Application and products of metabolic markers in the preparation of products for monitoring PB neurotoxicity

Abstract

Disclosed are the use of a metabolic marker in the preparation of a product for monitoring PB neurotoxicity and a product thereof, which can be used for the early diagnosis of PB neurotoxicity with high sensitivity, rapidity, convenience, and accurate and reliable results. The metabolic markers include LysoPE 20:4 / 0:0, creatine, SM d18:1 / 24:0, SM d18:0 / 24:1, LysoPC 18:0, palmitoyl sphingomyelin, phosphocholine, PC 14:0 / 22:1, creatinine, PC 18:3 / 16:0, SM d18:1 / 16:0, sphingosine, choline, LysoPC 18:1, L-carnitine, palmitic acid, PC 18:0 / 20:4, 3-hydroxy-2-octylglutaric acid, L-acetylcarnitine, LysoPC 20:4, LysoPC 16:0, and PC 20:1 / 14:1.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceutical testing, and in particular to the use of a metabolic marker in the preparation of a product for monitoring polymyxin B neurotoxicity, as well as a product that can effectively reduce the probability of a metabolic marker diagnostic model, which is used for the adjunctive treatment of polymyxin B neurotoxicity. Background Art

[0002] Polymyxin B (PB) entered clinical use in the 1950s for the treatment of Gram-negative bacterial infections. Due to frequent nephrotoxicity and neurotoxicity, the clinical use of PB gradually declined in the 1970s. In recent years, the incidence of multidrug-resistant Gram-negative bacterial (MDR-GNB) infections has been increasing, and PB has become a first-line drug for the treatment of MDR-GNB infections.

[0003] Clinically, the overall incidence of PB neurotoxicity is approximately 14-28%, with the main manifestations being confusion, drowsiness, dizziness, facial flushing, peripheral paresthesias, and even respiratory failure. For critically ill patients in a coma or deep sedation, the effects of PB neurotoxicity are more subtle and difficult to identify early. Failure to implement timely interventions can seriously threaten the patient's life. Therefore, it is particularly important and urgent to identify new, early, sensitive, specific, and non-invasive markers of PB neurotoxicity and strengthen monitoring of patients during PB use.

[0004] Pharmacometabolomics analyzes subtle changes in small molecule metabolites (specifically compounds with a molecular weight below 1000 Da) in biological fluids (including key samples such as blood, urine, and sweat) before and after drug administration, identifying metabolites with significant differences. This not only builds a solid bridge for early prediction and dynamic monitoring of pharmacological effects, but also becomes an indispensable tool in the fields of drug metabolism, efficacy evaluation, and toxicity research. Pharmacometabolomics technology is expected to efficiently and accurately discover PB neurotoxicity markers, enable early diagnosis of adverse drug reactions, and provide more accurate and safe guidance for clinical medication use. Summary of the Invention

[0005] In order to overcome the defects of the existing technology, the technical problem to be solved by the present invention is to provide an application of a metabolic marker in the preparation of a product for monitoring PB neurotoxicity, which can be used for the early diagnosis of PB neurotoxicity with high sensitivity, rapidity and convenience, and accurate and reliable results.

[0006] The technical solution of the present invention is: the use of such metabolic markers in the preparation of a product for monitoring PB neurotoxicity, wherein the metabolic markers include LysoPE 20:4 / 0:0, creatine, SM d18:1 / 24:0, SM d18:0 / 24:1, LysoPC 18:0, palmitoyl sphingomyelin, phosphocholine, PC 14:0 / 22:1, creatinine, PC 18:3 / 16:0, SM d18:1 / 16:0, sphingosine, choline, LysoPC 18:1, L-carnitine, palmitic acid, PC 18:0 / 20:4, 3-hydroxy-2-octylglutaric acid, L-acetylcarnitine, LysoPC 20:4, LysoPC 16:0, and PC 20:1 / 14:1.

[0007] The metabolic markers of the present invention can be effectively used for the early diagnosis of PB neurotoxicity with high sensitivity, rapidity and convenience, accurate and reliable results, which can provide a basis for clinical decision-making and a certain foundation for subsequent basic research and clinical research, and have great application and research value.

[0008] Also provided is a preparation of a metabolic marker for preparing a product for monitoring PB neurotoxicity, wherein the preparation is a composite preparation that reduces the probability of a metabolic marker diagnostic model in plasma, and the product is a food, a probiotic preparation, or a pharmaceutical preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Shown is the basic flow chart of the PB neurotoxicity metabolomics study of the present invention.

[0010] Figure 2 Shown is the detection result of high-resolution mass spectrometry of the sample in Example 1 of the present invention.

[0011] in, Figure 2 A is the total ion current of negative ion full scan obtained by high-resolution mass spectrometry of rat plasma samples; Figure 2 B is the total ion current of positive ion full scan obtained by high-resolution mass spectrometry of rat plasma samples; Figure 2 C is the total ion current of the full scan of negative ions obtained by high-resolution mass spectrometry of rat brain samples; Figure 2 D is the total ion current of the positive ion full scan obtained by high-resolution mass spectrometry of rat brain samples.

[0012] Figure 3 Shown is a multivariate statistical model diagram of rat plasma and brain samples in Example 1 of the present invention.

[0013] in, Figure 3 A is the plasma principal component analysis (PCA) model score graph, Figure 3B is a three-dimensional stereogram of the plasma partial least squares discriminant analysis (PLS-DA) model scores. The results showed that healthy control (HC) and PB neurotoxicity rats could be well distinguished; Figure 3 C is the brain PCA model score graph, Figure 3 D is a three-dimensional image of the brain PLS-DA model score. The results show that HC (healthy control) and PB neurotoxic rats can be well distinguished.

[0014] Figure 4 Shown is an intersection diagram of differentially neurotoxic metabolites found in rat plasma and rat brain, respectively, in Example 1 of the present invention.

[0015] Figure 5 Shown are the receiver operating characteristic (ROC) analysis results of the common metabolite markers in Example 2 of the present invention, including the ROC curve graph and the area under the curve (AUC) value.

[0016] Figure 6 Shown is the ROC curve and AUC analysis results of the shared metabolic marker fitting diagnostic model obtained by Logistic regression analysis in Example 3 of the present invention. DETAILED DESCRIPTION

[0017] Figure 1 Shown is the basic flow chart of the PB neurotoxicity metabolomics study of the present invention.

[0018] The application of this metabolic marker in the preparation of a product for monitoring PB neurotoxicity, wherein the metabolic marker includes LysoPE 20:4 / 0:0, creatine, SM d18:1 / 24:0, SM d18:0 / 24:1, LysoPC 18:0, palmitoyl sphingomyelin, phosphorylcholine, PC 14:0 / 22:1, creatinine, PC 18:3 / 16:0, SM d18:1 / 16:0, sphingosine, choline, LysoPC 18:1, L-carnitine, palmitic acid, PC 18:0 / 20:4, 3-hydroxy-2-octylglutaric acid, L-acetylcarnitine, LysoPC 20:4, LysoPC 16:0, PC 20:1 / 14:1.

[0019] The metabolic markers of the present invention can be effectively used for the early diagnosis of PB neurotoxicity with high sensitivity, rapidity and convenience, accurate and reliable results, which can provide a basis for clinical decision-making and a certain foundation for subsequent basic research and clinical research, and have great application and research value.

[0020] Preferably, the analysis method of the metabolic markers is to obtain a fitted regression curve through logistic regression analysis:

[0021] Y=Logit(p)=20.15a-14.21b+76.60c-51.09d-0.99e-4.34

[0022] Where Y is the diagnosis probability, Logit(p) is the logistic regression function, and ae are LysoPE

[0023] 20:4 / 0:0, Creatine, SM d18:1 / 24:0, SM d18:0 / 24:1, LysoPC 18:0.

[0024] Preferably, the biological sample is plasma, and the level of the metabolite marker in the biological sample is detected by one or more of the following methods: chromatography, spectroscopy, mass spectrometry, chemical analysis, and immunoassay.

[0025] Preferably, the chromatography includes high performance liquid chromatography, thin layer chromatography, and gas chromatography; the spectroscopy includes nuclear magnetic resonance spectroscopy, refractive index spectroscopy, ultraviolet spectroscopy, and near infrared spectroscopy; and the chemical analysis includes electrochemical analysis and radiochemical analysis.

[0026] Preferably, in the chromatography method, the mobile phase: mobile phase A is an aqueous solution containing 0.1% formic acid and 2.5 mmol / L ammonium formate, and the mobile phase D is acetonitrile; the gradient elution program for sample determination is: 0-1.0 min, 95% A; 1.0-5.0 min, 95%-40% A; 5.0-8.0 min, 40%-0% A; 8.0-11.0 min, 0% A; 11.0-14.0 min, 0%-40% A; 14.0-15.0 min, 40%-95% A; 15.0-18.0 min, 95% A, the analysis time is 0-18 min, 5 μL is injected each time, the flow rate is 0.25 mL / min, the chromatographic column is ACQUITY BEH C18 1.7 μm, 2.1×50 mm, and the column temperature is 30°C.

[0027] Preferably, the mass spectrometry is a high-resolution mass spectrometry method, which first uses a chromatographic column for gradient elution, and then collects data in an electrospray ion source ESI positive and negative ion Full scan-ddMS2 mode.

[0028] Preferably, in the mass spectrometry method, the spray voltage is 3000 V; the evaporation temperature is 350°C; the capillary temperature is 350°C; the S-lens RF is 50; the resolution of the first-level full scan is 70000, the scanning range is 70-1050 m / z; the second-level data-dependent scan has the following characteristics: resolution: 17500, AGC target: 1e5, MaximunTT: 50 ms, NCE: 20, 40, 60.

[0029] Preferably, the biological sample is pretreated before detection as follows: 20 μL of plasma is added with 180 μL of a precipitant containing an internal standard, the internal standard is dissolved in methanol and acetonitrile mixed in equal proportions, vortexed for 30 seconds, centrifuged at 12000 rpm for 10 minutes, and the supernatant is aspirated to obtain a test solution for quantitative analysis.

[0030] Also provided is a preparation of a metabolic marker for preparing a product for monitoring PB neurotoxicity, wherein the preparation is a composite preparation that reduces the probability of a metabolic marker diagnostic model in plasma, and the product is a food, a probiotic preparation, or a pharmaceutical preparation.

[0031] Preferably, the probability of the metabolic marker diagnostic model in the plasma of the candidate food, probiotic preparation or pharmaceutical preparation before and after intervention is detected, and screening is performed based on whether the probability is reduced.

[0032] The embodiments of the present invention are described in detail below.

[0033] Example 1

[0034] Screening of common metabolic markers in the plasma and brain of PB neurotoxicity rats and healthy controls (I) Animal studies and sample sources:

[0035] Male Sprague–Dawley rats (n = 49, 6–8 weeks old) were purchased from Sprague-Dawley Biotechnology Co., Ltd. (Beijing). Animal housing conditions included a temperature of 24 ± 2°C, a relative humidity of 50 ± 5%, and a 12-hour day / night cycle. Water and food were available ad libitum. After one week of acclimation to the experimental conditions, the rats were randomly divided into four experimental groups, 24 in each group. Polymyxin B sulfate for injection (Shanghai First Pharmaceuticals Co., Ltd.) was prepared in normal saline and subcutaneously injected twice daily with 0, 3, 6, or 12 mg / kg of polymyxin B sulfate (PB) according to common clinical doses. This treatment was continued for 10 consecutive days, and the rats were sacrificed on the 11th day. Blood was collected using anticoagulant (EDTA) tubes. Plasma was collected by centrifugation at 5000 × g for 5 min at 4°C and stored frozen at −80°C. Brains were quickly removed on ice and stored frozen at −80°C.

[0036] (2) Main reagents:

[0037] LC / MS-grade acetonitrile was purchased from Merck, HPLC-grade methanol was purchased from Merck, and formic acid was purchased from CNW. All other reagents were of commercial analytical grade. Deionized water was prepared using a Milli-Q ultrapure water system from Millipore.

[0038] (III) High-resolution mass spectrometry screening of differential metabolites in plasma and brain:

[0039] 3.1 Sample preparation:

[0040] Sample pretreatment: 20 μL of plasma (healthy control group and PB-treated group) was aspirated and added to 180 μL of precipitant (methanol:acetonitrile = 1:1) containing an internal standard. The mixture was vortexed for 60 seconds and centrifuged at 12,000 rpm for 10 minutes. 100 μL of the aliquot was used for metabolomics analysis. Brain tissue (0 mg / kg group and PB-treated group) was weighed and homogenized with ultrapure water (containing 50% methanol) at a mass-to-volume ratio of 1:10 (w / v) to obtain brain tissue fragments. The aliquots were centrifuged at 5,000 × g for 5 minutes at 4°C. 20 μL of the supernatant was aspirated and added to 180 μL of precipitant (methanol:acetonitrile = 1:1) containing an internal standard. The aliquots were vortexed for 60 seconds and centrifuged at 13,000 rpm for 10 minutes. 100 μL of the aliquots were used for metabolomics analysis.

[0041] 3.2 Chromatographic / mass spectrometry conditions:

[0042] The results were detected by high-resolution mass spectrometer QE-Orbitrap. The mobile phases were: A was an aqueous solution containing 0.1% formic acid and 2.5 mmol / L ammonium formate, and D was acetonitrile. The gradient elution program for sample determination was as follows: 0-1.0 min, 95% A; 1.0-5.0 min, 95%-40% A; 5.0-8.0 min, 40%-0% A; 8.0-11.0 min, 0% A; 11.0-14.0 min, 0%-40% A; 14.0-15.0 min, 40%-95% A; 15.0-18.0 min, 95% A, analysis time 0-18 min, 5 μL injection each time, flow rate 0.25 mL / min, chromatographic column: ACQUITY BEH C18 1.7 μm, 2.1×50 mm, column temperature 30°C, and the temperature of the autosampler was maintained at 4°C. Data were collected in positive and negative electrospray ionization (ESI) modes with a spray voltage of 3000 V, an evaporation temperature of 350°C, a capillary temperature of 350°C, an S-lens RF of 50, a full scan resolution of 70,000, and a scan range of 70–1050 m / z. The full MS / dd-MS2 data-dependent scan had a resolution of 17,500, an AGC target of 1e5, a maximum time interval of 50 ms, and NCEs of 20, 40, and 60.

[0043] High-resolution mass spectrometry results Figure 2 shown.

[0044] (IV) Data processing and statistical analysis:

[0045] Endogenous metabolites were identified using high-resolution mass spectrometry (mzCloud) to obtain the exact mass to five decimal places for each endogenous metabolite. Each endogenous metabolite was identified by its molecular formula. Compound Discover software then automatically searched a self-built library and publicly available online databases for metabolite name annotation. Subsequently, the metabolite matrix was analyzed using MetaboAnalyst 5.0, and PCA and PLS-DA models were plotted to identify differences in metabolic patterns and significant categorical trends between healthy controls and rats with PB neurotoxicity. Finally, differential metabolic markers were selected using a VIP > 1 criterion.

[0046] (V) Results:

[0047] 5.1 Establishment of PB neurotoxicity rat model

[0048] Approximately 10 minutes after subcutaneous injection of PB sulfate, rats developed redness and swelling throughout their bodies, most notably in the head, ears, limbs, and tail. They also developed ataxia, lethargy, and excessive drinking, with symptoms becoming more pronounced with higher doses. One rat in the high-dose PB sulfate group (12 mg / kg) died after the first dose, possibly due to respiratory failure induced by PB. These symptoms are consistent with PB neurotoxicity.

[0049] 5.2 Screening of shared markers in rat plasma and brain

[0050] Metabolomics total ion current chromatograms of rat plasma and brain are shown in Figure 2 MetaboAnalyst5.0 website was used to calculate PCA and PLS-DA. PCA( Figure 3 A) Results show the difference between the plasma of the 0 mg / kg group and the plasma of the PB neurotoxicity group, PLS-DA ( Figure 3 B) The results showed that the plasma metabolites of the 0 mg / kg group and the PB neurotoxicity group could be completely separated. PCA ( Figure 3 C) Results show the differences between the brain of the 0 mg / kg group and the brain of the PB neurotoxicity group, PLS-DA ( Figure 3 D) The results showed that the brain metabolites of the 0 mg / kg group and the PB neurotoxicity group were completely separated. At the same time, VIP>1 endogenous substances were selected as the main differential metabolites, and 56 differential metabolites were found in rat plasma and 86 differential metabolites in the brain. Plasma and brain common marker screening was performed, and a total of 23 common markers were obtained (see Figure 4 ).

[0051] Table 1 shows the change rates of common differential metabolites in plasma obtained using the PLS-DA model. The results in Table 1 show that in rat plasma, compared with the 0 mg / kg group, the proportions of 11 endogenous substances in the PB neurotoxicity group mainly increased, while the other 12 showed a downward trend. Table 2 shows the change rates of differential metabolites in the brain obtained using the PLS-DA model. The results in Table 2 show that in the rat brain, compared with the 0 mg / kg group, the proportions of 4 endogenous substances in the PB neurotoxicity group mainly increased, while the other 19 showed a downward trend. The results indicate that PB neurotoxicity affects the metabolic secretion of endogenous substances in plasma and brain, causing significant changes in their content. The main components that changed were lipids, organic acids, and carnitine substances.

[0052] Table 1

[0053]

[0054]

[0055] Table 2

[0056]

[0057]

[0058] Example 2

[0059] Confirmation of shared metabolic markers of PB neurotoxicity in plasma and brain

[0060] (1) Data statistics

[0061] In a healthy control group and a 12 mg / kg (high-dose) PB-administered group, 23 shared metabolite markers were further evaluated for their potential as early diagnostic biomarkers for PB neurotoxicity based on receiver operating characteristic (ROC) curves. Using SPSS software, group was set as the state variable and the target plasma metabolite intensity as the test variable. After calculating the ROC curve, SPSS generated an output report containing the ROC curve graph and area under the curve (AUC) value. An AUC greater than 0.6 was considered diagnostic.

[0062] (2) Results analysis

[0063] The ROC curve analysis results of the healthy control group and the high-dose PB administration group showed that the AUC values of 22 metabolites were all greater than 0.6, as shown in Table 3. Figure 5 , indicating that these 22 metabolites classified the data of healthy control and high-dose PB administration groups well, could accurately distinguish healthy and PB neurotoxic rats, and could serve as potential metabolic markers.

[0064] Table 3 ROC parameters of shared differential metabolites

[0065]

[0066]

[0067] Example 3

[0068] Establishment of PB neurotoxicity diagnostic model

[0069] (1) Data statistics

[0070] 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 indicated that the model fit was poor, otherwise the model fit was good.

[0071] (2) Results analysis

[0072] The common metabolic markers confirmed in Example 2 were subjected to logistic regression analysis to obtain a fitted regression curve, i.e., the diagnostic model: Y = Logit (p) = 20.15a-14.21b + 76.60c-51.09d-

[0073] 0.99e-4.34 (Y is the diagnosis probability, Logit(p) is the logistic regression function, ae is LysoPE 20:4 / 0:0, creatine, SM d18:1 / 24:0, SM d18:0 / 24:1, LysoPC 18:0. After fitting, the area under the ROC curve (AUC) in the comparison between the normal neurological function group and the PB neurotoxicity group was 1.00 ( Figure 6 ), with a 95% confidence interval of 0.86-1.00, which significantly improved the diagnostic efficacy of a single marker.

[0074] The Hosmer-Lemeshow test showed P = 0.60, indicating that the model fit was good.

[0075] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of protection of the technical solution of the present invention.

Claims

1. Application of a metabolic marker in the preparation of a product for monitoring PB neurotoxicity, characterized by: The metabolic markers include LysoPE 20:4 / 0:0, creatine, SMd18:1 / 24:0, SMd18:0 / 24:1, LysoPC 18:0, palmitoylsphingomyelin, phosphocholine, PC 14:0 / 22:1, creatinine, PC 18:3 / 16:0, SMd18:1 / 16:0, sphingosine, choline, LysoPC 18:1, L-carnitine, palmitic acid, PC 18:0 / 20:4, 3-hydroxy-2-octylglutaric acid, L-acetylcarnitine, LysoPC 20:4, LysoPC 16:0, PC 20:1 / 14:1; The analysis method of metabolic markers was performed by logistic regression analysis to obtain the fitted regression curve: Y = Logit (p) = 20.15a-14.21b + 76.60c-51.09d-0.99e-4.34 Where Y is the diagnosis probability, Logit(p) is the logistic regression function, ae are LysoPE 20:4 / 0:0, creatine, SMd18:1 / 24:0, SMd18:0 / 24:1, and LysoPC 18:0, respectively; the biological sample is plasma.

2. Use of the metabolic marker according to claim 1 in the preparation of a product for monitoring PB neurotoxicity, characterized in that: The levels of metabolic markers in biological samples are detected by one or more of the following methods: chromatography, spectroscopy, mass spectrometry, chemical analysis, and immunoassay.

3. Use of the metabolic marker according to claim 2 in the preparation of a product for monitoring PB neurotoxicity, characterized in that: The chromatography method includes high performance liquid chromatography, thin layer chromatography, and gas chromatography; the spectroscopy method includes nuclear magnetic resonance spectroscopy, refractive index spectroscopy, ultraviolet spectroscopy, and near infrared spectroscopy; and the chemical analysis method includes electrochemical analysis and radiochemical analysis.

4. Use of the metabolic marker according to claim 3 in the preparation of a product for monitoring PB neurotoxicity, characterized in that: The mobile phases in the chromatography method are: mobile phase A is an aqueous solution containing 0.1% formic acid and 2.5 mmol / L ammonium formate, and mobile phase D is acetonitrile; the gradient elution program for sample determination is: 0-1.0 min, 95% A; 1.0-5.0min, 95%-40%A; 5.0-8.0 min, 40%-0% A; 8.0-11.0 min, 0% A; 11.0-14.0min, 0%-40%A; 14.0-15.0min, 40%-95%A; 15.0-18.0 min, 95% A, analysis time 0-18 min, 5 μL injection each time, flow rate 0.25 mL / min, chromatographic column: ACQUITY BEH C18 1.7 μm, 2.1×50 mm, column temperature 30°C.

5. Use of the metabolic marker according to claim 2 in the preparation of a product for monitoring PB neurotoxicity, characterized in that: The mass spectrometry method is a high-resolution mass spectrometry method, which first uses a chromatographic column for gradient elution, and then collects data in an electrospray ion source ESI positive and negative ion Full scan-ddMS2 mode.

6. Use of the metabolic marker according to claim 5 in the preparation of a product for monitoring PB neurotoxicity, characterized in that: In the mass spectrometry, the spray voltage was 3000 V; the evaporation temperature was 350°C; the capillary temperature was 350°C; the S-lens RF was 50; and the first-stage full scan Resolution: 70,000, Scan range: 70-1050 m / z; Secondary data-dependent scan: Resolution: 17500, AGC target: 1e5, Maximun TT: 50ms, NCE: 20, 40, 60.

7. Use of the metabolic marker according to claim 1 in the preparation of a product for monitoring PB neurotoxicity, characterized in that: The biological sample was pretreated before testing as follows: 20 μL of plasma was added with 180 μL of a precipitant containing an internal standard, the internal standard was dissolved in methanol and acetonitrile mixed in equal proportions, vortexed for 30 seconds, centrifuged at 12,000 rpm for 10 minutes, and the supernatant was aspirated to obtain the test solution for quantitative analysis.

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