Liver reserve function assessment product and use of doxofylline in the assessment product

Abstract

The present application relates to the biomedical detection technical field, disclose the liver reserve function evaluation product and the application of doxofylline in the evaluation product, the method is continuously intravenous infusion doxofylline to the subject, collects at least three time point blood samples during infusion, adopts liquid chromatography-mass spectrometry to detect the concentration of its metabolites theophylline acetaldehyde, theophylline acetic acid, hydroxyethyl theophylline and theophylline acetic acid-2' hydroxyethyl ester, based on the change data of total molar concentration of metabolite with time, the linear slope is calculated in combination with enzyme kinetics principle, and then the liver metabolic reserve function is detected.The present application also provides a complete detection system, the related use and pharmaceutical composition of doxofylline.The scheme is high in liver specificity, fast in detection, convenient in operation, excellent in safety, and superior in sensitivity to classical ICG clearance test, can solve the limitation of existing evaluation method, is suitable for preoperative risk assessment of hepatobiliary surgery, chronic liver disease staging and other scenes, and has important clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical testing technology, specifically to a liver reserve function assessment product and the application of doxophylline in the assessment product. Background Technology

[0002] Accurate assessment of liver reserve function is a crucial step in the clinical diagnosis and treatment of liver diseases, playing a vital role in disease staging, surgical planning, prognosis, and individualized medication guidance. Currently used methods for assessing liver reserve function have several limitations: comprehensive assessment systems such as the Child-Pugh score rely on multiple clinical indicators, resulting in insufficient precision; static biochemical indicators such as serum albumin only reflect the immediate functional state of the liver and cannot reflect its functional potential; imaging examinations such as CT and MRI only reflect the structural volume of the liver, which is not equivalent to functional volume and cannot accurately assess metabolic function. The indocyanine green clearance test is considered a classic standard, but this method mainly reflects the liver's uptake and excretion functions, insufficiently assessing the liver's core metabolic capacity, and conflicts with its application in intraoperative fluorescence navigation for liver cancer, limiting its use in some clinical scenarios. Cytochrome P450 1A2 (CYP1A2) is a liver-specific metabolic enzyme, and studies have confirmed a significant correlation between CYP1A2 activity and liver metabolic reserve function. Clinical studies have shown that LiMAx, based on in vivo CYP1A2 enzyme activity assessment, has excellent predictive efficacy for liver failure and related mortality events, with a receiver operating characteristic (AUROC) of 0.99, making it one of the optimal methods for assessing liver reserve function. However, it suffers from high costs, potential metabolic toxicity, and insufficient population data, hindering its widespread adoption. Therefore, developing a safe, inexpensive, and rapid in vivo CYP1A2 activity assessment technology is crucial. This would improve the accuracy and specificity of liver reserve function assessment while reducing medical costs, providing a significant breakthrough for clinical translation in this field. It is particularly important for China, where liver cancer is highly prevalent, and holds urgent practical significance and clear research value.

[0003] The performance of the probe substrate is a core factor determining the accuracy, safety, and practicality of in vivo CYP1A2 activity assessment. An ideal in vivo CYP1A2 probe substrate must simultaneously meet two core criteria: high enzyme specificity and high safety. Currently, probes used clinically to assess in vivo CYP1A2 enzyme activity mainly include caffeine, melatonin, theophylline, and phenacetin. However, these probes all have significant defects and cannot simultaneously meet the above criteria, limiting their application in assessing liver reserve function. In addition to the inherent defects of probe substrates, existing in vivo enzyme activity assessment methods themselves also have fundamental flaws, failing to accurately characterize the true activity state of the enzyme. Currently, commonly used clinical methods for assessing in vivo CYP1A2 activity are mainly based on probe substrates and rely on single-point steady-state methods or the area under the plasma concentration-time curve (AUC) method. Both of these methods have fundamental flaws and are difficult to accurately characterize the true activity of the enzyme. While single-point methods are simple to operate, they rely on the ratio of metabolite to substrate concentration at a single time point, neglecting the dynamic and asynchronous nature of drug absorption, distribution, metabolism, and excretion in vivo. This ratio is easily affected by various factors at the moment of sampling and cannot reflect the overall or true state of enzyme activity. The AUC method calculates the AUC ratio of metabolite to substrate over a period of time, covering a longer observation window. However, its result is essentially a static cumulative measure of the combined effects of multiple processes such as drug distribution, metabolism, and excretion, rather than the dynamic metabolic rate itself. Especially when the excretion characteristics of substrates or products differ, the ratio is prone to bias and cannot analyze changes in metabolic rate over time. In summary, existing methods do not use the core "metabolite formation rate" as a direct indicator, leading to inherent limitations in assessment. Therefore, developing a novel evaluation method that directly and dynamically measures metabolic rate is of great significance for accurately reflecting CYP1A2 activity in vivo and accurately assessing liver reserve function. Summary of the Invention

[0004] This invention aims to provide a liver reserve function assessment product and the application of doxophylline in the assessment product, in order to solve the problems of insufficient specificity, long detection time, poor accuracy, and safety risks in existing liver metabolic reserve function detection methods. It provides a liver reserve function assessment product, related applications of doxophylline, a liver reserve function assessment system and its usage method, so as to achieve rapid, accurate and safe assessment of liver metabolic reserve function and meet the clinical needs for quantitative detection of liver metabolic reserve function.

[0005] A product for assessing liver reserve function, the product being a kit containing doxophylline as a probe for detecting liver reserve function.

[0006] Preferably, as an improvement, the kit further includes standards or detection reagents for the quantitative detection of doxophylline metabolites, wherein the metabolites are selected from at least one of theophylline acetaldehyde (TA), theophylline acetic acid (TAA), hydroxyethyl theophylline (ETO), and theophylline acetic acid-2'-hydroxyethyl ester (HET); and the detection reagents include reagents required for detection by liquid chromatography-tandem mass spectrometry.

[0007] Use of a doxophylline or a pharmaceutical composition containing doxophylline in the preparation of formulations for any of the following purposes: (i) Assess the patient’s liver function tolerance before performing hepatectomy; (ii) To guide individualized dosage adjustments for one or more drugs metabolized by hepatic cytochrome P450 enzymes; (iii) Assess liver metabolic reserve function during health checkups or screening of high-risk groups.

[0008] Preferably, as an improvement, the formulation is administered via continuous intravenous infusion, and liver reserve function is assessed during infusion by monitoring changes in the concentration of doxophylline metabolites, including theophylline acetaldehyde (TA), theophylline acetic acid (TAA), hydroxyethyl theophylline (ETO), and theophylline acetic acid-2'-hydroxyethyl ester (HET).

[0009] A liver reserve function assessment system, comprising: The infusion control module is used to continuously administer doxophylline to the subject via intravenous infusion; The analysis module is used to receive a series of biological samples from the subject and determine the concentration of theophylline metabolites therein, including theophylline acetaldehyde (TA), theophylline acetic acid (TAA), hydroxyethyl theophylline (ETO), and theophylline acetic acid-2'-hydroxyethyl ester (HET). The data processing module is used to receive the concentration data, calculate the kinetic parameters reflecting the rate of metabolite formation, and generate a liver reserve function assessment report based on the parameters.

[0010] Preferably, as an improvement, the infusion control module is an injection pump, the analysis module is a liquid chromatography-tandem mass spectrometry system, and the data processing module is built-in based on the relationship Slope=(1 / 2). CLint kin The algorithm for T is as follows: CLint is the intrinsic clearance rate of liver enzymes, kin is the infusion rate of doxophylline, and T is the infusion time.

[0011] A method of using a liver function assessment system according to any of the above includes the following steps: (1) Doxophylline is continuously infused into the subject via the infusion control module; (2) Collect biological samples at at least three different time points within the time interval between the start and end of the infusion; (3) The concentration of the theophylline metabolites in each biological sample is detected by the analysis module, and the total molar concentration is calculated; (4) Calculate the kinetic parameters through the data processing module and generate a liver reserve function assessment report.

[0012] Preferably, as an improvement, the duration of continuous intravenous infusion in step (1) is 1-15 minutes, and the doxophylline is administered at a detection-specific dosage (10 mg / min~500 mg / min); the time interval for collecting biological samples in step (2) is 1-5 minutes, and the biological samples are blood, plasma or serum.

[0013] Preferably, as an improvement, the total molar concentration in step (3) is the sum of the molar concentrations of theophylline acetaldehyde (TA), theophylline acetic acid (TAA), hydroxyethyl theophylline (ETO), and theophylline acetic acid-2'-hydroxyethyl ester (HET).

[0014] Preferably, as an improvement, the kinetic parameter in step (4) is the linear slope of the total molar concentration-time curve, which is obtained by least squares linear regression fitting.

[0015] Advantages of this invention: 1. A groundbreaking, safe, and specific probe: For the first time, doxophylline (DOXO), a respiratory drug already safely used clinically, has been innovatively developed into a specific diagnostic probe for assessing hepatic metabolic reserve function. This breakthrough fundamentally solves the problems of insufficient hepatic specificity, narrow therapeutic window, and potential toxicity commonly found in traditional probes (such as caffeine, phenacetin, and theophylline), while also avoiding the drawbacks of novel probes (such as...). 13 C-Methaxetine has limitations such as high cost and potential metabolic toxicity, achieving a balance between CYP1A2 metabolic specificity and excellent clinical safety.

[0016] 2. A new paradigm for dynamic and direct assessment was established: Abandoning the traditional indirect, static assessment model based on "candidate drug clearance," a novel direct and dynamic assessment method based on the "initial rate of metabolite formation" was pioneered. By measuring the linear slope of the total metabolite concentration during short-term infusion, the activity of hepatic CYP1A2 enzyme (characterized by CLint) can be directly and quantitatively reflected. This theoretically realizes a dynamic rate-based measurement of the liver's metabolic reserve capacity, with sensitivity superior to the classic ICG clearance assay.

[0017] 3. A rapid and efficient clinical assessment process has been achieved: the entire assessment process can be completed within a very short window (10 minutes), realizing a "rapid and quick" assessment of liver reserve function. This method is easy to operate and perfectly avoids conflicts with ICG fluorescence navigation during liver cancer surgery. At the same time, it has a significant cost advantage compared with similar foreign technologies (such as LiMAx), providing clinicians with a more efficient and economical new option.

[0018] 4. A complete and original technical system has been constructed: This invention is not an improvement on a single technical point, but rather the construction of a complete technical closed loop, from the original probe (a new application of doxophylline DOXO), to the core algorithm (slope kinetic model), and then to the integrated system (infusion-detection-analysis). This forms a standardized and scalable original solution for the precise assessment of liver reserve function, laying a solid foundation for applications in multiple scenarios such as hepatobiliary surgery, personalized medicine, and health management. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the metabolism of DOXO in microsomes of different human organs according to an embodiment of the present invention; Figure 2 This is a schematic diagram illustrating the metabolism of DOXO in homogenates from different organs of rats according to an embodiment of the present invention. Figure 3 The metabolite formation rates under different 1-ABT inhibitors in the embodiments of the present invention; Figure 4 This is an example of the changes in organ damage biochemical indicators in rats before and after administration of DOXO in this invention. Figure 5 For pathological observation of major organs in rats continuously administered saline and DOXO; Figure 6 Schematic diagram of the surgical sites for liver resection in different groups of rats; Figure 7 The results of liver reserve function assessment in rats with different degrees of hepatectomy were obtained using the metabolite slope method. Figure 8 The results of ICG assay for assessing liver reserve function in rats with different degrees of hepatectomy. Figure 9 Correlation analysis of the slope method and ICG clearance rate in liver reserve assessment in hepatectomized rats; Figure 10 Body weight changes during the rat liver fibrosis modeling process; Figure 11 Plasma biochemical parameters of the CCl4-treated group and the control group after 4 and 8 weeks of continuous intervention; Figure 12 The liver morphology and liver pathology were observed in the CCl4-treated group and the control group. Figure 13 The liver organ indexes of the CCl4-treated group and the control group were measured after 4 and 8 weeks of continuous intervention. Figure 14 The activity of CYP1A2 enzyme in rats in the control group and rats treated with CCl4 for 4 and 8 weeks was measured. Figure 15 The control group was compared with the rats in the CCl4 intervention groups for 4 weeks and 8 weeks for ICG-R15. Figure 16 Correlation analysis of slope and ICG-R15 in rats with liver fibrosis based on DOXO assessment. Detailed Implementation

[0020] The technical solutions and effects of the present invention are described in detail below through specific embodiments. All embodiments are based on the detection methods, detection systems and related principles disclosed in the present invention. Where no specific technology or conditions are specified in the experiment, the technology or conditions described in the literature in this field shall be followed. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased through formal channels.

[0021] Example 1: Organ attribution of doxophylline (DOXO) metabolism Experimental objective: To clarify the organ-specific metabolism of DOXO in vivo, verify whether it is specifically metabolized by the liver, and provide key evidence for using DOXO as a liver-specific functional probe.

[0022] Experimental materials: human liver microsomes (HLM), human intestinal microsomes (HIM), human kidney microsomes (HKM), healthy SD rats, doxophylline (DOXO), theophylline acetaldehyde (TA) standard, phosphate buffer, NADPH regeneration system, liquid chromatography-tandem mass spectrometry (LC-MS / MS), tissue homogenizer, high-speed refrigerated centrifuge.

[0023] Experimental methods: DOXO was incubated with human liver microsomes (HLM), human intestinal microsomes (HIM), and human kidney microsomes (HKM) for 60 minutes respectively. Homogenizes of major rat organs, including heart, liver, spleen, lung, kidney, stomach, intestine, and brain, were prepared, and DOXO was incubated with each organ homogenate for 60 minutes. After incubation, the reaction was terminated with acetonitrile, the supernatant was collected by centrifugation, and the formation of metabolite TA was detected by LC-MS / MS.

[0024] Experimental results: The generation of its characteristic initiating metabolite TA was detected only in the human liver microsome (HLM) incubation system. Figure 1 No DOXO metabolites were detected in human intestinal microsomes (HIM) or human kidney microsomes (HKM); In rat tissue homogenate incubation experiments, TA production was observed only in liver tissue homogenate; no related metabolites were detected in homogenates from other organs. Figure 2 ).

[0025] Experimental conclusion: The initiation of DOXO metabolism is strictly and specifically located in the liver, and other organs and tissues do not participate in this metabolic step, which provides the core basis for its use as a liver-specific functional probe.

[0026] Example 2: Establishment and Validation of Mathematical Model for Enzyme Activity Evaluation To verify the accuracy and specificity of the quantitative model of "linear slope of total metabolite concentration-time curve and intrinsic clearance rate of liver enzymes", this invention constructs a quantitative detection model of liver enzyme activity based on the kinetics of the initial stage of continuous intravenous injection, the derivation process of which is as follows: Simplified enzyme kinetics: According to the Michaelis-Menten equation, when the substrate concentration [S] in vivo is much lower than its K... m When the value is ([S]< <K m ), substrate enzymatic elimination rate (V elim This can be simplified to a first-order dynamic equation: V elim =(V max [S]) / K m , intrinsic clearance rate of enzymes (CL) int =V max / K m (This represents the catalytic efficiency of the enzyme), then: V elim =CL int S; Substrate accumulation kinetics: Within a short initial time window T following the initiation of continuous intravenous injection, the substrate concentration [S] in vivo can be approximated as linearly accumulating over time: [S]=k in t(Formula 2), Where, k in For a constant infusion rate, t is the time after the infusion begins; Metabolite formation kinetics: DOXO is specifically metabolized by liver enzymes to produce TA, which is then rapidly converted to TAA and ETO. Therefore, the rate of total metabolite formation, d[P_total] / dt, is numerically equal to the initial substrate metabolic rate V initiated by hepatic P450. elim Combining formulas 1 and 2, we get: d[P_total] / dt=V elim =CL int [S]=C Lint k in t(Formula 3); Total metabolite concentration integral: Integrating both sides of Equation 3, we obtain the total concentration of metabolites, P_total, at infusion time t: P_total=∫0 t (CL int k in dt = (1 / 2) CL int k in t 2 (Formula 4) Formula 4 shows that in the initial short period of infusion, the concentration of total metabolites is approximately proportional to the square of time.

[0027] Quantitative relationship between linear slope and enzyme activity: In practical applications, we collect blood samples at multiple time points within a preset short time window T. Within this finite time, the concentration-time curve of total metabolites (P_total~t) follows a square relationship and can be approximated as a straight line with high precision near the origin. The slope (Slpoe) of this line can be calculated using the following formula: Slpoe = ([P_total](T) - [P_total](0)) / (T-0) Substituting the value of Formula 4 at t=T, we obtain the core quantitative relationship of this invention: Slpoe=[(1 / 2) CL int k in T 2 ] / T=(1 / 2) CL int k in T(Formula 5) Since the infusion time T is a preset constant, Slpoe is related to the enzyme's intrinsic clearance rate CL. int and infusion rate k in The product of is directly proportional, that is: Slpoe∝CL int k in , Because doxophylline (DOXO) initiates the entire metabolic pathway through liver-specific metabolism, and k in T is a precisely controlled parameter; therefore, the Slpoe measured by the method of this invention can directly and quantitatively reflect the metabolic activity of the liver in vivo (i.e., CL). int This method enables rapid, dynamic, and highly sensitive assessment of enzyme activity by capturing the initial generation kinetics of total metabolites over a short time span.

[0028] Experimental objective: To verify the accuracy and specificity of the quantitative model of "the linear slope of the total metabolite concentration-time curve and the intrinsic clearance rate of liver enzymes".

[0029] Experimental materials: human liver microsomes, doxophylline (DOXO) and its metabolite standards, CYP broad-spectrum inhibitor 1-ABT, NADPH regeneration system, liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0030] Experimental methods: Set up a 1-ABT concentration gradient (0, 2, 20, 100, 200 mg / mg protein), and add doxophylline after pre-incubating different concentrations of 1-ABT with liver microsomes. After 60 minutes of incubation, the total concentration of the four metabolites was measured, and the dependence of the metabolite generation rate on the 1-ABT concentration was analyzed.

[0031] Experimental results: With increasing 1-ABT concentration, the total production rate of the four metabolites decreased significantly in a dose-dependent manner. Figure 3 When the concentration of 1-ABT reached 200 mg / mg protein, the rate of metabolite formation decreased to less than 5% of that in the control group (0 mg / mg 1-ABT).

[0032] Experimental conclusion: The detection signal is indeed specifically mediated by the CYP enzyme system, which verifies the accuracy and specificity of the mathematical model and shows that the method based on continuous intravenous injection of DOXO and calculation of metabolite slope can achieve dynamic and quantitative assessment of liver metabolic activity in vivo.

[0033] Example 3: Safety Evaluation Experimental objective: To evaluate the safety of DOXO as a probe drug and to examine its effects on the body at diagnostic doses.

[0034] Experimental materials: healthy SD rats, doxophylline (DOXO) injection, physiological saline, biochemical test reagents, pathological section reagents, infusion pump, biochemical analyzer, microscope.

[0035] Experimental methods: Rats were randomly divided into an experimental group (continuous intravenous infusion of DOXO) and a control group (continuous intravenous infusion of physiological saline). Rat serum was collected before and after drug administration to detect core indicators representing hepatocyte integrity (AST, ALT) and key parameters of renal function (BUN, UUN). After administration, the rats were sacrificed, and the major organs were grossly dissected and observed. Pathological sections were prepared to observe the morphological changes in the tissues.

[0036] Experimental results: Biochemical analysis showed that there were no statistically significant changes in the concentrations of AST, ALT, BUN, and UUN in rat serum before and after continuous intravenous injection of DOXO. Figure 4 ); Histopathological examination confirmed that the organs and tissues of the experimental group rats were structurally intact, with normal cell morphology, and no drug-related pathological changes (such as cell necrosis, inflammatory infiltration, etc.) were found, consistent with the organ pathological manifestations of the control group. Figure 5 ).

[0037] Experimental conclusion: Under the diagnostic dosage and administration regimen described in this invention, DOXO exhibits good safety characteristics, causes no significant damage to the body, and meets the safety requirements for in vivo probe drugs.

[0038] Example 4: Validation of rat hepatectomy reserve function assessment based on DOXO slope method Experimental objective: To verify the sensitivity of the method of the present invention in detecting the decline of liver reserve function, and to compare it with the traditional ICG clearance test to evaluate the reliability of the method.

[0039] Experimental materials: healthy SD rats, doxophylline (DOXO) injection, ICG reagent, liquid chromatography-tandem mass spectrometry (LC-MS / MS), ICG detector, surgical instruments.

[0040] Experimental methods: Rats were randomly divided into a sham-operated group, a 30% hepatectomy group, and a 38% hepatectomy group. In the hepatectomy group, resection was performed by ligating the corresponding hepatic lobe's vascular system (resection sites are shown in the diagram). Figure 6 ); After 10 days of postoperative feeding and once liver function had stabilized, the metabolite slope method of this invention was used for evaluation: Doxophylline was infused for 10 minutes, and blood samples were collected at multiple time points during the infusion. The total concentration of metabolites was detected by LC-MS / MS, and a linear slope was fitted. Simultaneously conduct ICG clearance experiments, calculate ICG clearance rates, and analyze the correlation between the two sets of data.

[0041] Experimental results: The method of this invention showed statistically significant differences in the linear slopes among the sham surgery group, the 30% resection group, and the 38% resection group (P<0.001 or P<0.05). Figure 7 This indicates that the method can sensitively distinguish between different degrees of decreased liver reserve function; ICG method: It can only distinguish between the sham surgery group and the resection group (P<0.001), but cannot distinguish the functional differences between the 30% and 38% resection groups. Figure 8 ); Correlation analysis: The slope method of this invention has a good negative correlation with ICG clearance rate (r=-0.8298, P<0.001). Figure 9 ).

[0042] Experimental conclusion: The assessment method provided by this invention is superior to the traditional ICG method in terms of detection sensitivity, can identify more subtle changes in liver reserve function, and the results are reliable, providing a new technical means for the accurate clinical assessment of liver reserve function.

[0043] Example 4: Validation of Liver Reserve Function Assessment in Liver Fibrosis Based on DOXO Slope Method Experimental objective: To verify the applicability and sensitivity of the DOXO slope method of this invention in quantitatively assessing liver reserve function in a liver fibrosis pathological model, and to conduct comparative and correlation analysis with the traditional ICG clearance test as a reference standard.

[0044] Experimental materials: healthy SD rats, carbon tetrachloride (CCl4, used to induce liver fibrosis), olive oil (solvent control), doxorubicin (DOXO) injection, indocyanine green (ICG) reagent, liquid chromatography-tandem mass spectrometry (LC-MS / MS), ICG detector, fully automated biochemical analyzer (detecting ALT and AST), histopathological staining reagents (HE, Masson's red, Sirius red), surgical and sampling instruments.

[0045] Experimental methods: Animal modeling and grouping: A rat liver fibrosis model was induced by subcutaneous injection of CCl4. A control group (olive oil solution), a 4-week intervention group (moderate fibrosis), and an 8-week intervention group (severe fibrosis) were established. Body weight was monitored weekly.

[0046] Endpoint indicator detection: At the end of the 4th and 8th weeks of modeling, blood was collected to detect plasma ALT and AST levels; after the animals were sacrificed, the liver was weighed to calculate the organ index, and macroscopic morphological observation and histopathological staining (HE, Masson, Sirius red) were performed to confirm the degree of fibrosis.

[0047] Liver reserve function assessment: The DOXO slope method of this invention: DOXO is intravenously infused, and a series of blood samples are collected during the infusion. The concentration of its specific metabolites is determined by LC-MS / MS, and the linear slope of the metabolite concentration-time curve is fitted to characterize the CYP1A2 enzyme activity.

[0048] Traditional ICG clearance test: ICG is injected intravenously, blood is collected at different time points after injection, plasma ICG concentration is measured, and the 15-minute retention rate (ICG-R15) is calculated.

[0049] Data analysis: The differences between DOXO metabolic slope and ICG-R15 among the groups were compared, and linear correlation analysis was performed on the two.

[0050] Experimental results: Model validation: Compared with the control group, the weight gain of rats in the model group was slower ( Figure 10 Plasma ALT and AST levels increased significantly in a time-dependent manner (reaching 2.9 / 2.3 times and 7.7 / 4.7 times the control level at 4 weeks and 8 weeks, respectively). Figure 11 The liver's macroscopic morphology deteriorated, and pathological staining confirmed moderate fibrosis at 4 weeks and severe fibrosis at 8 weeks. Figure 12 ), liver organ index increased ( Figure 13 ).

[0051] Functional assessment results: DOXO slope method: The CYP1A2 enzyme activity (expressed as slope) in the model group was significantly lower than that in the control group (P<0.001), and the activity in the 8-week group was further significantly lower than that in the 4-week group (P<0.001). Figure 14 This indicates that the method can sensitively distinguish between different degrees of liver reserve function impairment.

[0052] ICG clearance test: ICG-R15 in the model group was significantly higher than that in the control group (P<0.001), and the 8-week group was higher than that in the 4-week group (P<0.001). Figure 15 ).

[0053] Correlation analysis: The DOXO metabolic slope was highly significantly negatively correlated with ICG-R15 (r = -0.9257, P<0.0001). Figure 16 This indicates that the two methods are in good agreement when assessing liver fibrosis reserve function.

[0054] Experimental Conclusion: In a liver fibrosis pathological model, the DOXO slope method of this invention can effectively and sensitively quantitatively assess the progressive decline in liver reserve function as fibrosis worsens, and the results are highly correlated with the traditional ICG-R15 index. This confirms that this method is not only applicable to acute liver injury models, but also has reliable application value in chronic progressive liver diseases such as liver fibrosis, providing a new potential tool for clinically assessing liver reserve function in patients with chronic liver disease.

[0055] The above four embodiments comprehensively verify the feasibility, accuracy, and safety of the present invention from the perspective of core technology: Embodiment 1 confirms that DOXO has strict liver-specific metabolic characteristics, laying the foundation for its use as a probe for liver function testing; Embodiment 2 verifies the scientific validity of the quantitative model established in this invention, which relates the slope of the "metabolite concentration-time curve to the intrinsic clearance rate of liver enzymes," ensuring the accuracy of the detection principle; Embodiment 3 clarifies the excellent safety of DOXO at a specific detection dose, eliminating safety concerns in clinical applications; Embodiment 4, through a comparative experiment using a liver resection model, demonstrates that the detection method of this invention is significantly superior to the classic ICG clearance test in terms of sensitivity and reliability, and can accurately distinguish different degrees of changes in liver reserve function. In summary, the detection method, detection system, and related applications of this invention have undergone multi-dimensional experimental verification, are technically mature, and have sufficient data support, possessing the core conditions for clinical promotion and application, and can provide a precise, rapid, and safe new technical solution for assessing liver reserve function.

[0056] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A liver reserve function assessment product, characterized in that, The product is a kit containing doxophylline as a probe for detecting liver reserve function.

2. The liver function assessment product according to claim 1, characterized in that: The kit also includes standards or assays for the quantitative detection of doxophylline metabolites, wherein the metabolites are selected from at least one of theophylline acetaldehyde (TA), theophylline acetic acid (TAA), hydroxyethyl theophylline (ETO), and theophylline acetic acid-2'-hydroxyethyl ester (HET); and the assays include reagents required for detection by liquid chromatography-tandem mass spectrometry.

3. Use of a doxophylline or a pharmaceutical composition comprising doxophylline in the preparation of formulations for any of the following purposes: (i) Assess the patient’s liver function tolerance before performing liver resection surgery; (ii) To guide individualized dosage adjustments for one or more drugs metabolized by hepatic cytochrome P450 enzymes; (iii) Assess liver metabolic function during health checkups or screening of high-risk groups.

4. The use of the theophylline or a pharmaceutical composition containing theophylline according to claim 3, characterized in that: The formulation is administered via continuous intravenous infusion, and liver function is assessed by monitoring changes in the concentration of theophylline metabolites during infusion. These metabolites include theophylline acetaldehyde (TA), theophylline acetic acid (TAA), hydroxyethyl theophylline (ETO), and theophylline acetic acid-2'-hydroxyethyl ester (HET).

5. A liver reserve function assessment system, characterized in that, include: The infusion control module is used to continuously administer doxophylline to the subject via intravenous infusion; The sample processing and analysis module is used to receive a series of biological samples from the subjects and determine the concentration of theophylline metabolites therein, including theophylline acetaldehyde (TA), theophylline acetic acid (TAA), hydroxyethyl theophylline (ETO), and theophylline acetic acid-2'-hydroxyethyl ester (HET). The data processing module is used to receive the concentration data, calculate the kinetic parameters reflecting the rate of metabolite formation, and generate a liver function assessment report based on the parameters.

6. A liver function assessment system according to claim 5, characterized in that: The infusion control module is an injection pump, the sample analysis module is a liquid chromatography-tandem mass spectrometry system, and the data processing module is based on the relationship Slope=(1 / 2). CLint kin The algorithm for T is as follows: CLint is the intrinsic clearance rate of liver enzymes, kin is the infusion rate of doxophylline, and T is the infusion time.

7. A method of using a liver reserve function assessment system according to any one of claims 5-6, characterized in that, Includes the following steps: (1) Doxophylline is continuously infused into the subject via the infusion control module; (2) Collect biological samples at at least three different time points within the time interval between the start and end of the infusion; (3) The concentration of doxophylline metabolites in each biological sample is detected by the sample processing and analysis module, and the total molar concentration is calculated; (4) Calculate the kinetic parameters through the data processing module and generate a liver reserve function assessment report.

8. The method of using the liver reserve function assessment system according to claim 7, characterized in that: The duration of continuous intravenous infusion in step (1) is 1-15 minutes, and the doxophylline is administered at a dose specifically for testing. The time interval for collecting biological samples in step (2) is 1-5 minutes, and the biological samples are blood, plasma, or serum.

9. The method of using the liver reserve function assessment system according to claim 7, characterized in that: The total molar concentration in step (3) is the sum of the molar concentrations of theophylline acetaldehyde (TA), theophylline acetic acid (TAA), hydroxyethyl theophylline (ETO), and theophylline acetic acid-2'-hydroxyethyl ester (HET).

10. The method of using the liver reserve function assessment system according to claim 7, characterized in that: The kinetic parameter in step (4) is the linear slope of the total molar concentration-time curve, which is obtained by least squares linear regression fitting.

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