Development Of A Quantitative Method For Levonorgestrel In Rat Plasma And Its Inhibitory Effect On CYP450 Enzymes

Background In order to achieve the long-term and reversible contraception levonorgestrel sustained-release preparations have emerged continuously in recent years,such as levonorgestrel injectable long-acting drug delivery systems,transdermal patch,subcutaneous implant and so on.These dosage forms have the characteristics of less frequent use,longer action time and low plasma steady state concentration.Drug registration regulations require that preclinical pharmacokinetic study data of rats and other rodents should be provided before clinical trials of new slow and controlled release preparations,so as to support the formulation and implementation of clinical drug regimen.Blood drug concentration data at about 12 time points should be obtained to complete this study.Since the circulating blood volume of rats is generally 16 m L,the blood volume collected each time should not be higher than 0.3 m L.At the same time,for the analysis method of detecting biological samples,the lower limit of quantification should be the 1/10-1/20 of peak concentration(C_max).Currently,the analytical methods used in the research are to increase the amount of plasma to meet the requirements of the lower limit of quantification,but the large volume of blood collection is not only difficult to meet the relevant guidelines,but also easy to lead to inaccurate experimental results and fail to meet the new drug registration standards.In order to comprehensively evaluate the pharmacokinetic behavior of levonorgestrel in rats,it is necessary to establish a high-throughput quantitative levonorgestrel analytical method with high sensitivity and small plasma sample dosage.In addition,the new drug approval regulations also require that preclinical studies of new drugs should observe the inhibition of drug metabolic enzymes,especially cytochrome P450（CYP450）.The first compound preparation of levonorgestrel was published in 1960 without such regulation at that time.Over the years,studies have focused on the effect of levonorgestrel combined with other drugs on the activity of CYP450 enzyme,but no reports on the direct inhibitory effect of levonorgestrel on various CYP450 enzyme subtypes.Objective In order to meet the requirements of analytical methods in the study of new levonorgestrel preparation in vivo,a quantitative analytical method of levonorgestrel biological samples with low blood volume,high sensitivity and short analysis time was established in this study.At the same time,the Half Maximal inhibitory concentration(IC₅₀)of levonorgestrel against six major CYP450 enzyme subtypes,namely CYP1A2,CYP2D6,CYP2B6,CYP2C9,CYP2C19 and CYP3A4,was also investigated.Method This study used the technology of liquid chromatography-mass spectrometry（LC-MS/MS）and liquid-liquid extraction.The reduced consumption of biological samples was achieved via the addition of 0.1 mol/L sodium carbonate on the basis of incompatible liquid phase to increase hydrogen ion concentration to inhibit levonorgestrel ionization and enhance the extraction purification effect.The chromatography column selected ZORBAX SB-C₁₈（5μm,4.6×150 mm,Agilent）.The analytes and internal standard were separated on a via gradient elution with mobile phase consisting of acetonitrile-2 mmol/L ammonium acetate containing 0.1%formic acid aqueous solution.The mass spectrometry detection was performed in multiple reaction monitoring mode,electric spray ion source（ESI）and positive ion detection mode.The product ions used for quantitative analysis of mass-to-charge ratio（m/z）313.5→m/z 245.2 for levonorgestrel,m/z 285.2→m/z 193.1 for diazepam（internal standard）.The validated method was applied to study the plasma pharmacokinetics of levonorgestrel in rats to further verify the practicability of the method.The inhibition of levonorgestrel on a variety of CYP450 enzymes was studied.We established human liver microsomes incubation system（HLM）for levonorgestrel and LC-MS/MS analysis method for quantitation of the metabolites of CYP450 enzymes substrate to assess the inhibitory effect of levonorgestrel on CYP450 enzymes according IC₅₀ value.Results In validation of the bioanalytical method,the linear calibration curves for levonorgestrel were obtained in the concentration range of 0.025 ng/m L to 25 ng/m L in rat plasma.The lower limit of quantification was 25 pg/m L,the time of the method was3.5 min,and the blood volume was 50μL.The precision and accuracy of the samples to be measured were in line with the requirements of biological sample analysis.The pharmacokinetics results of levonorgestrel in rats after administration of 1.0 mg were as follows:the half-life(t_1/2)was 1.77 h,the peak concentration(C_max)was 36.53 ng/m L,and the area under curve（AUC）was 115.46 ng/m L·h.They are consistent with literature reports.The precision and accuracy of the LC-MS/MS analysis method established in this study for the metabolites of CYP450 enzyme substrate were in line with the requirements of biological sample analysis,and could be used for the quantitative analysis of the metabolites of CYP450 enzyme substrate.The IC₅₀ value of levonorgestrel for CYP1A2,CYP2D6,CYP3A4,CYP2C9,CYP2C19 and CYP2B6 is2.02E+09μmol/L,1.30E+08μmol/L,59.58μmol/L and 67.99μmol/L,3.94E+03μmol/L,1.10E+05μmol/L,1.33E+03μmol/L are all greater than 50μmol/L.Conclusion The high sensitivity LC-MS/MS analysis method established in this study has the characteristics of rapid,exclusive,accurate,high sensitivity and low blood consumption,thus achieving the high throughput quantitative determination of levonorgestrel in rat plasma.At the same time,levonorgestrel has no inhibitory effect on CYP1A2,CYP2D6,CYP3A4,CYP2C9,CYP2C19 and CYP2B6 metabolic enzymes,and the probability of drug-drug interaction is low.