A method for establishing a PK-PD model of Caesalpinia cambogia peel extract and its application

Abstract

The invention discloses a method for establishing a PK-PD model of a caesalpinia peel extract and an application thereof, belonging to the technical field of Chinese herbal medicines. The invention discloses a PK-PD model of a caesalpinia peel extract, wherein the extract PK-PD model comprises protosaccharin B, 3-deoxysaccharin chalcone and protosaccharin B-3-O-β-D-glucoside; a method for establishing a PK-PD model of a caesalpinia peel extract, which is used to establish the above-mentioned extract PK-PD model, and comprises the following steps: S1, preparation of a standard curve; S2, preparation of a caesalpinia peel extract; S3, treating AA model rats with the caesalpinia peel extract; S4, conducting pharmacokinetic experiments of the caesalpinia peel extract in normal and diseased animals; S5, PK sample pretreatment; S6, establishing a PK-PD model; the method can realize the establishment of a PK-PD model to determine the drug concentration-time-effect relationship of the caesalpinia peel extract components and cytokines in vivo, and can provide valuable information for the drug development and clinical application of the drug.

Description

Technical Field

[0001] The present invention relates to the technical field of Chinese herbal medicines, and more specifically to a method for establishing a PK-PD model of a Caesalpinia scoparia peel extract and an application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial tissue hyperplasia, synovial vascular membrane formation, and cartilage and bone erosion. The global prevalence of rheumatoid arthritis is as high as 0.18%-1.07%, and it is increasing year by year. The typical clinical features are joint swelling, pain, and irreversible joint destruction, which eventually lead to severe disability and endanger the patient's physical and mental health. Various animal models have been established to study the etiology, pathogenesis, and influencing factors of RA, as well as the study of new therapeutic targets and the evaluation of new therapies, including adjuvant-induced arthritis (AA), collagen-induced arthritis (CIA), and streptococcal cell wall-induced arthritis models. The AA model is similar to human rheumatoid arthritis in some pathological, histological, and immunological aspects. Therefore, the AA model was used as a disease model in this experiment.

[0003] The pathological mechanism of rheumatoid arthritis is complex, and inflammatory cytokines play a major role in rheumatoid arthritis. Studies have shown that IL-6 has a synergistic effect with IL-1β and TNF-α to produce vascular endothelial growth factor, which acts on vascular endothelial cells to increase their permeability, leading to increased joint exudation and swelling. These inflammatory factors have been widely used as biomarkers to evaluate the severity of inflammation and efficacy of drugs.

[0004] Caesalpinia decapetala (C. decapetala) is the dried root or root bark of Caesalpinia decapetala (Roxb.) Alston, a plant of the Leguminosae family. It is widely used in clinical practice to treat rheumatic arthritis, bronchitis, colds, etc. [13,14]. Modern pharmacological studies have shown that this medicinal material has a wide range of pharmacological activities, including anti-tumor, antioxidant, antipyretic and anti-inflammatory activities. There are several related preparations that use Caesalpinia decapetala as the main drug, such as Malan Ganhan Capsule and Caesalpinia decapetala Cold Mixture, which are used to treat inflammatory diseases such as fever, influenza, and colds. At present, the chemical components of Caesalpinia decapetala, such as terpenes, flavonoids, and coumarins, have been reported in the literature. In the early stage, the research group isolated components such as protosedinoside B (PTB) and 3-deoxysedinoside chalcone (3-DSC) from the anti-inflammatory active parts of Caesalpinia decapetala, indicating that these components may be the anti-inflammatory material basis of Caesalpinia decapetala. 3-DSC, PTB, and its glycoside PTD were structurally identified as high isoflavonoid compounds. It has been demonstrated that 3-DSC exerts anti-inflammatory activity in mouse macrophages by inducing the expression of HO-1 at the translational level. PTB has significant neuroprotective, anticancer, and anti-inflammatory activities. PTD shares the same parent nuclear structure as PTB, but its pharmacological activity and in vivo processes have not been reported.

[0005] The analysis of multiple biomarkers using a pharmacokinetic-pharmacodynamic (PK-PD) model is a promising method that has been widely used to evaluate the effects of drugs on pathological processes and scientifically reveal the relationship between the drug effect and duration of action of drugs in vivo, which is of great significance for drug screening and guiding reasonable clinical drug administration. However, the existing technology currently lacks a PK-PD research model for Caesalpinia scoparia extracts. Exploring the drug concentration-time-effect relationship of the multiple effect components of Caesalpinia scoparia extracts in vivo will help to clarify the mechanism of action of Caesalpinia scoparia and clarify its target of action, providing a theoretical basis for guiding its safe and effective clinical use. It has positive significance for the development of innovative drugs for Caesalpinia scoparia, giving full play to the advantages of special minority drug resources, and driving local economic development. Therefore, a PK-PD model for Caesalpinia scoparia extracts is urgently needed at this stage. Summary of the invention

[0006] 1. Technical issues to be solved

[0007] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for establishing a PK-PD model of a caesalpinia peel extract and its application, which can establish a PK-PD model to determine the drug concentration-time-effect relationship of the caesalpinia peel extract components and cytokines in vivo, and provide valuable information for the drug development and clinical application of the drug.

[0008] 2. Technical solution

[0009] To solve the above problems, the present invention adopts the following technical solutions.

[0010] A method for establishing a PK-PD model of a Caesalpinia scoparia extract comprises the following steps:

[0011] S1. Preparation of standard curve

[0012] 1) Weigh appropriate amounts of 3-DSC, PTB, and PTD respectively and dissolve them in methanol to prepare reference substance stock solutions;

[0013] 2) Take an appropriate amount of puerarin and dissolve it in methanol, dilute it into IS solution, and perform gradient dilution on the stock solution to obtain standard curve working solutions of 3-DSC, PTB and PTD respectively;

[0014] 3) Add 100 μL of standard curve working solution and 50 μL of IS solution to 100 μL of blank plasma to prepare standard curves for 3-DSC, PTB, and PTD;

[0015] S2. Preparation of Caesalpinia scoparia extract

[0016] The caesalpinia peel is dried and extracted twice with 70% ethanol, the extracts are mixed and filtered, and the ethanol is evaporated under reduced pressure to obtain the caesalpinia peel extract;

[0017] S3. Treatment of AA model rats with extract from Caesalpinia scoparia

[0018] 1) Adult male SD rats weighing 200-220 g were taken and raised for 1 week;

[0019] 2) The rats were randomly divided into a normal control group (CON), a model control group (MOD), a positive control group (POS) and a Caesalpinia scoparia extract (CDE) group; except for the CON group, the other groups were intradermally injected with CFA 0.1 mL on the right sole on the first day; the POS group and the CDE group were orally administered with Tripterygium wilfordii polyglycosides tablets (102 mg / kg) and the Caesalpinia scoparia extract (4.54 g / kg) in step S2, respectively; the CON group and the MOD group were given an equal volume of 0.5% CMC-Na; the four groups were given the same doses, twice a day, for 14 consecutive days;

[0020] 3) 2 hours after the last administration, the plasma of rats in each group was collected through the tail vein, and the plasma TNF-α, IL-6, IL-1β and RF levels of rats in each group were detected by ELISA; the rats were killed after anesthesia, the right posterior ankle joint was cut, the surrounding soft tissue was separated, and immersed in 4% paraformaldehyde solution for histopathological examination; the joint tissue of the rats was decalcified with EDTA-2Na, dehydrated step by step with different concentrations of ethanol, embedded in xylene for transparent section, with a thickness of about 4 μm, dewaxed, stained with hematoxylin-eosin, and the histopathological changes of the joints of rats in each group were observed under a microscope;

[0021] S4. Pharmacokinetics of Caesalpinia chamomile extract in normal and diseased animals

[0022] 1) Several male SD rats were randomly divided into three groups: single-dose normal group (Single-Norm), single-dose model group (SingleMod), and multiple-dose model group (Multiple-Mod);

[0023] 2) Multiple-dose model group (Multiple-Mod): The extract of Caesalpinia scoparia obtained in step S2 was administered by gavage, twice a day, for 14 consecutive days; 0.25 mL of blood was collected from the tail vein before administration and at 0.083, 0.167, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 24 h after the last administration, and the plasma samples were stored at 4°C;

[0024] 3) Take the above plasma sample, centrifuge at 6000-8000 rpm for 10-15 min, take the supernatant and divide it into two parts for PK and PD analysis, and store the supernatant sample in a -80-90°C refrigerator;

[0025] S5. PK sample pretreatment

[0026] Take 100 μL of the plasma sample in step S4-2, 20 μL of the IS solution in step S1-2, and 50 μL of 2% formic acid solution, add them to a 1.5 mL centrifuge tube, vortex mix for 1 min, add 400 μL of methanol, vortex mix for 1-3 min, ultrasonicate for 10-13 min, and centrifuge at 12000-15000 rpm for 10-12 min at 4-8° C.; take the supernatant and blow dry it with nitrogen at 37-40° C., dissolve the residue in 150 μL of 50% methanol, centrifuge at 12000-15000 rpm for 10-12 minutes to obtain the supernatant, and inject the obtained supernatant into the UPLC-MS / MS system for analysis;

[0027] S6. Establishment of PK-PD model

[0028] 1) Establishing the relationship between PK and PD by detecting the levels of PTB, 3-DSC, PTD, and the levels of TNF-α, IL-6, IL-1β, and RF in the plasma samples of the multiple-dose model group in step S4-2;

[0029] 2) Among them, Akaike's information criterion (AIC) was used to comprehensively evaluate the goodness of fit; the pharmacokinetic curve of Caesalpinia salsa in AA model rats conformed to the two-compartment model; by comparing different PK-PD models, it was found that the Sigmoid Imax model had the best fitting effect on PK-PD data, and its formula is as follows:

[0030] Furthermore, the stock solution in step S1-1 and the working solution in step S1-1 were both stored at 4°C, and the concentrations of 3-DSC, PTB, and PTD in step S1-1 were 0.5021 mg / mL, 1.0040 mg / mL, and 0.5021 mg / mL, respectively.

[0031] Furthermore, the concentration of the IS solution in step S1-2 was 20 ng / mL, and the concentrations of the standard curve working solutions of 3-DSC, PTB, and PTD were 0.20-50.21 ng / mL, 23.48-3006.00 ng / mL, and 93.94-48096.00 ng / mL.

[0032] Furthermore, in step S2, the addition ratios of the mass of the caesalpinia calamus peel to the volume of 70% ethanol for two extractions were 1:10 and 1:8, respectively, and the extraction times were 1.5 h and 1.0 h, respectively.

[0033] Furthermore, in step S3-1, the rat breeding environment is 20-25°C and 55-65% humidity.

[0034] Furthermore, the dosage in step S4 is 4.54 g / kg of the Caesalpinia scoparia extract in step S2.

[0035] Furthermore, the blood loss of the rats in step S4-2 was replaced with an equal volume of normal saline to maintain appropriate fluid levels and control potential hypovolemia.

[0036] Further, E in the formula in step S6-2 is the value of TNF-α, IL-6, IL-1β and RF in the plasma sample in step S4-2, E0 is the initial effect value in the plasma sample in step S4-2, and C represents the concentration of 3-DSC, PTB and PTD in the action site chamber; max stands for maximum inhibition fraction, IC 50 is the drug concentration required to produce 50% maximal inhibition; γ is the midpoint slope of the standard curve in step S1-3.

[0037] An application of a PK-PD model of a caesalpinia sylvestris peel extract, wherein the PK-PD model of a caesalpinia sylvestris peel extract is applied to the individualized medication of caesalpinia sylvestris peel in the treatment of inflammatory diseases.

[0038] 3. Beneficial effects

[0039] Compared with the prior art, the advantages of the present invention are:

[0040] This protocol uses a selective, sensitive and reliable UPLC-MS / MS method to simultaneously determine the three effective components in rat plasma of the caesalpinia sylvestris peel extract. By studying the pharmacokinetic behavior of these three active ingredients in normal rats and AA rats after single or multiple oral administration, the pharmacokinetic changes of the drug under disease conditions are predicted, and a PK-PD model is established to determine the drug concentration-time-effect relationship of these three main components and cytokines in vivo, which can provide a basis for formulating a more reasonable dosing regimen for the drug and improving the level of individualized use of caesalpinia sylvestris peel in the treatment of inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 HE staining images of the mouse ankle joint at different magnifications of the present invention

[0042] Figure 2 is a graph showing the levels of TNF-α, IL-6, IL-1β and RF in rat plasma according to the present invention;

[0043] Figure 3 It is the 3-DSC average blood drug concentration-time curve diagram of the present invention;

[0044] Figure 4 The average blood drug concentration-time curve of PTB of the present invention;

[0045] Figure 5 It is the average blood drug concentration-time curve of PTD of the present invention;

[0046] Figure 6 It is a time-concentration-effect curve diagram between the blood drug concentrations of DSC, PTB, PTD and the levels of TNF-α, IL-6, IL-1β and RF of the present invention;

[0047] Figure 7 The counterclockwise and clockwise hysteresis loops of DSC, PTB and PTD of the present invention show the delay plots between the effects and the distribution of the four compounds in plasma. DETAILED DESCRIPTION

[0048] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention; it is obvious that the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments, and all other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making creative work are within the scope of protection of the present invention.

[0049] Example:

[0050] See also Figure 1-7 , a method for establishing a PK-PD model of a Caesalpinia scoparia extract, comprising the following steps:

[0051] 2.1 Chemicals and reagents

[0052] Caesalpinia chaenomeles; the purity of all reference substances used was greater than 98%; 3-DSC, PTB, PTD standards; puerarin (internal standard, IS); methanol, formic acid and acetonitrile.

[0053] 2.2 Preparation of standard curve

[0054] Weigh appropriate amounts of 3-DSC, PTB, and PTD, respectively, and dissolve them in methanol to prepare reference stock solutions, with concentrations of 0.5021 mg / mL, 1.0040 mg / mL, and 0.5021 mg / mL, respectively. Dissolve appropriate amounts of puerarin in methanol and dilute to IS solution (20 ng / mL). Perform gradient dilutions on the stock solutions to obtain standard curve working solutions of 3-DSC (0.20-50.21 ng / mL), PTB (23.48-3006.00 ng / mL), and PTD (93.94-48096.00 ng / mL). Both the stock solution and the working solution were stored at 4°C for use.

[0055] 100 μL of standard curve working solution and 50 μL of IS solution were added to 100 μL of blank plasma to prepare standard curves for 3-DSC, PTB, and PTD.

[0056] 2.3 Preparation of Caesalpinia scoparia extract

[0057] Take 30kg of the dried caesalpinia peel and extract it twice with 70% ethanol (1:10, 1:8, W / V), extracting for 1.5h and 1.0h respectively. Mix the extracts and filter them, evaporate the ethanol under reduced pressure to obtain a 1g / mL crude substance. In order to calculate the dosage, the same extraction conditions as above were used to quantitatively determine the contents of the three components in the caesalpinia peel extract by the external standard method. The results showed that the 3-DSC content in the caesalpinia peel extract was 0.011%, the PTB content was 1.67%, and the PTD content was 0.906%.

[0058] 2.4 Treatment of AA model rats with Caesalpinia caesalpinia extract

[0059] Adult male SD rats weighing 200-220 g were raised in an environment of 20-25° C. and 55-65% humidity for 1 week before the experiment.

[0060] Twenty-four animals were randomly divided into normal control group (CON), model control group (MOD), positive control group (POS) and Caesalpinia scoparia extract (CDE) groups, with 6 animals in each group. Except for the CON group, the other groups were intradermally injected with CFA 0.1 mL on the right sole on the first day. The POS group and CDE group were orally administered with Tripterygium wilfordii polyglycosides tablets (102 mg / kg) and Caesalpinia scoparia extract (4.54 g / kg, equivalent to 0.03 mg / kg 3-DSC, 2.53 mg / kg PTB, 4.51 mg / kg PTD), respectively. Rats in the CON group and MOD group were given an equal volume of 0.5% CMC-Na. The four groups were given the above-mentioned doses twice a day for 14 consecutive days.

[0061] 2 hours after the last administration, plasma of rats in each group was collected through the tail vein, and the plasma levels of TNF-α, IL-6, IL-1β and RF of rats in each group were detected by ELISA. Rats were killed after anesthesia, the right posterior ankle joint was cut, the surrounding soft tissue was separated, and immersed in 4% paraformaldehyde solution for histopathological examination. The joint tissue of rats was decalcified with EDTA-2Na, dehydrated step by step with different concentrations of ethanol, embedded in xylene for transparent sections with a thickness of about 4μm, dewaxed, stained with hematoxylin-eosin, and the histopathological changes of the joints of rats in each group were observed under a microscope.

[0062] 2.5 Pharmacokinetics of Caesalpinia chamomile extract in normal and diseased animals

[0063] Eighteen male SD rats were randomly divided into three groups (n=6): a single-dose normal group (Single-Norm), a single-dose model group (SingleMod), and a multiple-dose model group (Multiple-Mod); the dosage of the extract of Caesalpinia scoparia was 4.54 g / kg.

[0064] Multiple-Mod: The extract of Caesalpinia scoparia was administered by gavage at a dose of 4.54 g / kg, twice a day, for 14 consecutive days. 0.25 mL of blood was collected from the tail vein before administration and at 0.083, 0.167, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 24 h after the last administration, and the plasma samples were stored at 4 °C. During the experiment, the blood loss of the rats was replaced by an equal amount of normal saline to maintain an appropriate fluid level and control potential hypovolemia.

[0065] The plasma samples were centrifuged at 6000 rpm for 10 min, and the supernatant was divided into two portions for PK and PD analysis. The supernatant samples were stored in a -80°C refrigerator.

[0066] 2.6PK sample pretreatment method

[0067] 100 μL of rat plasma sample, 20 μL of IS solution (20 ng / mL) and 50 μL of 2% formic acid solution were added to a 1.5 mL centrifuge tube and vortexed for 1 min, 400 μL of methanol was added, vortexed for 1 min, ultrasonicated for 10 min, and centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was blown dry with nitrogen at 37°C, the residue was dissolved in 150 μL of 50% methanol, centrifuged at 12,000 rpm for 10 min, and the supernatant was injected into the UPLC-MS / MS system for analysis.

[0068] 2.7 Multiple-dose pharmacokinetic-pharmacodynamic model

[0069] The relationship between PK and PD was established by detecting the levels of PTB, 3-DSC, PTD and TNF-α, IL-6, IL-1β, and RF in the plasma samples of the multiple-dose model group. The Akaike's information criterion (AIC) was used to comprehensively evaluate the goodness of fit. The pharmacokinetic curve of caesalpinia sylvestris in AA model rats conforms to the two-compartment model. By comparing different PK-PD models, it was found that the Sigmoid Imax model had the best fitting effect on PK-PD data, and its formula is as follows:

[0070]

[0071] where E is the value of TNF-α, IL-6, IL-1β and RF in plasma, E0 is the initial effect value in plasma, C represents the concentration of 3-DSC, PTB and PTD in the action site compartment; Imax represents the maximum inhibition fraction, IC50 is the drug concentration required to produce 50% maximum inhibition, which is used to describe the efficacy; γ is the midpoint slope of the standard curve, which is a shape coefficient that describes the sensitivity of the concentration-effect relationship.

[0072] 2.8 LC-MS / MS analysis conditions are as follows:

[0073] 2.8.1 UPLC conditions

[0074] The ACQUITY I UPLC system was equipped with an autosampler and an Acquity I UPLC BEH C18 column (2.1 mm × 100 mm, inner diameter 1.7 μm), and the pre-column was Waters Vanguard BEH C18 (2.1 mm × 5 mm, 1.7 μm). The elution gradient was 0-0.5 min (10% B), 0.5-3.0 min (10-90% B), 3.0-3.5 min (90% B), 3.5-4 min (90-10% B). The column temperature was 40°C and the injection volume was 1 μL.

[0075] 2.8.2MS conditions

[0076] Mass spectrometry was performed using a Waters Xevo TQS triple quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source. The ion source parameters were: ion source temperature 120°C, desolvation gas temperature 350°C, capillary voltage 3.0 kV, capillary ionization voltage 3 kV, and nebulizer gas (nitrogen) 650 L / h. Table 1 lists the optimal mass spectrometry parameters for the analytes and IS. Data acquisition and processing were performed on a MicroMass MassLynx v4.1 workstation.

[0077] Mass spectrometry parameters of analytes and IS

[0078]

[0079] 3 Results

[0080] 3.1 Pharmacodynamic evaluation of Caesalpinia scoparia extract in the treatment of AA model rats

[0081] Ankle joint histopathological sections and cytokine assay results are as attached to the instruction manual. Figure 1 and 2 As shown in the results, compared with the CON group, the joint synovium of MOD rats was extensively infiltrated with inflammatory cells, and the cartilage was severely damaged. The levels of plasma TNF-α, IL-6, IL-1β, and RF in the MOD group were significantly increased (P<0.001), and the differences were statistically significant compared with the CON group (P<0.05), indicating that the AA model was successfully established. After treatment, the above symptoms in the POS and CDE groups were significantly improved compared with the MOD group. The levels of the above four inflammatory factors in the CDE and POS groups were significantly reduced (P<0.05), indicating that the CDE and POS groups had a significant inhibitory effect on the secretion of inflammatory factors TNF-α, IL-6, IL-1β, and RF in rat plasma, and the degree of inhibition was similar.

[0082] 3.2 Standard curve and LLOQ

[0083] The following table summarizes the calibration curves, linear range, R2, and LLOQ for the three analytes. For all analytes, the R2 value was greater than 0.999 and the LLOQ was between 0.2 and 93.94 ng / mL. The results showed that these analytes showed good linearity within a certain range of concentrations in rat plasma.

[0084] Linear range, standard curve equation and correlation coefficient of the three compounds

[0085]

[0086] 3.3PK analysis

[0087] The average blood drug concentration-time curves of rats in the Single-Con group, Single-Mod group, and Multiple-Mod group are shown in the attached manual. Figure 3-5 The main PK parameters are shown in the table below. Compared with the Single-Con group, the Cmax and AUC of 3-DSC, PTB and PTD in the Single-Mod group were significantly increased, while CLZ / F and VZ / F were significantly decreased, indicating that the clearance and distribution of 3-DSC, PTB and PTD in the Single-Mod group were lower than those in the Single-Con group. The t1 / 2z of PTD in Single-Mod rats was shortened (P<0.05), and the MRT(0-t) was prolonged (P<0.01). The T1 / 2z and MRT(0-t) of PTB and 3-DSC were opposite, but the difference was not statistically significant. Compared with the Single-Mod group, the Cmax and AUC values ​​of PTD in the Multiple-Mod group were significantly reduced (P<0.01), and the MRT(0-∞) showed an upward trend. However, there were no significant differences in the PK parameters of PTB and 3-DSC between the single and multiple dosing model groups. These results suggest that the disease state may affect the absorption, distribution, and excretion of these bioactive components.

[0088] The following table shows the pharmacokinetic parameters of PTB, PTD and 3-DSC of rats in different groups after oral administration of Caesalpinia scoparia extract (Mean±SD, n=6)

[0089]

[0090]

[0091] Note: * P<0.001, ** P<0.01, *** P<0.001vs.Single-Norm group, # P<0.05, ## P<0.01, ### P<0.001 vs. Single-Mod group

[0092] 3.4PK-PD modeling

[0093] Finally, the two-compartment model of PK and the PD model with effect compartment are combined, as shown in the attached manual. Figure 6 As shown in the figure, a S-Imax PK-PD model with a high degree of fit was established to describe the time-concentration-effect relationship. After oral administration of Caesalpinia chamomile peel extract, the drug concentration and effect did not show a one-to-one correspondence, as shown in the attached manual. Figure 7As shown in the figure, the maximum effect obviously lags behind the peak blood drug concentration. The time-concentration-effect curves of TNF-α, IL-6, IL-1β, RF and plasma 3-DSC concentrations show counterclockwise hysteresis loops, while the time-concentration-effect curves of PTB, PTD and the four inflammatory cytokines show clockwise hysteresis loops. Therefore, a PK-PD model with a separate effect compartment was introduced to analyze the data set. The following table summarizes the PK-PD model parameters of PTD, 3-DSC and PTB calculated using WinNonLin8.2 software.

[0094] PK-PD parameters of three analytes in multiple-dose model rats

[0095]

[0096]

[0097] A model can be seen from the above that compared with the Single-Norm group, the AUC (0-t), AUC (0-∞), Cmax and MRT (0-t) of the three components in the Single-Mod group were significantly increased (P < 0.05), while the CLZ / F of the three components was significantly reduced (P < 0.05). The above results suggest that A increases the absorption of drugs in rats and prolongs the duration of action, which may be more beneficial to the treatment of rheumatoid arthritis.

[0098] Given that most drugs used in the clinic to treat chronic diseases require long-term administration, it is meaningful and necessary to study the pharmacokinetic characteristics of the extract from the bark of Caesalpinia caesalpiniae in multiple-dose model rats. Compared with single administration, after multiple administration, the Cmax and AUC of PTD in the plasma of AA rats decreased, the CLZ / F of PTD and PTB increased, and the 3-DSC parameters did not change significantly, indicating that there was no accumulation in the body of AA rats and they may be insensitive to toxic reactions.

[0099] The results of the preliminary experiment showed that compared with the model group, there was no significant change in the content of inflammatory cytokines such as TNF-α, IL-6, IL-1β and RF in the plasma of AA model rats after a single administration. Therefore, a pharmacodynamic study with multiple administrations was conducted. Compared with the control group, the expression of TNF-α, IL-6, IL-1β and RF were upregulated in the CFA-induced rat arthritis model. At the same time, histopathological analysis showed obvious inflammatory cell infiltration, synovial hyperplasia, cartilage and bone erosion in the ankle joint, proving that the model was successful. Compared with the model group, the extract of the calamus peel had an inhibitory effect on inflammatory markers, which may be one of the mechanisms of the calamus peel against RA, thereby alleviating the inflammatory response of AA rats and gradually reducing joint damage.

[0100] In pharmacokinetic and pharmacodynamic correlation analysis, a counterclockwise hysteresis loop is generally considered to be a process in which the effect increases over time at a given drug concentration, while a clockwise hysteresis loop means that the effect will decrease. In general, counterclockwise hysteresis is usually caused by the existence of an effect compartment or an indirect drug effect, such as the pharmacological effect of an ingredient is exerted by its metabolite, while clockwise hysteresis is caused by acute tolerance, the production of inhibitory metabolites, etc. The dose-effect relationship between 3-DSC and TNF-α, IL-6, IL-1β or RF showed a counterclockwise hysteresis loop, while PTB and PTD showed a clockwise hysteresis loop with the levels of the four inflammatory cytokines (as shown in the attached instructions). Figure 7 ). The lag between drug concentration and inflammatory cytokine levels may be due to the delay in drug transport from the circulatory system to the joint cavity. For this reason, the inhibitory effect SigmoidImax model was introduced to describe this process.

[0101] In this study, the AIC method was used to determine the compartment model. The smaller the AIC value, the better the fitting effect of the model. It was determined that the oral administration of Caesalpinia calamus peel extract conforms to the two-compartment model of extravascular administration. The main parameters of 3-DSC, PTB and PTD calculated by the PK-PD model are summarized in the above table. Ke0 is the rate constant of drug transport from the central to the effect chamber, also known as the equilibrium rate constant, which is of great significance to the PK-PD model. The Ke0 value determines the lag time of drug efficacy. The smaller the Ke0 value, the longer the lag time of drug efficacy to drug concentration. It can be seen that the Ke0 values ​​of various efficacy indicators are different, and the hysteresis phenomenon is also different. The reasons may be:

[0102] (1) Each efficacy indicator may belong to a different effect compartment. The time required for the equilibrium distribution process of the drug from the central compartment to each effect compartment is different.

[0103] (2) The binding activity of drugs to receptors is different. If the activity is high, the retention time is short, while if the activity is low, the retention time is long.

[0104] In addition, the γ value has important clinical significance when evaluating drug selectivity and sensitivity within the effective concentration range. In this study, the γ values ​​of the three components of the Caesalpinia calamus peel extract corresponding to TNF-α and IL-1β in the Imax model were all <1, indicating that the dose-effect curve was relatively flat and the safety window was wide. The PK-PD combined model helps to more comprehensively and accurately understand the changes in drug efficacy with dose and time, reveal the mechanism of drug action, and clarify the targeting effect. In addition, this model can also provide a basis for selecting clinically relevant doses for subsequent evaluation in disease models and clinical trials.

[0105] In summary, the caesalpinia peel can directly play an anti-inflammatory protective role by improving ankle joint injuries and significantly downregulating the secretion of proinflammatory cytokines. In addition, CFA-induced arthritis can change the pharmacokinetic behavior of the drug in rats, significantly improving its bioavailability in AA rats, thereby promoting the rapid and effective therapeutic concentration of RA, thereby exerting its pharmacological effects. Moreover, after multiple oral administrations, the caesalpinia peel extract did not accumulate in the body, indicating that the drug is rapidly metabolized and excreted in rats and is not prone to accumulation poisoning. A two-compartment Sigmoid Imax PK-PD combined model was successfully established to fit the relationship between the concentrations of the three components and the pharmacodynamic indicators. This model provides a basis for formulating a more reasonable dosing regimen and improving the level of individualized medication of caesalpinia peel in the treatment of inflammatory diseases.

[0106] The above is only a preferred specific implementation of the present invention; however, the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solution and its improved conception within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for establishing a PK-PD model of Caesalpinia cambogia peel extract, characterized in that: The following steps are involved: S1. Preparation of standard curve 1) Weigh appropriate amounts of 3-DSC, PTB, and PTD respectively and dissolve them in methanol to prepare reference stock solutions; 2) Take an appropriate amount of puerarin and dissolve it in methanol, dilute it into IS solution, and perform gradient dilution on the stock solution to obtain the standard curve working solutions of 3-DSC, PTB and PTD respectively; 3) Add 100 µL of standard curve working solution and 50 µL of IIS solution to 100 µL of blank plasma to prepare standard curves for 3-DSC, PTB, and PTD; S2. Preparation of Caesalpinia scoparia extract The caesalpinia scoparia peel was dried and extracted twice with 70% ethanol, the extracts were mixed and filtered, and the ethanol was evaporated under reduced pressure to obtain the caesalpinia scoparia peel extract; S3. Treatment of AA model rats with extract from Caesalpinia scoparia 1) Adult male SD rats weighing 200-220 g were obtained and raised for 1 week; 2) The rats were randomly divided into CON, MOD, POS and CDE groups. Except for the CON group, the other groups were intradermally injected with CFA 0.1 mL on the right sole on the first day. The POS and CDE groups were orally administered with Tripterygium wilfordii polyglycosides and the Caesalpinia chaenomeles extract in step S2, respectively. The CON and MOD groups were given an equal volume of 0.5% CMC-Na. The four groups were given the same doses, twice a day, for 14 consecutive days. 3) 2 hours after the last administration, the plasma of rats in each group was collected through the tail vein, and the plasma TNF-α, IL-6, IL-1β and RF levels of rats in each group were detected by ELISA. The rats were killed after anesthesia, the right posterior ankle joint was cut, the surrounding soft tissue was separated, and immersed in 4% paraformaldehyde solution for histopathological examination. The joint tissues of rats were decalcified with EDTA-2Na, dehydrated step by step with different concentrations of ethanol, embedded in xylene for transparent sections with a thickness of 4 μm, dewaxed, and stained with hematoxylin-eosin. The histopathological changes of the joints of rats in each group were observed under a microscope. S4. Pharmacokinetics of Caesalpinia chamomile extract in normal and diseased animals 1) Several male SD rats were randomly divided into 3 groups: single-dose normal group, single-dose model group, and multiple-dose model group; 2) Multiple-dose model group: The extract of Caesalpinia scoparia obtained in step S2 was administered by gavage, twice a day, for 14 consecutive days; 0.25 mL of blood was collected from the tail vein before administration and at 0.083, 0.167, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 24 h after the last administration, and the plasma samples were stored at 4 °C; 3) Take the above plasma samples, centrifuge at 6000-8000 rpm for 10-15 min, take the supernatant and divide it into two parts for PK and PD analysis, and store the supernatant samples in a -80-90℃ refrigerator; S5. PK sample pretreatment Take 100 μL of the plasma sample in step S4-2, 20 μL of the IS solution in step S1-2, and 50 μL of 2% formic acid solution, add them to a 1.5 mL centrifuge tube, vortex mix for 1 min, add 400 μL of methanol, vortex mix for 1-3 min, sonicate for 10-13 min, and centrifuge at 12000-15000 rpm for 10-12 min at 4-8°C; take the supernatant and blow dry it with nitrogen at 37-40°C, dissolve the residue in 150 μL of 50% methanol, centrifuge at 12000-15000 rpm for 10-12 minutes to obtain the supernatant, and inject the supernatant into the UPLC-MS / MS system for analysis; S6. Establishment of PK-PD model 1) Establishing the relationship between PK and PD by detecting the levels of PTB, 3-DSC, PTD, and the levels of TNF-α, IL-6, IL-1β, and RF in the plasma samples of the multiple-dose model group in step S4-2; 2) Among them, Akaike's information criterion was used to comprehensively evaluate the goodness of fit; the pharmacokinetic curve of Caesalpinia salsa in AA model rats conformed to the two-compartment model; by comparing different PK-PD models, it was found that the Sigmoid Imax model had the best fitting effect on PK-PD data, and its formula is as follows: ， Wherein E is the value of TNF-α, IL-6, IL-1β and RF in the plasma sample of step S4-2, E0 is the initial effect value in the plasma sample of step S4-2, and C represents the concentration of 3-DSC, PTB and PTD in the action site compartment; I max stands for maximum inhibition fraction, IC 50 is the drug concentration required to produce 50% maximal inhibition; γ is the midpoint slope of the standard curve in steps S1-3.

2. The method for establishing a PK-PD model of a Caesalpinia scoparia peel extract according to claim 1, characterized in that: The stock solution in step S1-1 and the working solution in step S1-1 were both stored at 4°C. The concentrations of 3-DSC, PTB, and PTD in step S1-1 were 0.5021 mg / mL, 1.0040 mg / mL, and 0.5021 mg / mL, respectively.

3. The method for establishing a PK-PD model of a Caesalpinia cambogia peel extract according to claim 1, characterized in that: The concentration of the IS solution in step S1-2 was 20 ng / mL, and the concentrations of the standard curve working solutions of 3-DSC, PTB, and PTD were 0.20−50.21 ng / mL, 23.48−3006.00 ng / mL, and 93.94−48096.00 ng / mL.

4. The method for establishing a PK-PD model of a Caesalpinia cambogia peel extract according to claim 1, characterized in that: The addition ratios of the mass of the caesalpinia scoparia in step S2 to the volume of 70% ethanol for two extractions were 1:10 and 1:8, respectively, and the extraction times were 1.5 h and 1.0 h, respectively.

5. The method for establishing a PK-PD model of a Caesalpinia cambogia peel extract according to claim 1, characterized in that: In step S3-1, the rats were kept in an environment with a temperature of 20-25°C and a humidity of 55-65%.

6. The method for establishing a PK-PD model of Caesalpinia cambogia peel extract according to claim 1, characterized in that: The dosage in step S4 is 4.54 g / kg of the Caesalpinia scoparia extract in step S2.

7. The method for establishing a PK-PD model of a Caesalpinia cambogia peel extract according to claim 1, characterized in that: The blood lost by the rats in step S4-2 was replaced with an equal volume of normal saline to maintain appropriate fluid levels and control potential hypovolemia.

8. An application of a PK-PD model of a Caesalpinia scoparia extract, characterized in that: The method for establishing a PK-PD model of a caesalpinia scoparia extract according to any one of claims 1 to 7 is applied to the individualized medication of caesalpinia scoparia to treat inflammatory diseases.

Images