Polymyxin B therapeutic drug monitoring method and application thereof

Abstract

The invention discloses a polymyxin B therapeutic drug monitoring method and application thereof, which can be used for monitoring PB renal toxicity, and has the advantages of high sensitivity, rapidness, convenience and accurate and reliable result. The method comprises the following steps: (1) acquiring the concentrations of the following components of polymyxin B in a biological sample: polymyxin B1, polymyxin B2, polymyxin B3 and isoleucine-polymyxin B1; (2) taking one or more of the following concentration ratios as monitoring data: (polymyxin B + polymyxin B3) / polymyxin B, (polymyxin B1 + isoleucine-polymyxin B1) / (polymyxin B + polymyxin B3), isoleucine-polymyxin B1 / (polymyxin B + polymyxin B3), polymyxin B1 / (polymyxin B + polymyxin B3), polymyxin B1 / (polymyxin B + polymyxin B3), polymyxin B1 / (polymyxin B + polymyxin B3), and polymyxin B1 / polymyxin B; and the isoleucine-polymyxin B1 / polymyxin B, and comparing the monitoring data with a cutoff value (Cuff).

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical detection, and particularly to a method for monitoring polymyxin B for therapeutic use, and an application of the method in preparing a product for monitoring the renal toxicity of PB. Background Art

[0002] The high morbidity and mortality caused by multi-drug resistant (MDR) Gram-negative bacteria have become a key challenge that needs to be addressed urgently worldwide. Polymyxin B (PB) is the last effective line of defense against MDR Gram-negative bacterial infections. However, the significant toxic side effects of PB, especially the strong renal toxicity, pose a severe challenge to clinical use. Therefore, implementing therapeutic drug monitoring (TDM) is crucial for PB, which helps clinicians accurately formulate dosing regimens, thereby minimizing the occurrence of adverse reactions while ensuring the therapeutic effect.

[0003] The "Chinese Consensus Guidelines for Therapeutic Drug Monitoring of Polymyxin B" recommends performing therapeutic drug monitoring of PB and predicting renal toxicity using PB exposure and PB plasma steady-state concentration. And the effective range of PB exposure (AUC ss , 24h ) is set at 50 - 100 mg·h·L -1 , and the PB plasma steady-state concentration is 2 - 4 μg·mL -1 . However, clinical practice has shown that such monitoring indicators have limited ability to indicate renal toxicity.

[0004] It should be noted that the traditional indicators for PB therapeutic drug monitoring - PB exposure and PB plasma steady-state concentration - are both based on the calculation results of the total PB components. However, PB is actually a complex mixture produced by fermentation of Paenibacillus polymyxa, and its main components are polymyxin B1 (PB1), isoleucine-polymyxin B1 (ILE-PB1), polymyxin B2 (PB2), polymyxin B3 (PB3), and polymyxin B6 (PB6). There are literature reports that the in vitro apoptotic effect of PB1 on human renal proximal tubular HK-2 cells is significantly higher than that of PB2 (>3 times), that is, the toxicity of PB1 and PB2 may be different. In view of this, if only relying on the monitoring of the overall PB components to evaluate renal toxicity, the toxic effects of each component may be ignored, resulting in deviation of the monitoring results. Therefore, it is urgent to finely distinguish and quantify each component of PB, and on this basis, construct a PB renal toxicity risk prediction with high diagnostic efficiency to monitor PB renal toxicity early and accurately. Summary of the Invention

[0005] To overcome the defects of the prior art, the technical problem to be solved by the present invention is to provide a method for monitoring polymyxin B therapeutic drugs, which can be used to monitor the renal toxicity of PB, has high sensitivity, is fast and convenient, and the results are accurate and reliable.

[0006] The technical solution of the present invention is as follows: This method for monitoring polymyxin B therapeutic drugs includes the following steps:

[0007] (1) Obtain the concentrations of the following components of polymyxin B in the biological sample: polymyxin B1, polymyxin B2, polymyxin B3, isoleucine-polymyxin B1;

[0008] (2) Use one or several of the following concentration ratios as monitoring data: (polymyxin B2 +

[0009] polymyxin B3) / polymyxin B, (polymyxin B1 + isoleucine-polymyxin B1)

[0010] / (polymyxin B2 + polymyxin B3), isoleucine-polymyxin B1 / (polymyxin B2 + polymyxin B3), polymyxin B1 / (polymyxin B2 + polymyxin B3), polymyxin B1 / polymyxin B, isoleucine-polymyxin B1 / polymyxin B, and compare the monitoring data with the group with normal renal function.

[0011] The method for monitoring polymyxin B therapeutic drugs of the present invention can be effectively used for the early diagnosis of PB renal toxicity, has high sensitivity, is fast and convenient, and the results are accurate and reliable. It can provide a basis for clinical decision-making, and at the same time provide a certain basis for subsequent basic research and clinical research, and has great application and research value.

[0012] It also provides the application of the method for monitoring polymyxin B therapeutic drugs in the preparation of products for monitoring PB renal toxicity. Description of the Drawings

[0013] Figure 1 It shows the overall flowchart of the method for monitoring polymyxin B therapeutic drugs of the present invention.

[0014] Figure 2 It shows the total ion chromatogram of the polymyxin B (PB) standard + blank plasma in Example 1 of the present invention. Among them, Figure 2 A is the total ion chromatogram of the standard polymyxin B2 (PB2) and polymyxin B3 (PB3); Figure 2 B is the total ion chromatogram of the standard isoleucine-polymyxin B1 (ILE-PB1) and polymyxin B1 (PB1); Figure 2 C is the total ion chromatogram of the standard polymyxin B6 (PB6).

[0015] Figure 3 Shown is the total ion current chromatogram of polymyxin B (PB) in a patient's body in the first embodiment of the present invention. Among them, Figure 3 A is the total ion current chromatogram of polymyxin B2 and polymyxin B3 (PB2 + PB3) in the patient's body; Figure 3 B is the total ion current chromatogram of isoleucine-polymyxin B1 (ILE-PB1) and polymyxin B1 (PB1) in the patient's body; Figure 3 C shows the total ion current chromatogram of polymyxin B6 (PB6) in the patient's body. The results show that the content of PB6 in the patient's body is extremely low and undetectable.

[0016] Figure 4 Shown is the distribution of each component of polymyxin B in the normal renal function group and the nephrotoxicity group in the first embodiment of the present invention. Among them, Figure 4 A is the half box plot of the total concentration of polymyxin B, a conventional index for therapeutic drug monitoring, in the normal renal function group and the nephrotoxicity group; Figure 4 B is the half box plot of the exposure amount of polymyxin B (AUC ss,24h ) in the normal renal function group and the nephrotoxicity group; Figure 4 C is the half box plot of the concentration of polymyxin B1 (PB1) in the normal renal function group and the nephrotoxicity group; Figure 4 D is the half box plot of the concentration of isoleucine-polymyxin B1 (ILE-PB1) in the normal renal function group and the nephrotoxicity group; Figure 4 E is the half box plot of the concentration of polymyxin B2 + polymyxin B3 (PB2 + PB3) in the normal renal function group and the nephrotoxicity group.

[0017] Figure 5 Shown is the distribution of the ratio of each component of polymyxin B in the normal renal function group and the nephrotoxicity group in the second embodiment of the present invention. Among them, Figure 5 A is the half box plot of the ratio of polymyxin B1 to the total concentration of polymyxin B (PB1 / PB) in the normal renal function group and the nephrotoxicity group; Figure 5 B is the half box plot of the ratio of polymyxin B2 + polymyxin B3 to the total concentration of polymyxin B ((PB2 + PB3) / PB) in the normal renal function group and the nephrotoxicity group; Figure 5 C is the half box plot of the ratio of isoleucine-polymyxin B1 to the total concentration of polymyxin B (ILE-PB1 / PB) in the normal renal function group and the nephrotoxicity group; Figure 5 D is the half box plot of the ratio of polymyxin B1 to the total concentration of polymyxin B2 + polymyxin B3 (PB1 / (PB2 + PB3)) in the normal renal function group and the nephrotoxicity group; Figure 5E is the half box plot of the ratio of isoleucine-polymyxin B1 to the total concentration of polymyxin B2 + polymyxin B3 (ILE-PB1 / (PB2+PB3)) in the normal renal function group and the nephrotoxicity group; Figure 5 F is the half box plot of the ratio of polymyxin B1 + isoleucine-polymyxin B1 to the total concentration of polymyxin B2 + polymyxin B3 ((PB1+ILE-PB1) / (PB2+PB3)) in the normal renal function group and the nephrotoxicity group.

[0018] Figure 6 It shows the receiver operating characteristic curve (ROC) analysis results of the conventional indicators for therapeutic drug monitoring of polymyxin B in Example 3 of the present invention. Figure 6 A shows the ROC curve graph and the area under the curve (AUC) value of the total concentration of polymyxin B; Figure 6 B shows the ROC curve graph and the AUC analysis results of the exposure amount of polymyxin B.

[0019] Figure 7 It shows the receiver operating characteristic curve (ROC) analysis results of the new indicators for therapeutic drug monitoring of polymyxin B in Example 3 of the present invention. Figure 7 A shows the ROC curve graph and the area under the curve (AUC) value of the concentrations of each component of polymyxin B; Figure 7 B shows the ROC curve graph and the AUC analysis results of the ratios between the concentrations of each component of polymyxin B; Figure 7 C shows the ROC curve graph and the AUC analysis results of the new diagnostic model of polymyxin B. Detailed implementation manners

[0020] This method for therapeutic drug monitoring of polymyxin B includes the following steps:

[0021] (1) Obtain the concentrations of the following components of polymyxin B in the biological sample: polymyxin B1, polymyxin B2, polymyxin B3, isoleucine-polymyxin B1;

[0022] (2) Use one or several of the following concentration ratios as monitoring data: (polymyxin B2 + polymyxin B3) / polymyxin B, (polymyxin B1 + isoleucine-polymyxin B1)

[0023] / (polymyxin B2 + polymyxin B3), isoleucine-polymyxin B1 / (polymyxin B2 + polymyxin B3), polymyxin B1 / (polymyxin B2 + polymyxin B3), polymyxin B1 / polymyxin B, isoleucine-polymyxin B1 / polymyxin B, and compare the monitoring data with the normal renal function group.

[0024] The method for therapeutic drug monitoring of polymyxin B of the present invention can be effectively used for the early diagnosis of PB nephrotoxicity, with high sensitivity, rapidity, convenience, and accurate and reliable results. It can provide a basis for clinical decision-making, and at the same time provide a certain foundation for subsequent basic research and clinical research, having great application and research value.

[0025] Preferably, the concentration ratio of the components of polymyxin B is analyzed, and the fitted regression curve is obtained through Logistic regression analysis:

[0026] Y = Logit(p) = 26.9a – 24.0b – 24.6

[0027] where Y is the diagnostic probability, Logit(p) is the logistic regression function, a is (polymyxin B1 +

[0028] isoleucine-polymyxin B1) / (polymyxin B2 + polymyxin B3), and b is polymyxin B1 / (polymyxin B2 + polymyxin B3).

[0029] Preferably, the biological sample is plasma, and the concentration ratio levels of each component of polymyxin B in the biological sample are detected by one or more of the following methods: chromatography, spectrometry, mass spectrometry, chemical analysis, immunoassay.

[0030] Preferably, the chromatography includes high performance liquid chromatography, thin layer chromatography, gas chromatography; the spectrometry includes nuclear magnetic resonance spectrometry, refractive index spectrometry, ultraviolet spectrometry, near-infrared spectrometry; the chemical analysis includes electrochemistry analysis, radiochemistry analysis.

[0031] Preferably, in the chromatography, the mobile phase: mobile phase A is an aqueous solution containing 1% formic acid, mobile phase D is a mixture of methanol and acetonitrile with a volume ratio of 1:1 and containing 1% formic acid; the gradient elution program for sample determination: 0 - 2.0 min, 75% A; 2.0 - 4.5 min, 75% - 70% A; 4.5 - 5.5 min, 70% - 5% A; 5.5 - 6.5 min, 5% - 5% A; 6.5 - 6.7 min, 5% - 75% A; 6.7 - 9.0 min, 75% - 75% A; the analysis time is 0 - 9 min, the injection volume is 5 μL each time, the flow rate is 0.20 mL / min, the chromatographic column: ACQUITY Peptide CSH C18 1.7 μm, 2.1×100 mm, and the column temperature is 40°C.

[0032] Preferably, the mass spectrometry is high resolution mass spectrometry. First, gradient elution is performed using a chromatographic column, and then data is collected in the positive ion PRM mode of the electrospray ionization source ESI.

[0033] Preferably, in the mass spectrometry, spray voltage: 2500 V; evaporation temperature: 400 °C; capillary temperature: 320 °C; S-lens RF: 50; resolution of full-scan in the first stage: 70000, detected ions: for polymyxin B2 and polymyxin B3, m / z 595.3926, for polymyxin B1 and isoleucine-polymyxin B1, m / z 602.4004, for polymyxin B6, m / z 610.3979; data-dependent scan in the second stage: resolution: 17500, AGC target: 2e 5 , Maximum TT: 100 ms, NCE: 22.

[0034] Preferably, the pretreatment method of the biological sample before detection is as follows: 150 μL of plasma is added with 150 μL of 5% trichloroacetic acid, vortexed and mixed evenly for 5 min, and centrifuged at 12000 rpm·min -1 for 10 min, 100 μL of the supernatant is aspirated, 100 μL of water is added, and vortexed and mixed evenly for 10 s to obtain a test solution for quantitative analysis.

[0035] The invention also provides the application of the polymyxin B therapeutic drug monitoring method in the preparation of products for monitoring PB nephrotoxicity.

[0036] Preferably, the application is a composite product for reducing the concentration ratio of each component of polymyxin B in plasma and the probability of the diagnostic model, which is a food, probiotic preparation or pharmaceutical preparation. The ratio of each component of polymyxin B in plasma and the probability of the diagnostic model before and after the intervention of the candidate food, probiotic preparation or pharmaceutical preparation are detected, and screening is carried out based on whether the probability decreases.

[0037] The overall process of the present invention is shown in Figure 1 , and the embodiments of the present invention will be described in detail below.

[0038] Example 1

[0039] Monitoring of each component of PB in patients with PB nephrotoxicity and the PB renal function normal group

[0040] (I) Case screening and sample collection

[0041] Collect 78 plasma samples from the normal renal function group (HC) of polymyxin B (PB) treatment, which are from 33 patients; and 47 plasma samples from the PB nephrotoxicity group, which are from 16 patients. ① Inclusion criteria: age > 18 years old, patients with severe infections; using PB; performing therapeutic drug monitoring of PB; diagnosis of PB nephrotoxicity: serum creatinine increased by 50% within 7 days, or estimated glomerular filtration rate (eGFR) decreased by 25%. ② Exclusion criteria: patients using continuous renal replacement therapy (CRRT) due to renal function injury or failure; patients using extracorporeal membrane oxygenation (ECMO) therapy. All participants are from China-Japan Friendship Hospital. The age and gender of HC are matched with those of PB nephrotoxic patients to exclude metabolic differences caused by gender and age.

[0042] Collect the routine indicators of therapeutic drug monitoring of PB from the subjects, namely PB exposure and total PB concentration, for statistical analysis; collect the remaining plasma samples after PB therapeutic drug monitoring from the subjects and store them in a -80 °C refrigerator for quantitative analysis of each component of PB.

[0043] (II) Sample detection and statistical analysis

[0044] 2.1 Sample preparation:

[0045] 2.1.1 Preparation of polymyxin B standard stock solution:

[0046] Accurately weigh 1 mg of standard polymyxin B1 (PB1), 5 mg of polymyxin B2 (PB2), 5 mg of polymyxin B3 (PB3), 5 mg of polymyxin B6 (PB6), and 0.5 mg of isoleucine-polymyxin B1 (IIE-PB1). Add 1 mL, 1 mL, 1 mL, 1 mL, and 0.5 mL of water (containing 1% formic acid) respectively to dilute completely to make stock solutions with concentrations of 1 mg·mL -1 , 5 mg·mL -1 , 5 mg·mL -1 , 5 mg·mL -1 , and 1 mg·mL -1 respectively. After aliquoting 50 μL each, store them at -80 °C.

[0047] 2.1.2 Preparation of standard curve:

[0048] Pipette 15 μL, 3 μL, 3 μL, 3 μL, and 15 μL of PB1, PB2, PB3, PB6, and ILE-PB1 stock solutions into centrifuge tubes, and add 961 μL of blank plasma to prepare solution S with a concentration of 15 μg·mL for each component of PB -1 . Add appropriate blank plasma to S and serially dilute it to obtain a standard curve with a linear range of 0.46875 μg·mL -1 ~15 μg·mL -1 containing 6 concentration points.

[0049] 2.1.3 Sample pretreatment:

[0050] For 150 μL of patient plasma / standard curve concentration points, add 150 μL of 5% trichloroacetic acid, vortex mix for 5 min, and centrifuge at 12,000 rpm·min -1 for 10 min. Pipette 100 μL of the supernatant, add 100 μL of water, and vortex mix for 10 s to obtain the test solution for quantitative analysis.

[0051] 2.2 Chromatography / mass spectrometry conditions:

[0052] Detection was performed using a high-resolution mass spectrometer QE-Orbitrap. Mobile phase A was an aqueous solution containing 1% formic acid, and mobile phase D was a 1:1 (v / v) mixture of methanol and acetonitrile containing 1% formic acid. The gradient elution program for sample determination was as follows: 0 - 2.0 min, 75% A; 2.0 - 4.5 min, 75% - 70% A; 4.5 - 5.5 min, 70% - 5% A; 5.5 - 6.5 min, 5% - 5% A; 6.5 - 6.7 min, 5% - 75% A; 6.7 - 9.0 min, 75% - 75% A. The analysis time was 0 - 9 min, the injection volume was 5 μL each time, the flow rate was 0.20 mL / min, the chromatographic column was ACQUITY Peptide CSH C18 1.7 μm, 2.1×100 mm, and the column temperature was 40°C. Data were collected in the positive ion mode of the electrospray ionization source (ESI). Spray voltage: 2500 V; evaporation temperature: 400°C; capillary temperature: 320°C; S-lens RF: 50; resolution of the first-stage full scan: 70,000, detected ions: m / z 595.3926 (PB2, PB3), m / z 602.4004 (PB1, ILE-PB1), m / z 610.3979 (PB6); second-stage data-dependent scanning: resolution: 17,500, AGC target: 2e5, Maximum TT: 100 ms, NCE: 22.

[0053] 2.3 Statistical analysis:

[0054] The Thermo Fisher Xcalibur liquid chromatography-mass spectrometry analysis software was used to perform peak extraction and integration for each target PB component. All target PB components were analyzed by the standard addition method (subtracting the matrix background peak area from the standard peak area), and a standard curve (intercept 0, weighting factor 1 / X 2 ) was plotted for quantitative analysis. The independent samples t-test in the SPSS 25.0 statistical analysis software was used to compare the differences between groups. p < 0.05 indicates a significant difference in the results; p < 0.01 indicates a very significant difference; p < 0.001 indicates an extremely significant difference; p < 0.0001 indicates an extremely significant difference.

[0055] (III) Result Analysis

[0056] 3.1 Standard Curves and Linear Ranges of Each Component of PB

[0057] The total ion current chromatogram of blank plasma added with PB standard is shown in Figure 2 , and the results show that the chromatographic peaks of PB1 (retention time 5.34 min, Figure 2 B), ILE-PB1 (retention time 5.10 min, Figure 2 B), PB2 (retention time 4.60 min, Figure 2 A), PB3 (retention time 4.88 min, Figure 2 A), and PB6 (retention time 4.02 min, Figure 2 C) can be baseline separated, indicating strong specificity of the analytical method. Samples with a series of concentrations of the PB standard curve were continuously injected from low to high concentration. Using the standard addition method, the added concentration of each analyte was used as the abscissa (X), and the peak area of the analyte minus the background peak area was used as the ordinate (Y) to calculate the standard curve, obtaining the linear equation of the standard curve and the linear correlation coefficient value R 2 (Table 1). The results show that for PB1, ILE-PB1, PB2, PB3, and PB6, within their respective concentration ranges, the peak area has a good linear relationship with the concentration, and the correlation coefficient R 2 is greater than 0.99 for all of them.

[0058] Table 1 Standard Curve Equations and Linear Correlation Coefficients of Each Component of PB

[0059] Each component of PB Standard curve <![CDATA[R 2 > PB1 Y = -319759 + 12532.1*X 0.9995 ILE-PB1 Y = -148244 + 8940.63*X 0.9993 PB2 + PB3 Y = -253141 + 11384.8*X 0.9995 PB6 Y = -68298.7 + 11333.5*X 0.9985

[0060] 3.2 Sample Analysis of PB Nephrotoxic Patients and PB Patients with Normal Renal Function

[0061] Quantitative analysis was performed on each component of PB in the plasma samples of PB nephrotoxic patients and PB patients with normal renal function. The total ion current chromatograms of each component of PB in the patient samples are shown in Figure 3 . The results show that 5PB2 and PB3 cannot be baseline separated, so sum analysis of PB2 + PB3 was performed ( Figure 3 A); at the retention time of PB6 of 4.02 min, there is no chromatographic peak ( Figure 3 C), indicating that the level of PB6 in the patient body is below the detection limit, so no analysis was performed.

[0062] According to the standard curve, the concentrations of PB1, ILE-PB1, and PB2 + PB3 components in the bodies of PB nephrotoxic patients and PB patients with normal renal function were calculated, and further t-tests were performed on the concentrations of each component of PB between groups. 10 At the same time, the routine indicators of therapeutic drug monitoring for patients were collected and statistically analyzed - the total concentration of PB and the PB exposure amount,

[0063] Perform the t-test analysis between groups. Figure 4 A-4B shows that there are no significant differences in the conventional indicators of clinical therapeutic drug monitoring, namely the total PB component concentration and PB exposure, compared with PB patients with normal renal function; while there are extremely significant differences in both PB1 and ILE-PB1, and very significant differences in PB2+PB3, as shown in Figure 4 C-4E. The specific parameters of the t-test are shown in Table 2.

[0064] Table 2 Comparison of t-tests for conventional indicators of PB therapeutic drug monitoring and concentrations of each component between groups

[0065]

[0066] Note: p < 0.05 represents a significant difference in the results; p < 0.01 represents a very significant difference in the results; p < 0.001 represents an extremely significant difference in the results; p < 0.0001 represents an extremely significant difference in the results.

[0067] Compared with the conventional indicators of clinical therapeutic drug monitoring, namely the total PB component concentration and PB exposure, the concentrations of each component PB1, ILE-PB1, and PB2+PB3 may be more helpful for the monitoring of PB nephrotoxicity.

[0068] Example 2

[0069] Analysis of the ratio of each PB component in PB patients with nephrotoxicity and the PB group with normal renal function

[0070] (I) Data statistics

[0071] For the concentrations of each PB component in PB patients with normal renal function and PB patients with nephrotoxicity, perform ratio analysis, and use the independent samples t-test in the SPSS 25.0 statistical analysis software to compare the differences between groups. p < 0.05 represents a significant difference in the results; p < 0.01 represents a very significant difference in the results; p < 0.001 represents an extremely significant difference in the results; p < 0.0001 represents an extremely significant difference in the results.

[0072] (II) Result analysis

[0073] The ratios of each component of PB in the plasma samples of patients with PB nephrotoxicity and patients with normal PB renal function were respectively analyzed by t-test, namely, the ratio of PB1 to the total PB component concentration (PB1 / PB), the ratio of PB2 + PB3 to the total PB component concentration ((PB2 + PB3) / PB), the ratio of ILE-PB1 to the total PB component concentration (ILE-PB1 / PB), the ratio of PB1 to the concentration of PB2 + PB3 (PB1 / (PB2 + PB3)), the ratio of ILE-PB1 to the concentration of PB2 + PB3 (ILE-PB1 / (PB2 + PB3)), and the ratio of the sum of the concentrations of PB1 and ILE-PB1 to the concentration of PB2 + PB3 ((PB1 + ILE-PB1) / (PB2 + PB3)). The results showed that there were extremely significant differences in PB1 / PB, (PB2 + PB3) / PB, PB1 / (PB2 + PB3), ILE-PB1 / (PB2 + PB3), and (PB1 + ILE-PB1) / (PB2 + PB3), as shown in Figure 5 A, 5B, 5D - 5F; there was a very significant difference in ILE-PB1 / PB, as shown in Figure 5 C. The specific parameters of the t-test are shown in Table 3.

[0074] Table 3 t-test comparison of the concentration ratios of each component of PB between groups

[0075]

[0076] Note: p < 0.05 represents a significant difference in the results; p < 0.01 represents a very significant difference in the results; p < 0.001 represents an extremely significant difference in the results; p < 0.0001 represents an extremely significant difference in the results.

[0077] Compared with the conventional indicators of clinical therapeutic drug monitoring, namely, the total PB component concentration and PB exposure, the component ratios PB1 / PB, (PB2 + PB3) / PB, ILE-PB1 / PB, PB1 / (PB2 + PB3), ILE-PB1 / (PB2 + PB3), and (PB1 + ILE-PB1) / (PB2 + PB3) may be more significant for the diagnosis and treatment of PB nephrotoxicity.

[0078] Example 3

[0079] Establishment of PB nephrotoxicity diagnosis model

[0080] (I) Data statistics

[0081] The Receiver Operating Characteristic (ROC) curve is an important means to determine the diagnostic test criteria (i.e., the cut-off value for distinguishing normal and abnormal), and measures the ability of the test to identify diseases through sensitivity (true positive rate) and misdiagnosis rate (false positive rate, 1 - specificity). In this study, the sensitivity and specificity of monitoring PB nephrotoxicity by each component of PB and its ratio were comprehensively investigated through ROC curve analysis.

[0082] For patients with normal PB renal function and PB nephrotoxicity, further based on the ROC curve, it was determined whether the concentrations and ratios of 9 polymyxin B components such as PB1, ILE - PB1, PB2 + PB3, PB1 / PB, (PB2 + PB3) / PB, ILE - PB1 / PB, PB1 / (PB2 + PB3), ILE - PB1 / (PB2 + PB3), (PB1 + ILE - PB1) / (PB2 + PB3) screened out in Example 1 and Example 2 could be used as new indicators for monitoring PB nephrotoxicity.

[0083] Using SPSS software, the group was set as the status variable, and the concentrations and ratios of polymyxin B components were the test variables. After calculating the ROC curve, SPSS generated an output report containing the ROC curve graph and the Area under curve (AUC) value. Logistic regression analysis in SPSS was used to construct the diagnostic model, and the Hosmer - Lemeshow test was used to evaluate the goodness of fit of the model. When P > 0.05, it indicates that the model has a good fitting effect, otherwise the model fitting effect is poor; when AUC > 0.7, the indicator can be considered to have good diagnostic ability. The Cutoff value, that is, the judgment standard, is the cut-off value for determining whether a sample is positive or negative.

[0084] (II) Result analysis

[0085] The results of the ROC curve analysis of patients with normal renal function and PB nephrotoxicity showed ( Figure 6 A - 6B), for the conventional indicators of clinical therapeutic drug monitoring, that is, the total PB component concentration and PB exposure, the AUC values were 0.566 and 0.589 respectively, both less than 0.7. This indicates that the above two conventional indicators have limited ability to distinguish between patients with normal PB renal function and nephrotoxic patients. (PB2 + PB3) / PB: AUC = 0.930; (PB1 + ILE - PB1) / (PB2 + PB3): AUC = 0.930; ILE - PB1 / (PB2 + PB3): AUC = 0.925; PB1 / (PB2 + PB3): AUC = 0.919; PB1 / PB: AUC = 0.885; PB1: AUC = 0.751; ILE - PB1: AUC = 0.745, all greater than 0.7, seeFigure 7 A-B and Table 4 indicate that the concentrations and ratios of these 7 PB components can distinguish between patients with normal PB renal function and those with renal toxicity.

[0086] ROC parameters of the concentration ratios of each PB component between groups in Table 4

[0087]

[0088]

[0089] Furthermore, through Logistic regression analysis, the regression curve fitted with the concentrations and ratios of each component of polymyxin B was obtained, that is, the diagnostic model: Y = Logit(p) = 26.9a - 24.0b - 24.6

[0090] Among them, Y is the diagnostic probability, Logit(p) is the logistic regression function, a is polymyxin B1 + isoleucine-polymyxin B1 / polymyxin B2 + polymyxin B3 (abbreviation (PB1 + ILE - PB1) / (PB2 + PB3)); b is polymyxin B1 / polymyxin B2 + polymyxin B3 (abbreviation PB1 / (PB2 + PB3)).

[0091] After fitting, the area under the ROC curve AUC was 0.943 ( Figure 7 B), and the 95% confidence interval was 0.885 - 0.977, which improved the diagnostic efficiency of a single marker. The Hosmer-Lemeshow test showed that P = 0.4389, indicating that the model fitting effect was good.

[0092] The above is only a preferred embodiment of the present invention, and does not impose any form of limitation on the present invention. Any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for therapeutic drug monitoring of polymyxin B, characterized in that: It includes the following steps: (1) Obtain the concentrations of the following components of polymyxin B in a biological sample: polymyxin B1, polymyxin B2, polymyxin B3, isoleucine-polymyxin B1; (2) Use one or more of the following concentration ratios as monitoring data: (polymyxin B2 + polymyxin B3) / polymyxin B, (polymyxin B1 + isoleucine-polymyxin B1) / (polymyxin B2 + polymyxin B3), isoleucine-polymyxin B1 / (polymyxin B2 + polymyxin B3), polymyxin B1 / (polymyxin B2 + polymyxin B3), polymyxin B1 / polymyxin B, isoleucine-polymyxin B1 / polymyxin B, and compare the monitoring data with that of the normal renal function group.

2. The method for monitoring the therapeutic drug of polymyxin B according to claim 1, wherein: Analyze the concentration ratios of the components of polymyxin B, and obtain a fitted regression curve through Logistic regression analysis: Y = Logit(p) = 26.9a – 24.0b – 24.6 Where Y is the diagnostic probability, Logit(p) is the logistic regression function, a is (polymyxin B1 + isoleucine-polymyxin B1) / (polymyxin B2 + polymyxin B3), and b is polymyxin B1 / (polymyxin B2 + polymyxin B3).

3. The method for monitoring polymyxin B therapeutic drugs according to claim 2, characterized in that: The biological sample is plasma, and the concentration ratio levels of each component of polymyxin B in the biological sample are detected by one or more of the following methods: chromatography, spectroscopy, mass spectrometry, chemical analysis, immunoassay.

4. The method for monitoring polymyxin B therapeutic drugs according to claim 3, wherein: The chromatography includes high performance liquid chromatography, thin layer chromatography, gas chromatography; the spectroscopy includes nuclear magnetic resonance spectroscopy, refractive index spectroscopy, ultraviolet spectroscopy, near-infrared spectroscopy; the chemical analysis includes electrochemistry analysis, radiochemical analysis.

5. The method for monitoring the therapeutic drug of polymyxin B according to claim 4, characterized in that: In the chromatography, the mobile phase: mobile phase A is an aqueous solution containing 1% formic acid, mobile phase D is a mixture of methanol and acetonitrile with a volume ratio of 1:1 and containing 1% formic acid; the gradient elution program for sample determination: 0 - 2.0 min, 75% A; 2.0 - 4.5 min, 75% - 70% A; 4.5 - 5.5 min, 70% - 5% A; 5.5 - 6.5 min, 5% - 5% A; 6.5 - 6.7 min, 5% - 75% A; 6.7 - 9.0 min, 75% - 75% A; the analysis time is 0 - 9 min, the injection volume is 5 μL each time, the flow rate is 0.20 mL / min, the chromatographic column: ACQUITY Peptide CSH C18 1.7 μm, 2.1×100 mm, and the column temperature is 40°C.

6. The method for monitoring the therapeutic drug of polymyxin B according to claim 3, wherein: The mass spectrometry is high-resolution mass spectrometry. First, gradient elution is performed using a chromatographic column, and then data is collected in the positive ion PRM mode of the electrospray ionization source ESI.

7. The method for monitoring polymyxin B therapeutic drugs according to claim 6, wherein: In the mass spectrometry, the spray voltage: 2500 V; the evaporation temperature: 400°C; the capillary temperature: 320°C; S-lens RF: 50; Resolution of the first-level full scan: 70000, detected ions: for polymyxin B2 and polymyxin B3, m / z 595.3926, for polymyxin B1 and isoleucine-polymyxin B1, m / z 602.4004, for polymyxin B6, m / z 610.3979; Second-level data-dependent scan: Resolution: 17500, AGC target: 2e 5 , Maximum TT: 100 ms, NCE:

22.

8. The method for monitoring the therapeutic drug of polymyxin B according to claim 2, characterized in that: The pretreatment method of the biological sample before detection is as follows: 150 μL of plasma is added with 150 μL of 5% trichloroacetic acid, vortexed and mixed evenly for 5 min, and centrifuged at 12,000 rpm·min -1 for 10 min, 100 μL of the supernatant is aspirated, 100 μL of water is added, and the mixture is vortexed and mixed evenly for 10 s to obtain a test solution for quantitative analysis.

9. Use of the polymyxin B therapeutic drug monitoring method according to any one of claims 2 - 8 in the preparation of a product for monitoring PB nephrotoxicity.

10. Use of the method for monitoring polymyxin B therapeutic drug according to claim 9 in the preparation of a product for monitoring PB nephrotoxicity, characterized in that: The application is a composite product for reducing the concentration ratio of each component of polymyxin B in plasma and the probability of the diagnostic model, which is a food, probiotic preparation or pharmaceutical preparation. The ratio of each component of polymyxin B in plasma and the probability of the diagnostic model are detected before and after the intervention of the candidate food, probiotic preparation or pharmaceutical preparation, and screening is carried out based on whether the probability decreases.

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