to its parent compound tryptophan is calculated from the measured level. The measurement means is not particularly limited and any known method can be used, such as liquid chromatography, mass spectrometry, ligand binding, nuclear magnetic resonance spectroscopy, thin layer chromatography, colorimetry, and combinations thereof.

In the examples described below, the inventors used an analytical method that they developed independently using liquid chromatography and a mass spectrometer. To briefly explain the method, the target biomarkers were extracted by mixing an internal standard substance not present in living bodies and an extract with a blood or urine sample, and then separated using a two-liquid gradient and a reversed-phase column, or a potassium hydroxide gradient and an anion exchange column. The biomarker molecules were ionized by the HESI (heated electrospray ionization) method, and selected reaction monitoring (SRM) was performed using precursor ions and product ions for each biomarker ion in positive ion mode or negative ion mode, or full scan measurement was performed using accurate mass.

The level of a molecule to be measured in a sample derived from a subject may be one type or multiple types. By referring to the measurement data of multiple molecules, a more accurate evaluation may be possible. In addition, for the molecular groups involved in the kynurenine pathway, an accurate evaluation may also be possible by referring to the ratio to the parent compound tryptophan obtained from the measurement data.

The level (e.g., concentration) of at least one LPC molecule selected from the group consisting of LPC(14:0), LPC(15:0), LPC(16:0), LPC(16:1), LPC(17:0), LPC(18:0), LPC(18:1), LPC(18:2), LPC(19:0), LPC(20:0), LPC(20:3), LPC(20:4), LPC(20:5) and LPC(22:6) in a sample from a subject is measured, and if the level is low, it is determined that there is a high possibility that drug-induced interstitial pneumonia has developed, and if the level is high, it is determined that there is a low possibility that drug-induced interstitial pneumonia has developed. The same applies to LPC(16:1), LPC(18:2), LPC(20:3), LPC(20:4), LPC(20:5) and LPC(22:6).

The levels (e.g., concentrations) of kynurenine and molecules downstream of its metabolic pathway (e.g., 3-hydroxykynurenine, 3-hydroxyanthranilic acid, 2-amino-3-carboxymuconic acid semialdehyde, quinolinic acid, picolinic acid) in a sample from a subject are measured, and if the levels are high, it can be determined that there is a high possibility that drug-induced interstitial pneumonia has developed, and if the levels are low, it can be determined that there is a low possibility that drug-induced interstitial pneumonia has developed.
The level (e.g., concentration) of tryptophan in a sample from a subject is measured, and if the level is low, it is determined that there is a high possibility that drug-induced interstitial pneumonia has developed, and if the level is high, it is determined that there is a low possibility that drug-induced interstitial pneumonia has developed.
Furthermore, when the ratio of kynurenine and molecules downstream of its metabolic pathway to tryptophan is calculated, a high level can be determined to indicate a high possibility of drug-induced interstitial pneumonia, whereas a low level can be determined to indicate a low possibility of drug-induced interstitial pneumonia.

Therefore, the method of the present invention can assist in the diagnosis of drug-induced interstitial pneumonia (determination of the presence or absence of onset of drug-induced interstitial pneumonia).
a1. Obtaining a sample from a subject;
b1. Measuring the level of at least one molecule selected from the group of lysophosphatidylcholine (LPC) molecules and/or at least one molecule selected from the group of molecules involved in the kynurenine pathway in a sample from a subject; and
c1. The method includes determining whether or not drug-induced interstitial pneumonia has developed based on the measured value of b1 and/or a ratio calculated from the measured values of b1 of the two molecules.

As an example of the present invention, the diagnosis of drug-induced interstitial pneumonia can be performed according to the following criteria. The level (e.g., concentration) of at least one of the above molecules in a sample collected from a subject is measured, and the ratio is calculated. If a value higher (or lower) than a preset cutoff value or reference value is obtained, the subject is evaluated as having drug-induced interstitial pneumonia. The preset cutoff value or reference value can be appropriately set by a person skilled in the art. For example, the 95% confidence interval of the quantitative value of a healthy subject who does not develop drug-induced interstitial pneumonia or a case of non-development of drug-induced interstitial pneumonia who takes a similar drug can be used as the reference value, or the cutoff value can be set from an ROC curve. In one embodiment, for the diagnosis of drug-induced interstitial pneumonia using plasma or serum samples, the following cutoff value can be set based on the concentration at which Youden's Index [sensitivity - (100 - specificity)] is maximized (the value at which sensitivity and specificity are highest) as an index, based on the ROC curve analysis for discriminating between all acute cases of drug-induced interstitial pneumonia (A) in the Examples described below and cases that did not develop drug-induced interstitial pneumonia due to taking the same type of drug (C). LPC(14:0): 332.7-391.2 ng/ml ±15% (preferably 370.9 ng/ml ±15%), LPC(15:0): 196.3-235.6 ng/ml ±15% (preferably 209.8 ng/ml ±15%), LPC(16:0): 36.98-39.77 μg/ml ±15% (preferably 38.88 μg/ml ±15%), LPC(17:0): 403.0-479.5 ±15% (preferably 439.7 ng/ml ±15%), LPC(18:0): 9.21-11.37 μg/ml ±15% (preferably 10.01 μg/ml ±15%), LPC(18:1): 6.04-7.20 μg/ml ±15% (preferably 6.64 μg/ml ±15%), LPC(19:0): 39.13 to 45.89 ng/ml ±15% (preferably 41.5 ng/ml ±15%), LPC(20:0): 36.85 to 42.79 ng/ml ±15% (preferably 41.1 ng/ml ±15%), kynurenine: 445.9 to 500.5 ng/ml ±15% (preferably 466.5 ng/ml ±15%), quinolinic acid: 74.7 to 97.2 ng/ml ±15% (preferably 88.6 ng/ml ±15%), tryptophan: 9.54 to 10.35 μg/ml ±15% (preferably 10.00 μg/ml ±15%), kynurenine/tryptophan ratio: 0.058-0.069 ±15% (preferably 0.064 ±15%). The above ±15% is a variation value taking into consideration the variability of the analysis system, etc., and there may be a variation of ±15% for each of the upper and lower limits of the numerical range of the above cutoff value, as well as for the preferred value. Alternatively, if at least one of the above molecules shows an upward (or downward) trend compared with past measured values, the possibility of onset of drug-induced interstitial pneumonia is suspected. By changing the cutoff value or the reference value to a value for diagnosing DAD-type drug-induced interstitial pneumonia, it is possible to diagnose DAD-type drug-induced interstitial pneumonia in a similar manner. In one embodiment, for the diagnosis of DAD-type drug-induced interstitial pneumonia, the following cutoff value can be set based on the concentration at which Youden's Index [sensitivity - (100 - specificity)] is maximized (the value at which sensitivity and specificity are highest) as an index, based on the ROC curve analysis for discriminating between acute-phase cases of DAD-type drug-induced interstitial pneumonia (A-DAD) in the Examples described below and cases (C) where no drug-induced interstitial pneumonia has developed despite taking the same type of drug: LPC(14:0): 194.1-379.6 ng/ml ±15% (preferably 267.0 ng/ml ±15%), LPC(15:0): 136.4-215.5 ng/ml ±15% (preferably 177.6 ng/ml ±15%), LPC(16:0): 29.49-39.29 μg/ml ±15% (preferably 33.78 μg/ml ±15%), LPC(17:0): 320.7-479.5 ng/ml ±15% (preferably 383.3 ng/ml ±15%), LPC(18:0): 8.34-11.82 μg/ml ±15% (preferably 9.86 μg/ml ±15%), LPC(18:1) : 7.43 to 9.93 μg/ml ±15% (preferably 8.23 μg/ml ±15%), LPC(19:0): 30.92 to 49.9 ng/ml ±15% (preferably 40.98 ng/ml ±15%), LPC(20:0): 22.84 to 33.82 ng/ml ±15% (preferably 26.13 ng/ml ±15%), kynurenine: 456.6 to 649.4 ng/ml ±15% (preferably 537.9 ng/ml ±15%), quinolinic acid: 63.9 to 162.8 ng/ml ±15% (preferably 109.9 ng/ml ±15%), tryptophan: 8.02 to 10.61 μg/ml ±15% (preferably 9.33 μg/ml ±15%), kynurenine/tryptophan ratio: 0.055 to 0.082 ±15% (preferably 0.064 ±15%). The above ±15% is a variation value taking into consideration the variability of the analytical system, etc., and the upper and lower limits of the numerical range of the above cutoff value, as well as the preferred value, may vary by ±15%.
In the Examples described later, all plasma LPC molecules were significantly decreased in acute DAD-type drug-induced interstitial pneumonia cases (A-DAD) compared with cases not developing drug-induced interstitial pneumonia taking the same drug (C) (Figure 1, A-DAD vs C). Also, significant decreases were observed in all acute drug-induced interstitial pneumonia cases (A) compared with cases not developing drug-induced interstitial pneumonia taking the same drug (C) (Figure 1, A vs C).
Serum kynurenine pathway molecules (kynurenine and quinolinic acid) and tryptophan molecules were significantly increased and decreased in acute-phase DAD-type drug-induced interstitial pneumonia cases (A-DAD) compared with cases not developing drug-induced interstitial pneumonia taking the same drugs (C) (Fig. 2, A-DAD vs C). Also, in all acute-phase drug-induced interstitial pneumonia cases (A), serum kynurenine pathway molecules (kynurenine and quinolinic acid) and tryptophan molecules were significantly increased and decreased, respectively, compared with cases not developing drug-induced interstitial pneumonia taking the same drugs (C) (Fig. 2, A vs C).

The method of the present invention can also be used for the specific diagnosis of drug-induced interstitial pneumonia. In this specification, "specific diagnosis" refers to determining whether or not a subject has drug-induced interstitial pneumonia. The method of the present invention can also be used for the specific diagnosis of DAD-type drug-induced interstitial pneumonia, and in this case, "specific diagnosis" refers to determining whether or not a subject has DAD-pattern interstitial pneumonia. Since DAD is a particularly severe disease type among interstitial pneumonia disease types, the use of the method for the specific diagnosis of DAD is of great clinical significance. The level (e.g., concentration) of at least one LPC molecule selected from the group consisting of LPC(14:0), LPC(15:0), LPC(16:0), LPC(16:1), LPC(17:0), LPC(18:0), LPC(18:1), LPC(18:2), LPC(19:0), LPC(20:0), LPC(20:3), LPC(20:4), LPC(20:5) and LPC(22:6) in a sample from a subject is measured, and if the level is lower than a preset cutoff value or reference value, it is determined that the disease is highly likely to be drug-induced interstitial pneumonia, and if the level is higher than the cutoff value or reference value, it is determined that the disease is low likely to be drug-induced interstitial pneumonia. The preset cutoff value and reference value can be appropriately set by a person skilled in the art. For example, the 95% confidence interval of the quantitative value of cases of lung diseases other than drug-induced interstitial pneumonia can be used as the reference value, or the cutoff value can be set from the ROC curve. By changing the cutoff value or reference value to a value for the specific diagnosis of DAD-type drug-induced interstitial pneumonia, it is possible to perform a specific diagnosis of DAD-type drug-induced interstitial pneumonia in the same way.
In the Examples described later, all plasma LPC molecules were significantly decreased in cases of acute DAD-type drug-induced interstitial pneumonia (A-DAD) compared with cases of lung cancer (D), pulmonary nontuberculous mycobacterial disease (G), idiopathic interstitial pneumonia (H), collagen vascular lung disease (I), COPD (J), and bronchial asthma (K) (Figure 1, A-DAD vs DK). In all acute drug-induced interstitial pneumonia cases (A), significant decreases were observed in all LPC molecule groups in lung cancer cases (D), LPC(19:0) and LPC(20:0) in pulmonary nontuberculous mycobacterial disease cases (G), LPC(14:0), LPC(15:0), LPC(16:0), LPC(17:0), LPC(18:0), LPC(18:1), and LPC(20:0) in idiopathic interstitial pneumonia cases (H), all LPC molecule groups in connective tissue disease lung cases (I), LPC(14:0), LPC(16:0), LPC(18:0), LPC(18:1), and LPC(20:0) in COPD cases (J), and LPC(14:0) in asthma cases (K) (Fig. 1, A vs DK).

The levels (e.g., concentrations) of kynurenine and molecules downstream of its metabolic pathway (e.g., 3-hydroxykynurenine, 3-hydroxyanthranilic acid, 2-amino-3-carboxymuconic acid semialdehyde, quinolinic acid, picolinic acid) in a sample from a subject are measured, and the ratio to tryptophan is calculated. If the level is higher than a preset cutoff value or reference value, it is determined that the disease is highly likely to be drug-induced interstitial pneumonia, and if the level is lower than the cutoff value or reference value, it is determined that the disease is low likely to be drug-induced interstitial pneumonia. The preset cutoff value or reference value can be appropriately set by a person skilled in the art. For example, the 95% confidence interval of the quantitative value of a case of developing a lung disease other than drug-induced interstitial pneumonia can be used as the reference value, or the cutoff value can be set from an ROC curve. By changing the cutoff value or reference value to a value for specific diagnosis of DAD-type drug-induced interstitial pneumonia, a specific diagnosis of DAD-type drug-induced interstitial pneumonia can be similarly performed.
In the Examples described later, serum kynurenine and quinolinic acid molecules were significantly increased in acute DAD-type drug-induced interstitial pneumonia cases (A-DAD) compared with lung cancer cases (D), bacterial infection cases (F), pulmonary nontuberculous mycobacterial disease cases (G), idiopathic interstitial pneumonia cases (H), collagen disease lung cases (I), COPD cases (J), and bronchial asthma cases (K) (Figure 2, A-DAD vs DK). Also, a significant increase was observed in all acute drug-induced interstitial pneumonia cases (A) in relation to lung disease, similar to the DAD type (Figure 2, A vs DK).

The level (e.g., concentration) of tryptophan in a sample from a subject is measured, and if the level is lower than a preset cutoff value or reference value, it is determined that the disease is highly likely to be drug-induced interstitial pneumonia, and if the level is higher than the cutoff value or reference value, it is determined that the disease is low likely to be drug-induced interstitial pneumonia. The preset cutoff value or reference value can be appropriately set by a person skilled in the art. For example, the 95% confidence interval of the quantitative value of a case of a lung disease other than drug-induced interstitial pneumonia can be set as the reference value, or the cutoff value can be set from an ROC curve. By changing the cutoff value or reference value to a value for specific diagnosis of DAD-type drug-induced interstitial pneumonia, specific diagnosis of DAD-type drug-induced interstitial pneumonia can be performed in the same manner.
In the Examples described later, a significant decrease in serum tryptophan molecules was observed in all cases except for bacterial infection (F) (Figure 2, A-DAD vs DK). In addition, a significant decrease was observed in all acute drug-induced interstitial pneumonia cases (A) in lung disease, similar to the DAD type (Figure 2, A vs DK).
In one embodiment, for specific diagnosis of drug-induced interstitial pneumonia using plasma or serum samples, the following cutoff values can be set based on the concentration at which Youden's Index [sensitivity-(100-specificity)] is maximized (the value at which sensitivity and specificity are highest) of the metabolite cutoff value from ROC curve analysis for discrimination of all acute drug-induced interstitial pneumonia cases (A) and lung cancer cases (D), fungal pneumonia cases (E), bacterial infection cases (F), pulmonary nontuberculous mycobacterial disease cases (G), idiopathic interstitial pneumonia cases (H), collagen disease lung cases (I), COPD cases (J), or bronchial asthma cases (K) in the examples described below. Upper row: appropriate value, lower row: preferred value. The ±15% in the table below is a variation value taking into account the variation of the analysis system, etc., and there may be a ±15% variation in each of the upper and lower limits of the numerical range of the appropriate value and the preferred value.

In another embodiment, for specific diagnosis of DAD-type drug-induced interstitial pneumonia using plasma or serum samples, the following cutoff values can be set based on the concentration at which Youden's Index [sensitivity - (100 - specificity)] is maximized (the value at which sensitivity and specificity are highest) for the metabolite cutoff value from ROC curve analysis for discrimination between all acute-phase DAD-type drug-induced interstitial pneumonia cases (A-DAD) and lung cancer cases (D), fungal pneumonia cases (E), bacterial infection cases (F), pulmonary nontuberculous mycobacterial disease cases (G), idiopathic interstitial pneumonia cases (H), collagen disease lung cases (I), COPD cases (J), or bronchial asthma cases (K) in the Examples described below. Upper row: appropriate value, lower row: preferred value. The ±15% in the table below is a variation value that takes into account variability in the analytical system, etc., and there may be a ±15% variation in the upper and lower limits of the numerical range of the appropriate value, as well as in the preferred value.

Therefore, the values measured in the method of the present invention can assist in the specific diagnosis of drug-induced interstitial pneumonia.

It is preferable that subjects undergoing a specific diagnosis of drug-induced interstitial pneumonia are in the acute phase of the disease. In this specification, the "acute phase of the disease" refers to the period near the worst stage of the disease. The acute phase is diagnosed by a respiratory specialist based on a combination of clinical symptoms, physical examination findings, blood test findings, chest image findings, respiratory function tests, pathology patterns, etc.

The method of the present invention can also be used to diagnose the progression of drug-induced interstitial pneumonia. In this specification, "disease progression diagnosis" refers to the assessment of the degree or change in the pathology (severity, treatment effect, etc.) of a subject diagnosed with drug-induced interstitial pneumonia. The method of the present invention can also be used to diagnose the progression of DAD-type drug-induced interstitial pneumonia. The level (e.g., concentration) of at least one LPC molecule selected from the group consisting of LPC(14:0), LPC(15:0), LPC(16:0), LPC(16:1), LPC(17:0), LPC(18:0), LPC(18:1), LPC(18:2), LPC(19:0), LPC(20:0), LPC(20:3), LPC(20:4), LPC(20:5) and LPC(22:6) in a sample from a subject is measured, and if the level is lower than a preset cutoff value or reference value, it is determined that there is a high possibility that the subject is in the acute phase of drug-induced interstitial pneumonia, and if the level is higher than the cutoff value or reference value, it is determined that there is a low possibility that the subject is in the acute phase of drug-induced interstitial pneumonia. These preset cutoff values and reference values can be appropriately set by those skilled in the art. For example, the 95% confidence interval of the quantitative value of the recovery-stage case of drug-induced interstitial pneumonia can be used as the standard value, and the cutoff value can be set from the ROC curve. By changing the cutoff value or standard value to a value for diagnosing the disease progression of DAD-type drug-induced interstitial pneumonia, the disease progression of DAD-type drug-induced interstitial pneumonia can be similarly diagnosed.
In one embodiment, for diagnosis of the progression of drug-induced interstitial pneumonia using plasma samples, the following cutoff value can be set based on the concentration at which Youden's Index [sensitivity - (100 - specificity)] is maximized (the value at which sensitivity and specificity are highest) as the cutoff value for the metabolite from the ROC curve analysis for discriminating between all acute-phase cases of drug-induced interstitial pneumonia (A) and all convalescent-phase cases of drug-induced interstitial pneumonia (B) in the Examples described below. LPC(14:0): 284.1-325.7 ng/ml ±15% (preferably 291.0 ng/ml ±15%), LPC(15:0): 187.3-208.8 ng/ml ±15% (preferably 200.5 ng/ml ±15%), LPC(16:0): 39.39-42.34 μg/ml ±15% (preferably 40.89 μg/ml ±15%), LPC(17:0): 410.0-481.6 ng/ml ±15% (preferably 439.8 ng/ml ±15%), LPC(18:0): 8.97-9.72 μg/ml ±15% (preferably 9.26 μg/ml ±15%), LPC(18:1): 8.04-8.64 μg/ml ±15% (preferably 8.28 μg/ml ±15%), LPC(19:0): 48.34 to 51.83 ng/ml ±15% (preferably 49.46 ng/ml ±15%), LPC(20:0): 30.48 to 33.40 ng/ml ±15% (preferably 32.47 ng/ml ±15%). The above ±15% is a variation value taking into consideration the variability of the analysis system, etc., and there may be a variation of ±15% for each of the upper and lower limits of the numerical range of the above cutoff value, as well as for the preferred value.
By changing the cutoff value or the reference value to a value for diagnosing DAD-type drug-induced interstitial pneumonia, it is possible to diagnose DAD-type drug-induced interstitial pneumonia in a similar manner. In one embodiment, for diagnosing the progression of DAD-type drug-induced interstitial pneumonia using a plasma sample, the following cutoff value can be set using the concentration at which Youden's Index [sensitivity - (100 - specificity)] is maximized (the value at which sensitivity and specificity are highest) as an index, based on the ROC curve analysis for discriminating between acute-phase DAD-type drug-induced interstitial pneumonia cases (A-DAD) and all convalescent-phase drug-induced interstitial pneumonia cases (B) in the Examples described below. LPC(14:0): 226.9-299.1 ng/ml ±15% (preferably 260.2 ng/ml ±15%), LPC(15:0): 157.9-217.5 ng/ml ±15% (preferably 182.6 ng/ml ±15%), LPC(16:0): 29.62-38.29 μg/ml ±15% (preferably 33.53 μg/ml ±15%), LPC(17:0): 315.3-444.5 ng/ml ±15% (preferably 381.5 ng/ml ±15%), LPC(18:0): 9.02-11.12 μg/ml ±15% (preferably 9.67 μg/ml ±15%), LPC(18:1) : 7.39 to 8.70 μg/ml ±15% (preferably 8.23 μg/ml ±15%), LPC(19:0): 34.63 to 48.94 ng/ml ±15% (preferably 40.69 ng/ml ±15%), LPC(20:0): 23.20 to 32.97 ng/ml ±15% (preferably 28.05 ng/ml ±15%). The above ±15% is a variation value taking into consideration the variability of the analysis system, etc., and there may be a variation of ±15% for each of the upper and lower limits of the numerical range of the above cutoff value, as well as for the preferred value.
In the Examples described later, all plasma LPC molecules were significantly decreased in cases of DAD-type acute-phase drug-induced interstitial pneumonia (Fig. 1, A-DAD) compared with all cases of recovery phase drug-induced interstitial pneumonia (B) (Fig. 1, A-DAD vs. B). Also, a significant decrease was observed in all cases of acute-phase drug-induced interstitial pneumonia (A) compared with all cases of recovery phase drug-induced interstitial pneumonia (Fig. 1, A vs. B).

The levels (e.g., concentrations) of kynurenine and molecules downstream of its metabolic pathway (e.g., 3-hydroxykynurenine, 3-hydroxyanthranilic acid, 2-amino-3-carboxymuconic acid semialdehyde, quinolinic acid, picolinic acid) in a sample from a subject are measured, and in some cases, the ratio to tryptophan is further obtained. If the level or ratio is higher than a preset cutoff value or reference value, it is determined that there is a high possibility that the subject is in the acute phase of drug-induced interstitial pneumonia, and if the level is lower than the cutoff value or reference value, it is determined that there is a low possibility that the subject is in the acute phase of drug-induced interstitial pneumonia. The preset cutoff value or reference value can be appropriately set by a person skilled in the art. For example, the 95% confidence interval of the quantitative value of a case of drug-induced interstitial pneumonia in the recovery phase can be used as the reference value, or the cutoff value can be set from an ROC curve. By changing the cutoff value or reference value to a value for diagnosing the disease progression of DAD-type drug-induced interstitial pneumonia, the disease progression of DAD-type drug-induced interstitial pneumonia can be similarly diagnosed.
In one embodiment, for diagnosis of the disease progression of drug-induced interstitial pneumonia using serum samples, the following cutoff values can be set using the concentration at which Youden's Index [sensitivity-(100-specificity)] is maximized (the value at which sensitivity and specificity are highest) as an index based on the ROC curve analysis for discrimination between all acute-phase cases of drug-induced interstitial pneumonia (A) and all convalescent-phase cases of drug-induced interstitial pneumonia (B) in the Examples described below: kynurenine: 508.5-555.8 ng/ml ±15% (preferably 522.3 ng/ml ±15%), quinolinic acid: 84.4-96.1 ng/ml ±15% (preferably 90.7 ng/ml ±15%), kynurenine/tryptophan ratio: 0.053-0.061 ±15% (preferably 0.056 ±15%). The above ±15% is a variation value taking into consideration variability in the analytical system, etc., and the upper and lower limits of the numerical range of the above cutoff value, as well as the preferred value, may vary by ±15%.
By changing the cutoff value or the reference value to a value for diagnosing DAD-type drug-induced interstitial pneumonia, it is possible to diagnose DAD-type drug-induced interstitial pneumonia in a similar manner. In one embodiment, for diagnosing the progression of DAD-type drug-induced interstitial pneumonia using a serum sample, the following cutoff value can be set using the concentration at which Youden's Index [sensitivity - (100 - specificity)] is maximized (the value at which sensitivity and specificity are highest) as an index, based on the ROC curve analysis for discriminating between acute-phase DAD-type drug-induced interstitial pneumonia cases (A-DAD) and all convalescent-phase drug-induced interstitial pneumonia cases (B) in the Examples described below. Kynurenine: 649.5 to 788.3 ng/ml ±15% (preferably 707.1 ng/ml ±15%), quinolinic acid: 93.4 to 155.9 ng/ml ±15% (preferably 125.1 ng/ml ±15%), kynurenine/tryptophan ratio: 0.055 to 0.075 ±15% (preferably 0.064 ±15%). The above ±15% is a variation value taking into consideration the variability of the analysis system, etc., and there may be a variation of ±15% for each of the upper and lower limits of the numerical range of the above cutoff value, as well as for the preferred value.
In the Examples described later, serum kynurenine molecules and quinolinic acid molecules were significantly increased in acute-phase DAD-type drug-induced interstitial pneumonia cases (A-DAD) compared with all convalescent-phase drug-induced interstitial pneumonia cases (B) (Figure 2, A-DAD vs. B and Figure 3, A-DAD vs. B). Also, serum kynurenine molecules and quinolinic acid molecules were significantly increased in all acute-phase drug-induced interstitial pneumonia cases (A) compared with all convalescent-phase drug-induced interstitial pneumonia cases (Figure 2, A vs. B and Figure 3, A-DAD vs. B).

The level (e.g., concentration) of tryptophan in a sample derived from a subject is measured, and if the level is lower than a preset cutoff value or reference value, it is determined that the subject is likely to be in the acute phase of drug-induced interstitial pneumonia, and if the level is higher than the cutoff value or reference value, it is determined that the subject is unlikely to be in the acute phase of drug-induced interstitial pneumonia. The preset cutoff value or reference value can be appropriately set by a person skilled in the art. For example, the 95% confidence interval of the quantitative value of a case of drug-induced interstitial pneumonia in the recovery phase can be set as the reference value, or the cutoff value can be set from an ROC curve. By changing the cutoff value or reference value to a value for diagnosing the disease progression of DAD-type drug-induced interstitial pneumonia, the disease progression of DAD-type drug-induced interstitial pneumonia can be similarly diagnosed.
In one embodiment, for the diagnosis of the disease progression of drug-induced interstitial pneumonia using serum samples, the following cutoff values can be set using the concentration at which Youden's Index [sensitivity-(100-specificity)] is maximized (the value at which sensitivity and specificity are highest) as an index based on the ROC curve analysis for discriminating between all acute-phase cases of drug-induced interstitial pneumonia (A) and all convalescent-phase cases of drug-induced interstitial pneumonia (B) in the Examples described below: Tryptophan: 9.05-9.57 μg/ml ±15% (preferably, 9.32 μg/ml ±15%). The above ±15% is a variation value taking into consideration the variability of the analysis system, etc., and there may be a variation of ±15% for each of the upper and lower limits of the numerical range of the above cutoff value, as well as for the preferred value.
By changing the cutoff value or the reference value to a value for diagnosing DAD type drug-induced interstitial pneumonia, it is possible to diagnose DAD type drug-induced interstitial pneumonia in a similar manner. In one embodiment, for the diagnosis of the progression of DAD type drug-induced interstitial pneumonia using a serum sample, the following cutoff values can be set using the concentration at which Youden's Index [sensitivity - (100 - specificity)] is maximized (the value at which sensitivity and specificity are highest) as an index, based on the ROC curve analysis for discriminating between acute phase cases of DAD type drug-induced interstitial pneumonia (A-DAD) and all convalescent phase cases of drug-induced interstitial pneumonia (B) in the Examples described below. Tryptophan: 7.62 to 8.94 μg/ml ±15% (preferably 8.44 μg/ml ±15%). The above ±15% is a variation value taking into consideration variability in the analytical system, etc., and the upper and lower limits of the numerical range of the above cutoff value, as well as the preferred value, may vary by ±15%.
In the Examples described later, serum tryptophan molecules were significantly decreased in acute-phase DAD-type drug-induced interstitial pneumonia cases (A-DAD) compared with all convalescent-phase drug-induced interstitial pneumonia cases (B) (Figure 2, A-DAD vs B). Also, a significant decrease was observed in all acute-phase drug-induced interstitial pneumonia cases (A) compared with all convalescent-phase drug-induced interstitial pneumonia cases (Figure 2, A vs B).

Therefore, the values measured in the method of the present invention can assist in diagnosing the progression of drug-induced interstitial pneumonia.
a1. Obtaining a sample from a subject;
b1. Measuring the level of at least one molecule selected from the group of lysophosphatidylcholine (LPC) molecules and/or at least one molecule selected from the group of molecules involved in the kynurenine pathway in a sample from a subject; and
c1. The method further comprises determining the progression of drug-induced interstitial pneumonia based on the measured value of b1 and/or a ratio calculated from the measured values of b1 of the two molecules.

Furthermore, the levels of at least one molecule selected from the lysophosphatidylcholine (LPC) molecule group and/or at least one molecule selected from the molecule group included in the kynurenine pathway in a sample from a subject determined to be likely to be in the acute phase of drug-induced interstitial pneumonia are measured once or multiple times at different times, and in some cases, the ratio of the measured values of the two molecules (for example, the ratio of one molecule selected from the molecule group included in the kynurenine pathway excluding tryptophan to tryptophan) is obtained. If the level and/or ratio is close to a cutoff value or a reference value, or has changed to above or below the cutoff value or reference value, it is determined that the subject has recovered from drug-induced interstitial pneumonia due to treatment, and if the level remains higher or lower than the cutoff value or reference value, it is determined that the subject has not recovered from drug-induced interstitial pneumonia due to treatment, or that the recovery is insufficient. The method of the present invention can be used not only to diagnose the disease progression of drug-induced interstitial pneumonia, but also to examine the prognosis and confirm the effectiveness of treatment.

If it is determined that the subject is highly likely to have developed drug-induced interstitial pneumonia, the suspected drug is discontinued immediately. Next, a combination of tests, such as infectious disease tests of sputum and serum, imaging tests, bronchoalveolar lavage tests, and pathological tests, are performed to confirm the diagnosis of drug-induced interstitial pneumonia. In severe cases of drug-induced interstitial pneumonia such as DAD, corticosteroids are started immediately. The Japanese Respiratory Society's treatment guidelines recommend pulse therapy with 500-1000 mg/day of methylprednisolone for three days, followed by a continued dose of 0.5-1.0 mg/kg/day in prednisolone equivalent, followed by gradual tapering. There is no set standard for the rate of tapering, and the dosage should be reduced while monitoring the patient's response to treatment. For lung damage that is resistant or intractable to steroid treatment, multidisciplinary treatment is performed by combining immunosuppressants (cyclosporine, tacrolimus, etc.), neutrophil elastase inhibitors (sivelestat), polymyxin B immobilized fiber column (PMX) therapy, etc. These treatments have little evidence for drug-induced lung damage and are not covered by insurance, so they are actually used in accordance with acute exacerbation of IPF (idiopathic pulmonary fibrosis) and ARDS (acute respiratory distress syndrome). For respiratory failure, measures are taken according to the severity, such as high-flow oxygen administration, noninvasive positive pressure ventilation, or artificial respiration management under endotracheal intubation.

In the United States, the incidence of drug-induced interstitial pneumonia is not as high as in Japan (RESPIRATORY INVESTIGATION, Dec 2013, 51(4):260-77), and idiopathic interstitial pneumonia is the first type of interstitial pneumonia to be mentioned, not drug-induced. Although the American Thoracic Society has published a statement on diagnosis (disease type, etc.) (AM J RESPIR CRIT CARE MED. Sep 2013;188(6):733-48), it does not have treatment guidelines for general disease types, and treatment is basically left to the discretion of each medical facility. For example, at the Mayo Clinic, drug therapy, oxygen therapy, pulmonary rehabilitation, and lung transplants are performed as treatments for general interstitial pneumonia (https://www.mayoclinic.org/diseases-conditions/interstitial-lung-disease/diagnosis-treatment/drc-20353113). Drug treatments include: 1) corticosteroids (prednisolone, etc.) and their combination with immunosuppressants, which are expected to slow or stabilize the progression of interstitial pneumonia; 2) drugs for treating idiopathic pulmonary fibrosis (pirfenidone and nintedanib, which are expected to slow the progression of interstitial pneumonia); and 3) gastric acid secretion inhibitors (lansoprazole, omeprazole, etc., which prevent reflux esophagitis and reduce aspiration of gastric juices and prevent the worsening of lung damage). Oxygen therapy cannot prevent lung damage, but it is expected to make breathing easier, prevent and reduce complications due to low blood oxygen levels, and reduce blood pressure on the right side of the heart. Pulmonary rehabilitation improves quality of life, such as the ease of daily activities. Lung transplantation is the last resort for patients with severe interstitial pneumonia who have no other treatment options.

On the other hand, for IPF, a type of interstitial pneumonia, there are many patients and the prognosis is poor, so the American Thoracic Society has published treatment guidelines based on the results of clinical research (AMERICAN JOURNAL OF RESPIRATORY AND CRINICAL CARE MEDICINE, JULY 2015. 192(2):e3-19). Medications that are conditionally recommended as treatments for IPF include nintedanib (an inhibitor of tyrosine kinases such as vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), and platelet-derived growth factor receptor (PDGFR)), pirfenidone (anti-fibrotic effect), and gastric acid secretion inhibitors (prevention of reflux esophagitis and suppression of worsening lung damage). However, the efficacy of warfarin (an anticoagulant), imatinib (a selective tyrosine kinase inhibitor of platelet-derived growth factor (PDGF)), the combination of prednisone, azathioprine, and N-acetylcysteine, and ambrisentan (a selective endothelin receptor antagonist) has been questioned, and their use is not recommended, but their use is not excluded.
In the present invention, the above-mentioned treatment methods for interstitial pneumonia may be used.

The present invention relates to a method for diagnosing and treating drug-induced interstitial pneumonia,
a1. Obtaining a sample from a subject;
b1. Measuring the level of at least one molecule selected from the group of lysophosphatidylcholine (LPC) molecules and/or at least one molecule selected from the group of molecules involved in the kynurenine pathway in a sample from a subject;
c1. Determining the presence or absence of drug-induced interstitial pneumonia based on the measured value of b1 and/or the ratio calculated from the measured values of b1 of the two molecules; and
d1. The method includes administering treatment for drug-induced interstitial pneumonia to a subject determined to be highly likely to have developed drug-induced interstitial pneumonia.

The present invention also provides a method for diagnosing and treating drug-induced interstitial pneumonia, comprising:
a1. Obtaining a sample from a subject;
b1. Measuring the level of at least one molecule selected from the group of lysophosphatidylcholine (LPC) molecules and/or at least one molecule selected from the group of molecules involved in the kynurenine pathway in a sample from a subject;
c1. Determining the severity of drug-induced interstitial pneumonia based on the measured value of b1 and/or the ratio calculated from the measured values of b1 of the two molecules; and
d1. The method includes administering treatment for drug-induced interstitial pneumonia to a subject determined to be likely to be in the acute phase of drug-induced interstitial pneumonia.

The present invention also provides a kit for testing for drug-induced interstitial pneumonia, comprising a reagent capable of measuring the level in a sample from a subject of at least one molecule selected from the lysophosphatidylcholine (LPC) molecule group and/or at least one molecule selected from the molecule group included in the kynurenine pathway.

As an example, the kit of the present invention may include reagents (e.g., methanol, acetonitrile, isopropanol, chloroform, and mixtures thereof) for extracting at least one molecule selected from the lysophosphatidylcholine (LPC) molecule group and/or at least one molecule selected from the molecule group included in the kynurenine pathway from a sample derived from a subject, internal standards (e.g., LPC(12:0), LPC(13:0), LPC(18:1-d7), kynurenine-d6, quinolinic acid-d3, tryptophan-d5), derivatization reagents (e.g., 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS)), and instructions. The instructions may also include, in addition to instructions on how to use the kit, criteria for evaluation and/or differentiation (discrimination) of acute exacerbation cases of drug-induced interstitial pneumonia and DAD-type drug-induced interstitial pneumonia. In addition, the kit should preferably include a column and a pretreatment filter in the case of LC/MS analysis, and standard substances and antibodies in the case of the ligand binding method.

The kit of the present invention can be used as a pharmaceutical for diagnosing diseases.

The present invention will now be described in more detail with reference to examples.
Example 1
(1) Samples Human interstitial pneumonia samples used in the analysis were collected and analyzed with the approval of the research ethics committees at four base hospitals (Shinshu University, Nippon Medical School, Chiba University, and Hiroshima University), the National Institute of Health Sciences, the Kihara Foundation, Astellas Pharma, and Daiichi Sankyo.

Blood samples were taken from patients suspected of developing drug-induced interstitial pneumonia at the above-mentioned base hospitals during the acute phase (near the worst stage) and recovery phase of drug-induced interstitial pneumonia. Blood and urine samples were taken early in the morning on an empty stomach during hospitalization, and at any time during outpatient care. Blood samples were taken at each base hospital with the patient's consent using a 7 mL EDTA-2K blood collection tube for plasma collection, and the blood was promptly mixed and centrifuged at 1,300 g x 10 minutes (15°C to 20°C). Blood samples were also taken using a silica microparticle blood collection tube for accelerating coagulation for serum collection, and the blood was promptly mixed, left at room temperature for 60 minutes, and then centrifuged at 1,300 g x 10 minutes (15°C to 20°C). The collected plasma and serum were frozen and stored at -80°C. Urine samples were taken from midstream urine using a paper cup for urine collection, centrifuged at 1,300 g x 10 minutes (15°C to 20°C), and frozen and stored at -80°C.

Plasma samples from patients with drug-induced interstitial pneumonia were collected for analysis of LPC molecules from 21 cases with diffuse alveolar damage (DAD) pattern in diagnostic imaging, 83 cases with other patterns, 65 cases collected during the recovery phase of drug-induced interstitial pneumonia, 31 cases from patients who were administered the same drugs as the patients who developed drug-induced interstitial pneumonia but did not develop drug-induced interstitial pneumonia for more than 3 months, 74 cases from lung cancer patients, 2 cases from fungal/viral pneumonia patients, 17 cases from bacterial pneumonia patients, 20 cases from nontuberculous mycobacterial disease patients, 41 cases from idiopathic interstitial pneumonia patients, 27 cases from collagen disease lung patients, 15 cases from chronic obstructive pulmonary disease (COPD) patients, and 12 cases from bronchial asthma patients.
Serum samples from patients with drug-induced interstitial pneumonia were collected for analysis of kynurenine pathway molecules from 21 cases with diffuse alveolar damage (DAD) pattern in diagnostic imaging, 81 cases with other patterns, 62 cases with drug-induced interstitial pneumonia in the recovery phase, 30 cases from patients who were administered the same drugs as the patients who developed drug-induced interstitial pneumonia but did not develop drug-induced interstitial pneumonia for more than 3 months, 70 cases from lung cancer patients, 15 cases from bacterial infection patients, 21 cases from nontuberculous mycobacterial disease patients, 45 cases from idiopathic interstitial pneumonia patients, 26 cases from collagen disease patients, 15 cases from chronic obstructive pulmonary disease (COPD) patients, and 13 cases from bronchial asthma patients.
Urine samples from patients with drug-induced interstitial pneumonia included 10 samples taken in the acute phase of cases that showed a diffuse alveolar damage (DAD) pattern in diagnostic imaging, 33 samples taken in the acute phase of cases that showed other patterns, and 32 samples taken in the recovery phase of drug-induced interstitial pneumonia, and were subjected to quinolinic acid analysis.

(2) Selection of candidate metabolites for drug-induced interstitial pneumonia biomarkers Candidate metabolites for drug-induced interstitial pneumonia were selected through search and validation using lipidomics analysis, which comprehensively analyzes hydrophobic metabolites, and metabolomics analysis, which comprehensively analyzes hydrophilic metabolites. In addition, 45 acute-phase cases of drug-induced interstitial pneumonia, including 9 cases with DAD patterns, and 33 convalescent-phase cases of drug-induced interstitial pneumonia, including 16 cases with DAD patterns, were used for the search, and 60 acute-phase cases of drug-induced interstitial pneumonia and 34 convalescent-phase cases of drug-induced interstitial pneumonia, including 16 cases with DAD patterns, were used for validation, with some samples overlapping with the search. As a result of the search, 117 molecules were identified as promising biomarker candidates for hydrophobic metabolites in cases showing DAD pattern, with a significance probability of p < 0.05 or less and an absolute effect size of 0.8 or more. In particular, the LPC molecule group showed a low significance probability p and a high effect size. As a result of verification, six LPC molecules (LPC(14:0), LPC(16:1), LPC(17:0), LPC(18:0), LPC(18:2), LPC(20:5)) showed a significance probability of p < 0.05 or less and an absolute effect size of 0.8 or more. Furthermore, a similar tendency was observed for all other detectable LPC molecules (LPC(16:0), LPC(18:1), LPC(20:3), LPC(20:4), LPC(22:6)). Therefore, we quantified and evaluated eight LPC molecules for which standard samples are available and abundant as biomarkers for drug-induced interstitial pneumonia. On the other hand, as a result of the search for hydrophilic metabolites, 14 molecules were identified as promising biomarker candidates in cases showing DAD pattern with a significance probability of p < 0.05 or less and an absolute effect size of 0.8 or more. As a result of verification, six molecules (phenylalanyl-tryptophan, tryptophan, kynurenine, citrulline, glycerophosphocholine, histidine) showed a significance probability of p < 0.05 or less and an absolute effect size of 0.8 or more. Furthermore, a similar tendency was observed for quinolinic acid, which is a molecule that belongs to the kynurenine pathway like tryptophan and kynurenine. Therefore, we quantified and evaluated tryptophan, kynurenine, and quinolinic acid, which are available as standard samples and abundant as biomarkers for drug-induced interstitial pneumonia.

(3) Biomarker quantification methods
Measurement and analysis of LPC molecular groups were performed using a ThermoFisher high-performance liquid chromatography (LC; U3000)/mass spectrometer (MS; TSQ Quantiva) with a proprietary analytical method. Sample preparation consisted of mixing 5 μl of plasma with MeOH containing 2 ng/ml of an internal standard (LPC (12:0), which is not detectable in living organisms), and passing the mixture through a 0.22 um filter plate to remove protein components. 3 μl of the sample solution was introduced into the LC/MS. The LC conditions were as follows: mobile phase A was 10 mM aqueous ammonium formate, mobile phase B was water with 10 mM aqueous ammonium formate: isopropanol (1:100), and the separation column was an InertCore C18 (2.4 μm pore, 2.1 x 50 mm, GL Science) heated to 50°C. The initial condition was set to 35% B, which increased to 100% B in 2.5 minutes, was maintained at 100% B until 3.4 minutes, was decreased to 35% B at 3.41 minutes, and was maintained at 100% B until 5 minutes. The equilibration was performed while maintaining the pH at 35%. The MS was introduced from 0 to 0.1 min, 0.5 to 2.9 min, and 4.3 to 5 min during the analysis time, and the liquid was discharged by switching to the waste line using a valve during the other time periods. The MS conditions were as follows: the ion source type was HESI (heated electrospray ionization), the ion mode was positive ion mode, the spray voltage was 3500 V, the sheath gas was 40 Arb, the Aux gas was 10 Arb, the sweep gas was 1 Arb, the ion transfer tube temp was 350 °C, and the vaporizer temp was 250 °C. SRM measurements were performed using the precursor ion and production ion for each ion in Table 1. The cycle time was 1 sec, the RF lens was 100 V, the Q1 resolution was 0.7 FWHM, the Q3 resolution was 0.7 FWHM, the CID gas was 1.5 mTorr, and the Chrom Filter was 3 sec.
The calibration curve ranges of the measured molecules were set as follows: 1 μg/mL - 200 μg/mL for LPC(16:0), 0.4 μg/mL - 80 μg/mL for LPC(18:0) and LPC(18:1), 20 ng/mL - 4000 ng/mL for LPC(14:0), LPC(15:0) and LPC(17:0), and 2 ng/mL - 400 ng/mL for LPC(19:0) and LPC(20:0). Four types of Quality Control (QC) samples were used for each analytical unit to calculate the relative accuracy and precision, and the data for analytical units that were less than 15% were used.

Kynurenine and tryptophan molecules were measured and analyzed using a proprietary analytical method developed using a ThermoFisher high-performance liquid chromatography (LC; U3000)/mass spectrometer (MS; TSQ Quantiva). Sample preparation consisted of mixing 50 μl of serum with 50 μl of water, and then mixing with MeOH containing internal standards (stable isotopes of kynurenine and tryptophan (kynurenine-d6 and tryptophan-d5) at 12.5 and 100 ng/ml, respectively), and passing the mixture through a 0.22 um filter plate to remove protein components. The mixture was then passed through a lipid-removing filter plate packed with C18 to remove lipid components. The collected sample liquid was diluted with three volumes of water, and 5 μl was introduced into the LC/MS. The LC conditions were as follows: mobile phase A was 0.3% formic acid water, mobile phase B was 0.3% formic acid methanol, and the separation column was a Triart Phenyl (1.9 μm pore, 2.1 x 100 mm, YMC) heated to 45°C. The initial conditions were set to 20% B, maintained for 0.5 min, and then B was diluted for 2.5 min. The concentration of B was increased to 100%, maintained at 100% for 4 min, decreased to 20% B at 4.1 min, and then maintained at 20% B for 5 min for equilibration. The sample was introduced into the MS from 1.2 to 3.5 min during the analysis time, and was discharged using a valve to the waste line during the other time periods. The MS conditions were as follows. The ion source type was HESI (heated electrospray ionization), the ion mode was positive ion mode, the spray voltage was 3500 V, the sheath gas was 40 Arb, the Aux gas was 10 Arb, the sweep gas was 1 Arb, the ion transfer tube temp was 350 °C, and the vaporizer temp was 250 °C. SRM measurements were performed using the precursor ion and production ion for each ion in Table 2. The cycle time was 0.5 sec, the RF lens was the calibrated value, the Q1 resolution was 0.7 FWHM, the Q3 resolution was 0.7 FWHM, and the CID gas was 1.5 mTorr, Chroma Filter was used for 3 sec.
Quinolinic acid was measured and analyzed using a proprietary analytical method using ThermoFisher's ion chromatography (IC; Dionex ICS-5000+)/high-resolution mass spectrometer (HRMS; Q-Exactive). In this blood concentration measurement system, a significant matrix effect due to serum components was confirmed, so normal human pooled serum was used to prepare the calibration curve samples instead of alternative matrices such as bovine serum albumin solution. In addition, since a certain amount of quinolinic acid is present in human serum, in this measurement method, quinolinic acid-d3, which has the same ionization efficiency as natural quinolinic acid, was used as an alternative molecule to prepare the calibration curve samples, and the blood concentration of quinolinic acid was calculated by substituting the measured values of natural quinolinic acid into the calibration curve prepared with the measured values of quinolinic acid-d3. Sample preparation consisted of mixing 20 μl of serum with 80 μl of water, then mixing with 400 μl of MeOH containing 18.4 ng/ml of internal standard (hippuric acid-d5), and passing the mixture through a 0.22 μm filter plate to remove protein components. The mixture was then passed through a lipid-removing filter plate packed with C18 to remove lipid components. 100 μl of the collected sample was completely dried in an evaporator, redissolved in 80 μl of distilled water, passed through a 0.22 μm PVDF filter column, and 5 μl was introduced into the IC/MS. The IC conditions were as follows: potassium hydroxide (KOH) aqueous solution was used as the mobile phase, and a Dionex IonPac AS11-HC-4 μm IC column (2 × 250 mm, Thermo Fisher Scientific) was used as the separation column, heated to 30°C. The initial condition was a KOH concentration of 10 mM, which was increased to 100 mM in 7 minutes, maintained at 100 mM until 8 minutes, decreased to 10 mM until 8.1 minutes, and then maintained at 10 mM until 10 minutes for equilibration. The MS conditions were as follows: ion source type was HESI (heated electrospray ionization), ion mode was negative ion mode, spray voltage was -2000 V, sheath gas was 40 Arb, Aux gas was 10 Arb, sweep gas was 0 Arb, AUX gas heater temp was 300 °C, capillary temp was 350 °C, and RF lens was set to 35. SRM measurements were performed using precursor ions and production ions for each ion in Table 2. The Automatic Gain Control (AGC) Target was 2× ¹⁰⁵ , the Maximum Injection time (IT) was 100 msec, the Isolation Window was 1.0 m/z, the Fixed First Mass was 50.0 m/z, and the Resolution was 17,500. The instruments and main analytical conditions for measuring the urinary concentrations were the same as those for blood concentrations, and full scan measurements were performed using the exact mass of the precursor ion. The difference in urinary concentration due to the difference in water intake before sample collection was corrected by using the probabilistic quotient normalization (PQN) method for the area value of each extracted component corrected with the internal standard substance.
The calibration curve ranges for the measured molecules were set as follows: kynurenine 20 ng/mL - 4000 ng/mL, tryptophan 0.15 μg/mL - 30 μg/mL, and quinolinic acid 3.3 ng/mL - 836 ng/mL. Four types of Quality Control (QC) samples were used for each analytical unit to calculate the relative accuracy and precision, and the data for analytical units that were less than 15% were used.

(4) Performance of biomarkers for drug-induced interstitial pneumonia (comparison with recovered cases)
Figure 1 shows the plasma concentrations of eight hydrophobic biomarkers for drug-induced interstitial pneumonia (LPC molecule group; LPC(14:0), LPC(15:0), LPC(16:0), LPC(17:0), LPC(18:0), LPC(18:1), LPC(19:0), LPC(20:0)) in dot plots. All LPC molecules were significantly decreased in acute-phase DAD-type drug-induced interstitial pneumonia cases (A-DAD) compared with all convalescent-phase drug-induced interstitial pneumonia cases (B) (A-DAD vs B). In addition, all acute-phase drug-induced interstitial pneumonia cases (A) were significantly decreased compared with all convalescent-phase drug-induced interstitial pneumonia cases (A vs B).
Figure 2 shows the serum concentrations of three hydrophilic biomarkers of drug-induced interstitial pneumonia (kynurenine pathway molecules; kynurenine, tryptophan, and quinolinic acid) in dot plots. Kynurenine, tryptophan, and quinolinic acid molecules were significantly increased (kynurenine and quinolinic acid) and (tryptophan) in acute-phase DAD-type drug-induced interstitial pneumonia cases (A-DAD) compared with all convalescent-phase drug-induced interstitial pneumonia cases (B) (A-DAD vs B). In addition, all acute-phase drug-induced interstitial pneumonia cases (A) were significantly increased and decreased, respectively, compared with all convalescent-phase drug-induced interstitial pneumonia cases (A vs B).
Figure 3 shows the urinary concentration of quinolinic acid in a dot plot. Quinolinic acid molecules were significantly increased in acute-phase DAD-type drug-induced interstitial pneumonia cases (A-DAD) compared with all convalescent-phase drug-induced interstitial pneumonia cases (B) (A-DAD vs B). In addition, a significant increase was observed in all acute-phase drug-induced interstitial pneumonia cases (A) compared with all convalescent-phase drug-induced interstitial pneumonia cases (A vs B).
Table 3 summarizes the AUC values obtained by ROC analysis to evaluate the performance of these biomarkers for drug-induced interstitial pneumonia, using plasma or serum concentrations, to distinguish between acute DAD-type drug-induced interstitial pneumonia cases (A-DAD), which are particularly severe, and all cases of drug-induced interstitial pneumonia in the recovery phase (B) (B). The LPC molecule group showed good discrimination performance, with AUCs of 0.903-0.938, kynurenine molecule of 0.912, tryptophan molecule of 0.800, quinolinic acid molecule of 0.884, and kynurenine/tryptophan of 0.943. Table 4 also summarizes the AUC values obtained by ROC analysis to distinguish between all cases of drug-induced interstitial pneumonia in the acute phase (A) and all cases of drug-induced interstitial pneumonia in the recovery phase (B) using plasma or serum concentrations (B). The LPC molecule group showed 0.714-0.821, kynurenine molecules 0.827, tryptophan molecules 0.747, quinolinic acid molecules 0.808, and kynurenine/tryptophan 0.870, showing good discrimination performance for some of the LPC molecules, kynurenine, quinolinic acid, and kynurenine/tryptophan.

(5) Performance of biomarkers for drug-induced interstitial pneumonia (comparison with non-disease cases)
All plasma LPC molecules were significantly decreased in acute-phase DAD-type drug-induced interstitial pneumonia cases (A-DAD) compared with cases not developing drug-induced interstitial pneumonia taking the same drugs (C) (Fig. 1, A-DAD vs C). Also, all acute-phase drug-induced interstitial pneumonia cases (A) were significantly decreased compared with cases not developing drug-induced interstitial pneumonia taking the same drugs (C) (Fig. 1, A vs C).
Serum kynurenine, tryptophan, and quinolinic acid molecules were significantly increased (kynurenine and quinolinic acid) and decreased (tryptophan) in patients with acute DAD-type drug-induced interstitial pneumonia (A-DAD) compared with patients without drug-induced interstitial pneumonia who were taking the same drugs (C) (Fig. 2, A-DAD vs C). In addition, in all acute drug-induced interstitial pneumonia cases (A), serum kynurenine and quinolinic acid were significantly increased (kynurenine and quinolinic acid) and decreased (tryptophan) compared with patients without drug-induced interstitial pneumonia who were taking the same drugs (C) (Fig. 2, A vs C).
The AUC values of the receiver operating characteristic curve (ROC) analysis for distinguishing between acute-phase DAD-type drug-induced interstitial pneumonia cases (A-DAD) using plasma or serum concentrations were 0.929-0.971 for the LPC molecule group, 0.938 for kynurenine molecules, 0.802 for tryptophan molecules, 0.955 for quinolinic acid molecules, and 0.963 for kynurenine/tryptophan, demonstrating good discrimination performance (Table 3, C). The AUC values of the receiver operating characteristic curve (ROC) for discriminating between acute-phase drug-induced interstitial pneumonia cases and all cases (A) using plasma or serum concentrations were 0.743-0.849 for the LPC molecule group, 0.851 for kynurenine molecules, 0.760 for tryptophan molecules, 0.901 for quinolinic acid molecules, and 0.895 for kynurenine/tryptophan, showing good discrimination performance for some of the LPC molecules, kynurenine, quinolinic acid, and kynurenine/tryptophan (Table 4, C).

(6) Performance of biomarkers for drug-induced interstitial pneumonia (comparison with lung diseases)
All plasma LPC molecules were significantly decreased in patients with acute DAD-type drug-induced interstitial pneumonia (A-DAD) compared with patients with lung cancer (D), pulmonary nontuberculous mycobacterial disease (G), idiopathic interstitial pneumonia (H), collagen vascular disease (I), COPD (J), and asthma (K) (Figure 1, A-DAD vs DK). In all acute drug-induced interstitial pneumonia cases (A), significant decreases were observed in all LPC molecule groups in lung cancer cases (D), LPC(19:0) and LPC(20:0) in pulmonary nontuberculous mycobacterial disease cases (G), LPC(14:0), LPC(15:0), LPC(16:0), LPC(17:0), LPC(18:0), LPC(18:1), and LPC(20:0) in idiopathic interstitial pneumonia cases (H), all LPC molecule groups in connective tissue disease lung cases (I), LPC(14:0), LPC(16:0), LPC(18:0), LPC(18:1), and LPC(20:0) in COPD cases (J), and LPC(14:0) in asthma cases (K) (Fig. 1, A vs DK).
Kynurenine and quinolinic acid molecules in serum were significantly increased in acute DAD-type drug-induced interstitial pneumonia cases (A-DAD) compared with lung cancer cases (D), bacterial infection cases (F), pulmonary nontuberculous mycobacterial disease cases (G), idiopathic interstitial pneumonia cases (H), collagen disease lung cases (I), COPD cases (J), and bronchial asthma cases (K). Tryptophan molecules were also significantly decreased in all cases except bacterial infection cases (F) (Fig. 2, A-DAD vs DK). In addition, in all acute drug-induced interstitial pneumonia cases (A), significant increases and decreases were observed in relation to lung disease, similar to DAD-type cases (Fig. 2, A vs DK).
The AUC values of the receiver operating characteristic curve (ROC) analysis for the discrimination of acute-phase DAD-type drug-induced interstitial pneumonia cases (A-DAD) using plasma or serum concentrations of the LPC molecule group were 0.880-0.923 (D), 0.900-0.978 (G), 0.916-0.96 (H), 0.892-0.944 (I), 0.937-0.981 (J), and 0.942-0.972 (K) for lung cancer cases (D), pulmonary nontuberculous mycobacterial disease cases (G), idiopathic interstitial pneumonia cases (H), collagen disease lung cases (I), COPD cases (J), and bronchial asthma cases (K). The kynurenine molecule group showed The IL-10001-related ... The acid molecule showed 0.937 (D), 0.918 (F), 0.892 (G), 0.872 (H), 0.902 (I), 0.908 (J), and 0.980 (K) in lung cancer (D), bacterial infection (F), pulmonary nontuberculous mycobacterial disease (G), idiopathic interstitial pneumonia (H), collagen disease (I), COPD (J), and bronchial asthma (K). An showed good discrimination performance for cases of lung cancer (D), bacterial infection (F), pulmonary nontuberculous mycobacterial disease (G), idiopathic interstitial pneumonia (H), collagen vascular lung disease (I), COPD (J), and bronchial asthma (K) with scores of 0.925 (D), 0.910 (F), 0.969 (G), 0.923 (H), 0.908 (I), 0.900 (J), and 0.977 (K) (Table 3, DK).
Regarding the AUC values of the receiver operating characteristic curve (ROC) analysis for discriminating acute-phase drug-induced interstitial pneumonia cases from all cases (A) using plasma or serum concentrations, the LPC molecule group showed good discrimination performance for some molecules for cases of pulmonary nontuberculous mycobacterial disease (G), idiopathic interstitial pneumonia (H), collagen vascular lung disease (I), COPD (J), and bronchial asthma (K) (Table 4, DK). On the other hand, the kynurenine molecule showed 0.820 (D), 0.866 (G), 0.816 (H), and 0.864 (K) in the lung cancer case (D), pulmonary nontuberculous mycobacterial disease case (G), idiopathic interstitial pneumonia case (H), and bronchial asthma case (K), respectively, while the tryptophan molecule showed 0.866 (K) in the bronchial asthma case (K), and the quinolinic The acid molecule showed 0.861 (D) and 0.925 (K) for lung cancer cases (D) and bronchial asthma cases (K), while kynurenine/tryptophan showed 0.847 (D), 0.817 (F), 0.902 (G), 0.854 (H), 0.827 (I), 0.827 (J), and 0.919 (K) for lung cancer cases (D), bacterial infection cases (F), pulmonary nontuberculous mycobacterial disease cases (G), idiopathic interstitial pneumonia cases (H), collagen disease lung cases (I), COPD cases (J), and bronchial asthma cases (K), demonstrating good discrimination performance (Table 4, DK).

B, All cases of drug-induced interstitial pneumonia in the recovery stage, C, Non-disease cases of drug-induced interstitial pneumonia due to use of similar drugs, D, Lung cancer cases, E, Fungal pneumonia cases, F, Bacterial infection cases, G, Pulmonary nontuberculous mycobacterial disease cases, H, Idiopathic interstitial pneumonia cases, I, Connective tissue disease cases, J, COPD cases, K, Bronchial asthma cases

The cutoff value for distinguishing and discriminating between A and BK obtained from the ROC curve analysis is determined from the results of each ROC curve analysis by using the concentration at which the Youden's Index [sensitivity - (100 - specificity)] is maximized (the value at which sensitivity and specificity are highest). The values in the table below are: upper: appropriate value, lower: preferred value. The ±15% in the table is a variation value that takes into account variability in the analysis system, etc., and there may be a ±15% variation in the upper and lower limits of the numerical range of the appropriate value, as well as in the preferred value.

The cutoff value for distinguishing and discriminating between A and BK obtained from the ROC curve analysis is determined from the results of each ROC curve analysis by using the concentration at which the Youden's Index [sensitivity - (100 - specificity)] is maximized (the value at which sensitivity and specificity are highest). The values in the table below are: upper: appropriate value, lower: preferred value. The ±15% in the table is a variation value that takes into account variability in the analysis system, etc., and there may be a ±15% variation in the upper and lower limits of the numerical range of the appropriate value, as well as in the preferred value.
All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

The present invention can be used for in vitro diagnostics, clinical testing, etc.

Claims (8)

A method for testing for drug-induced interstitial pneumonia, comprising measuring the level of at least one molecule selected from the group consisting of tryptophan and/or lysophosphatidylcholine (LPC) molecules in a sample derived from a subject. A method for testing for drug-induced interstitial pneumonia, comprising measuring the levels in a sample from a subject of at least one molecule selected from a group of molecules included in the kynurenine pathway and/or at least one molecule selected from a group of lysophosphatidylcholine (LPC) molecules, wherein the measured values assist in diagnosing the progression of the disease by indicating whether the drug-induced interstitial pneumonia is in the acute phase or the recovery phase . The method according to claim 1, wherein the measured value aids in the specific diagnosis of drug-induced interstitial pneumonia. The method according to any one of claims 1 to 3, wherein the drug-induced interstitial pneumonia is DAD-type drug-induced interstitial pneumonia. The method according to claim 2 , wherein the molecular group involved in the kynurenine pathway includes kynurenine, quinolinic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, 2-amino-3-carboxymuconic acid semialdehyde, picolinic acid and tryptophan. The method according to any one of claims 1 to 5, wherein the LPC molecule group includes LPC(14:0), LPC(15:0), LPC(16:0), LPC(16:1), LPC(17:0), LPC(18:0), LPC(18:1), LPC(18:2), LPC(19:0), LPC(20:0), LPC(20:3), LPC(20:4), LPC(20:5) and LPC(22:6). A kit for testing for drug-induced interstitial pneumonia, comprising a reagent capable of measuring the level of at least one molecule selected from the tryptophan and/or lysophosphatidylcholine (LPC) molecule group in a sample derived from a subject. A kit for diagnosing whether a patient is in the acute or recovery phase of drug-induced interstitial pneumonia, comprising a reagent capable of measuring the levels in a sample from a subject of at least one molecule selected from a group of molecules involved in the kynurenine pathway and/or at least one molecule selected from a group of lysophosphatidylcholine (LPC) molecules.