A novel ligustrazine film coating agent and a method for detecting blood concentration after administration of the preparation

Abstract

The application belongs to the field of medicine, and particularly relates to a novel ligustrazine film coating agent and a method for detecting blood drug concentration after preparation administration. The self-made ligustrazine film coating agent can maintain a relatively stable and high blood drug concentration for a long time in the body, and under the premise of equivalent administration dose, the self-made TMP film coating agent can exceed the AUC of intravenous injection and has a higher drug peak concentration than gavage at the corresponding time. The prescription of the film coating agent is reasonable and has good permeability. Meanwhile, the HPLC method provided by the application is accurate, sensitive, rapid and can be used for the determination of ligustrazine blood drug concentration.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals, specifically relating to a novel tetramethylpyrazine coating agent and a method for detecting the blood drug concentration after administration. Background Technology

[0002] Tetramethylpyrazine (TMP), with the chemical structure of tetramethylpyrazine, is the active ingredient in the rhizome of *Ligusticum chuanxiong* Hort., a plant in the Apiaceae family. It is also widely found in *Curcuma zedoaria* (Zingiberaceae) and *Jatropha curcas* (Euphorbia pekinensis). Literature reports that TMP has vasodilatory, microcirculation-improving, and platelet aggregation-inhibiting effects, making it a commonly used drug in my country for the clinical treatment of ischemic cerebrovascular diseases. In addition, clinical applications have shown that TMP is effective in treating gestational hypertension, asthma, and chronic pulmonary heart disease. Currently, commonly used tetramethylpyrazine preparations include tablets, capsules, and injections. Tetramethylpyrazine phosphate tablets and capsules are included in Part II of the 2015 edition of the Chinese Pharmacopoeia. However, TMP is mainly distributed in tissues with rich blood flow, metabolizes rapidly in the body, has a short half-life, low bioavailability, and a high hepatic uptake rate. Furthermore, oral and injectable administration can damage the gastrointestinal system, accompanied by various adverse reactions. Therefore, sustained-release and controlled-release formulations of TMP have become one of the main research directions for TMP.

[0003] Transdermal drug delivery systems (TMPs) are simple to prepare, convenient to use, avoid the first-pass effect of the liver, and allow for discontinuation of administration at any time. They are a relatively advanced transdermal drug delivery system developed in recent years. Tetramethylpyrazine has a small molecular weight, low melting point, and is easily volatile, and has been proven to have good transdermal properties. Therefore, this experiment conducted a preliminary study on the blood concentration and pharmacokinetics of a TMP film-coating agent to provide a reference for the development of new TMP formulations. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a novel tetramethylpyrazine coating agent and a method for detecting blood drug concentration after administration.

[0005] The technical solution of the present invention is as follows: a ligustrazine coating agent, comprising ligustrazine, anhydrous ethanol, glycerin, Tween, azone, and polyvinyl alcohol.

[0006] This invention provides a method for preparing the ligustrazine coating agent, comprising the following steps: weighing 1650 mg of TMP raw material, adding 10 mL of anhydrous ethanol, 2 mL of glycerol, 0.5 mL of Tween 80, and 0.5 mL of azone, stirring to dissolve, and then adding polyvinyl alcohol that has swollen to a gel state (taking 5 g of polyvinyl alcohol, adding an appropriate amount of water, heating to swell to 30 mL), and stirring at 2000 r·min -1Vortex for 5 minutes to mix thoroughly. The coating solution contains 50 mg / mL of the drug. -1 , stored at 4°C.

[0007] This invention also provides a method for detecting the plasma drug concentration of the aforementioned ligustrazine coating agent after administration. This method employs high-performance liquid chromatography (HPLC) using a Symmetry-C18 column (4.6 x 250 mm, 5 μm). Mobile phase A is water, and mobile phase B is acetonitrile, with gradient elution: 0 min to 20 min, 5% B to 41% B. During gradient elution, the sum of the percentages of mobile phase A and mobile phase B is 100%. The flow rate is 1.0 mL / min. -1 The detection wavelength was 280 nm; the column temperature was 35℃; and the injection volume was 5 μL.

[0008] In some embodiments, the method for detecting the plasma drug concentration after administration of the tetramethylpyrazine film-coated agent includes the following steps:

[0009] 1) Plasma sample processing: Take 2 mL of drug-containing blood and place it in a 3 mL heparin sodium anticoagulant blood collection tube, and stir at 3500 r·min -1 Centrifuge for 10 min, collect the supernatant and add 90% acetonitrile at a 1:1 ratio to precipitate the protein, vortex to mix, and then centrifuge at 10000 r·min. -1 Centrifuge for 10 min and collect the supernatant;

[0010] 2) Accurately weigh 10.0 mg of tetramethylpyrazine reference standard, dissolve it in 90% acetonitrile, and dilute to volume in a 50 mL volumetric flask to prepare a solution with a mass concentration of 200 μg·mL⁻¹. -1 The standard stock solution of ligustrazine was prepared by serially diluting the above-mentioned ligustrazine standard stock solution with blank plasma to 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 μg·mL. -1 After processing according to the method in step 1), HPLC determination was performed. The TMP concentration C was plotted on the x-axis and the peak area A was plotted on the y-axis. Linear regression was then performed, and the regression equation was calculated to obtain A = 12.856C + 16.055.

[0011] 3) Perform HPLC analysis on the plasma sample from step 1) to obtain the peak area of ​​tetramethylpyrazine, and then use the peak area to calculate the concentration of tetramethylpyrazine in the plasma sample using the regression equation in step 2).

[0012] In some embodiments, the blood sample in step 1) is taken from the abdominal aorta after administration of ligustrazine film-coated agent.

[0013] The beneficial effects of this invention are as follows: The self-made tetramethylpyrazine film-coated agent of this invention can maintain a relatively stable and high blood drug concentration in vivo for a long time. Under the premise of equivalent dosage, the self-made TMP film-coated agent can exceed the AUC of intravenous injection and the peak drug concentration is higher than that of gavage in the corresponding time. The formulation of this film-coated agent is reasonable and has good permeability. At the same time, the HPLC method provided by this invention is accurate, sensitive and rapid for determining blood drug concentration and can be used for the determination of tetramethylpyrazine blood drug concentration. Attached Figure Description

[0014] Figure 1 HPLC chromatograms: A - blank plasma; B - blank plasma + TMP; C - plasma sample

[0015] Figure 2 TMP plasma concentration-time curve Detailed Implementation

[0016] The following embodiments are intended to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the invention to the scope of the embodiments described.

[0017] 1. Instruments and Reagents

[0018] 1.1 Instruments

[0019] Agilent 1260 Infinity high-performance liquid chromatograph (Agilent Technologies, USA); TCL16M benchtop high-speed refrigerated centrifuge (Hunan Xiangli Scientific Instruments Co., Ltd.); VORTEX-5 vortex mixer (Haimen Qilin Bell Instrument Manufacturing Co., Ltd.).

[0020] 1.2 Drug Testing

[0021] TMP reference standard (China National Institutes for Food and Drug Control, batch number: 110817-201608); TMP active pharmaceutical ingredient (Shenzhen Lijing Biochemical Technology Co., Ltd.); film-forming agent matrix provided by the National-Local Joint Engineering Research Center for Medicinal Special Insects of Dali University; acetonitrile (chromatographic grade, TEDIA, USA); all other reagents were of analytical grade, and water was ultrapure water.

[0022] 1.3 Animals

[0023] Clean-grade SD rats, male, weighing 250-280 g, provided by Hunan Slack Jingda Experimental Animal Co., Ltd., license number: SCXK (Yunnan) 2016-0002.

[0024] 2. Experimental Methods and Results

[0025] 2.1 Chromatographic conditions

[0026] Chromatographic column: Symmetry-C18 (4.6 x 250 mm, 5 μm, 120 Å) column; mobile phase: water (A) - acetonitrile (B); gradient elution: 0 min to 20 min, 5% - 41% B; flow rate: 1.0 mL / min -1 Detection wavelength: 280 nm; Column temperature: 35℃; Injection volume: 5 μL.

[0027] 2.2 Preparation of coating agent

[0028] Weigh 1650 mg of TMP raw material, add 10 mL of anhydrous ethanol, 2 mL of glycerol, 0.5 mL of Tween 80, and 0.5 mL of azone. After stirring to dissolve, add polyvinyl alcohol that has swollen to a gel state (take 5 g of polyvinyl alcohol, add an appropriate amount of water, heat to swell to 30 mL), and stir at 2000 r·min. -1 Vortex for 5 minutes to mix thoroughly. The coating solution contains 50 mg / mL of the drug. -1 , stored at 4°C.

[0029] 2.3 Plasma Sample Processing

[0030] Take 2 mL of blood containing the drug and place it in a 3 mL heparin sodium anticoagulant blood collection tube. Stir at 3500 r·min -1 Centrifuge for 10 min, collect the supernatant and add 90% acetonitrile at a 1:1 ratio to precipitate the protein, vortex to mix, and then centrifuge at 10000 r·min. -1 Centrifuge for 10 min, collect the supernatant and inject it for analysis.

[0031] 2.4 Specificity Examination

[0032] Blank plasma, plasma with added TMP standard, and plasma treated in section "2.3" were injected separately, and their HPLC chromatograms were determined. (See attached figures.) Figure 1 The results showed that under these chromatographic conditions, the TMP peak shape in the blood sample was good, the resolution was good, and the elution time was moderate. Plasma and the coating matrix did not interfere with the determination of TMP, proving that the method has good specificity.

[0033] 2.5 Preparation of standard curve and determination of detection limit and quantitation limit

[0034] Accurately weigh 10.0 mg of TMP reference standard, dissolve it in 90% acetonitrile, and dilute to volume in a 50 mL volumetric flask to prepare a solution with a mass concentration of 200 μg·mL⁻¹. -1 The standard stock solution.

[0035] The above TMP standard stock solution was serially diluted with blank plasma to 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 μg·mL.-1 After processing according to the method in section "2.1", the concentration of TMP (C) was plotted on the x-axis and the peak area (A) on the y-axis, and linear regression was performed. The regression equation was calculated as A = 12.856C + 16.055 (n = 5, r = 0.999). Based on the signal-to-noise ratio S / N ≥ 3, the detection limit is 0.0977 μg·mL. -1 The limit of quantification for an S / N ≥ 10 is 0.3906 μg·mL. -1 The results showed that TMP was effective in the range of 1.5625-100 μg·mL⁻¹. -1 The internal linear relationship is good.

[0036] 2.6 Precision Test

[0037] Take an appropriate amount of blank plasma and prepare standard stock solutions at concentrations of 100, 50, and 3.125 μg / mL using the solutions described in section "2.5". -1 Three concentration samples (high, medium, and low) were processed according to the method in section "2.3" and measured five times within a day for three consecutive days. The intra-day and inter-day precision were calculated and shown in Table 1. The results showed that the deviation of this method was low, and the intra-day and inter-day precision RSDs were both <2%, which met the requirements for biological sample analysis.

[0038] Table 1. Precision results of HPLC determination of TMP in rat plasma

[0039]

[0040] 2.7 Recovery Rate Examination

[0041] Take an appropriate amount of blank plasma and prepare standard stock solutions at concentrations of 100, 50, and 3.125 μg / mL using the solutions described in section "2.5". -1 Three concentration samples (high, medium, and low) were prepared according to the method described in section "2.3" and then analyzed. Separate standard stock solutions were prepared to concentrations of 100, 50, and 3.125 μg·mL. -1 The standard solutions were injected in equal volumes, and the relative recovery and extraction recovery were calculated, as shown in Table 2. The results showed that the recoveries were all >85%, RSD <4%, indicating good reproducibility and meeting the requirements for biological sample analysis.

[0042] Table 2. Results of HPLC determination of TMP in rat plasma

[0043]

[0044] 2.8 Stability Test

[0045] Take an appropriate amount of blank plasma and prepare standard stock solutions at concentrations of 100, 50, and 3.125 μg / mL using the solutions described in section "2.5". -1 Three concentrations of samples (high, medium, and low) were processed according to the method described in section "2.3" and their stability was investigated under room temperature, frozen storage, and repeated freeze-thaw cycles. Five replicates of each concentration were performed. The results showed that after 24 hours at room temperature, the TMP concentration deviation in the blood samples was <2%; after 5 days at -20℃, the TMP concentration deviation was <4%; and after three freeze-thaw cycles, the TMP concentration deviation was <5%, demonstrating good sample stability.

[0046] 2.9 Dosing regimen and sample collection

[0047] Five male SD rats were fasted for 12 hours before administration but allowed free access to water. The rats' back hair was shaved before administration, and 400 μL of the drug was applied to the skin of their backs, covering an area of ​​2 × 3 cm². 2 Blood samples were collected from the abdominal aorta at 15, 30, 45, 60, 75, 90, 120, 150, and 180 minutes after drug administration. The blood samples were processed and injected according to the method described in section "2.3". Peak areas were measured, and concentrations were calculated. A blood drug concentration-time curve was plotted with blood drug concentration (C) on the ordinate and drug administration time (t) on the abscissa. (See figure...) Figure 2 Data processing was performed using DAS 2.0 software. Based on AIC and goodness of fit, the model was determined to be compatible with a two-compartment model, with a weight of 1 / C. 2 The main pharmacokinetic parameters were obtained, as shown in Table 3.

[0048] Table 3. Main pharmacokinetic parameters

[0049]

[0050] 3. Conclusion

[0051] Previous literature has reported on intravenous injection and gavage administration of 80 mg / kg to rats. -1In this study, the TMP in vivo pharmacokinetics of TMP was investigated. The invention employs a transdermal application of a film-like formulation, with each rat receiving 20 mg of TMP. The dosages are similar to those used in intravenous administration, making them comparable. This study found that the self-prepared TMP film-like formulation surpasses the AUC of intravenous injection within a certain timeframe, with a higher peak concentration than gavage, demonstrating the rationality of the formulation and its good permeability. Clinically, due to TMP's short half-life and rapid metabolism, high doses are often administered to achieve therapeutic effects, leading to adverse reactions in the nervous, respiratory, and digestive systems. Frequent administration also results in significant fluctuations in blood drug concentrations, posing a significant risk, especially to elderly patients with cardiovascular and cerebrovascular diseases. The TMP film-like formulation developed in this invention maintains a relatively stable blood drug concentration over a prolonged period. Furthermore, the drug content in vivo after transdermal administration is generally positively correlated with the administration area; a larger administration area results in a higher drug content. The smaller administration area used in this invention allows for a higher drug loading capacity in the film-like formulation, which is expected to meet clinical treatment needs.

[0052] Research on transdermal drug delivery (TMP) has progressed from compound decoctions to drop pills, nasal preparations, and sustained-release formulations, with developers continuously improving its efficacy and reducing adverse reactions. Our method has been examined and found to meet the methodological requirements for quantitative drug analysis in biological samples. This transdermal drug delivery system, using a film-coated agent as a carrier, is low-cost, convenient to administer, and provides slow-release with high concentrations, demonstrating broad development prospects.

Claims

1. A tetramethylpyrazine coating agent comprising tetramethylpyrazine, anhydrous ethanol, glycerin, Tween, azone and polyvinyl alcohol.

2. The preparation method of the tetramethylpyrazine film-forming agent according to claim 1, comprising the following steps: weighing 1650 mg of TMP raw material, adding 10 mL of anhydrous ethanol, 2 mL of glycerol, 0.5 mL of Tween 80, and 0.5 mL of azone, stirring to dissolve, and then adding polyvinyl alcohol swollen to a gel state (taking 5 g of polyvinyl alcohol, adding an appropriate amount of water, heating to swell to 30 mL), at 2000 r·min -1 Vortex for 5 minutes to mix thoroughly. The coating agent contains 50 mg / mL of the drug. -1 , stored at 4°C.

3. The method for detecting the plasma drug concentration of ligustrazine film-coated agent after administration according to claim 1, wherein the method employs high performance liquid chromatography (HPLC) using a Symmetry-C18 column with dimensions of 4.6 x 250 mm and a diameter of 5 μm; mobile phase A is water, mobile phase B is acetonitrile, and gradient elution is used: 0 min to 20 min, 5% B to 41% B, wherein the sum of the percentages of mobile phase A and mobile phase B during gradient elution is 100%; the flow rate is 1.0 mL / min. -1 The detection wavelength was 280 nm; the column temperature was 35℃; and the injection volume was 5 μL.

4. The detection method according to claim 3, characterized in that, The method for detecting the plasma drug concentration after administration of the ligustrazine film-coated agent includes the following steps: 1) Plasma sample processing: Take 2 mL of drug-containing blood and place it in a 3 mL heparin sodium anticoagulant blood collection tube, and stir at 3500 r·min -1 Centrifuge for 10 min, collect the supernatant and add 90% acetonitrile at a 1:1 ratio to precipitate the protein, vortex to mix, and then centrifuge at 10000 r·min. -1 Centrifuge for 10 min and collect the supernatant; 2) Accurately weigh 10.0 mg of tetramethylpyrazine reference standard, dissolve it in 90% acetonitrile, and dilute to volume in a 50 mL volumetric flask to prepare a solution with a mass concentration of 200 μg·mL⁻¹. -1 The standard stock solution of ligustrazine was prepared by serially diluting the above-mentioned ligustrazine standard stock solution with blank plasma to 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 μg·mL. -1 After processing according to the method in step 1), HPLC determination was performed. The TMP concentration C was plotted on the x-axis and the peak area A was plotted on the y-axis. Linear regression was then performed, and the regression equation was calculated to obtain A = 12.856C + 16.

055. 3) Perform HPLC analysis on the plasma sample from step 1) to obtain the peak area of ​​tetramethylpyrazine, and then use the peak area to calculate the concentration of tetramethylpyrazine in the plasma sample using the regression equation in step 2).

5. The detection method according to claim 4, characterized in that, The blood sample in step 1) is taken from the abdominal aorta after administration of ligustrazine film-coated agent.

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