Development of simultaneous determination method of a pregnan steroid with gestagenic activity – gestobutanoil and two its metabolites in rat serum

Introduction. Gestobutanoil is a synthetic pregnane steroid with gestagenic activity. Gestobutanoil has two pharmacologically active metabolites (AMOL and megestrol acetate). This implies the need for a detailed study of the kinetics of metabolites. It is rational to combine the study of the pharmacokinetics of gestobutanoil and its metabolites (AMOL and megestrol acetate). The simultaneous determination of several analytes in the rats’ serum can be carried out using chromatography-mass-spectrometry. Aim. Development of an analytical method for the simultaneous determination of gestobutanoil and two its metabolites in a biomatrix (rat serum). Materials and methods. The following methods were used to determine gestobutanoyl and two its metabolites in a biological matrix: GC-MS, HPLC-ESI-MS, HPLC-ESI-MS with derivatization, HPLC-APCI-MS. Results and discussion. When working with GC-MS, the chromatographic peaks of gestobutanoyl, AMOL, and megestrol acetate were strongly blurred and superimposed on each other, which is apparently due to the thermolability of the substances. The GC-MS method was abandoned in favor of HPLC. Analytes were separated by HPLC gradient elution on a C18 column. ESI ionization did not give typical protonated ions of gestobutanoyl and AMOL, and the intense signals of their cationized ions and fragment ions, which were observed in the spectra of AMOL and gestobutanoyl, could not ensure the reproducibility of the spectra, since the conditions of their formation are not suitable for routine analysis. Derivatization of analytes to form oximes and substituted hydrazones did not give the expected reaction products for HPLC-ESI-MS. APCI made it possible to remove intense cationized ions from the spectra of gestobutanoyl and AMOL and to increase the reliability of the method. The HPLC-APCI-MS technique was reproduced on model rat blood serum. Conclusion. An HPLC-MS method was developed for the simultaneous determination of gestobutanoyl, megestrol acetate, and AMOL. The technique was tested on a model rat blood serum containing all three analytes. © Stepanova E. S., Makarenkova L. M., Goryainov S. V., Fedotcheva T. A., Shimanovsky N. L., 2021

Metabolism of gestobutanoil, a novel drug of progestin group

The aim of the study is to evaluate the metabolism of progestin drug Gestobutanoil in the experiment with administration of tablet dosage form containing Gestobutanoil (2 mg), to experimental animals (rats and rabbits). Materials and Methods. There was performed analysis of biomatrix obtained from different species of animals: female rats weighing 200.0±60.0 g and female rabbits weighing 3.0±0.2 kg, which were administered different doses of the drug, single or multiple. Metabolites were identified using high performance liquid chromatography-mass spectrometry (HPLC-MS). Results. The analysis shows that Gestobutanoil is rapidly metabolized into 17α-acetoxy-3β-hydroxy-6-methylpregna-4,6-dien-20-one (AMP-17) and 17-hydroxy-6-methylpregna-1,4-diene-3,20-dione in the form of acetate (MA). The steroid core of Gestobutanoil has the butyric acid radical in the 3β position. This radical cleavage underlies biotransformation of Gestobutanoil. The obtained pharmacokinetic parameters for metabolites have demonstrated that Gestobutanoil has a stepwise nature of metabolism: the time to reach the maximum concentration of AMP-17 is 1.5 h, MA — 3 h. Also AMP-17 proves to penetrate into the peripheral tissues better than MA. Conclusion. The data obtained speak of a unique, different from other gestagens, metabolism of Gestobutanoil. Unlike the known progestogen medroxyprogesterone acetate whose main route of transformation is hydroxylation of the steroid nucleus of the molecule with rather high bioavailability in an unchanged state, Gestobutanoil shows rapid biotransformation into metabolites AMP-17 and MA manifesting their own gestagenic activity with release of butyric acid, which, in turn, may produce a calming effect on the central nervous system. © 2019, Privolzhsky Research Medical University. All rights reserved

Evaluation of the toxicity of new progestogen gestobutanoil in experiments on rats and mice

A toxicological study of gestobutanoil tablets containing 0.002 g of progestogen 17a-acetoxy-3b-hydroxy-6-methylpregna-4,6-dien-20-one in the form of butyl ester as an active pharmaceutical substance for peroral administration was carried out on mice and rats of both sex. It was found that LD 50 of the drug exceeded 5 g/kg, which indicated low toxicity of gestobutanoil tablets. Chronic intragastric administration of gestabutanoil in doses of 2.5 and 25 mg/kg for 30 days did not cause clinically significant changes in the integral state of animals, biochemical and hematological parameters, except for an increase in the concentration of direct bilirubin in the blood by 37-40% (p - 0,01), which is a characteristic biochemical feature of gestagen-containing drugs. The observed tendency to decrease in the locomotor, orientation, and research activity and emotional behavior of female rats upon the administration of gestabutanoil for a month indicates its possible antistress effect on the central nervous system of rats. A specific effect of the drug in the studied doses 2.5 and 25 mg/kg on the target organs (uterus and ovaries) was revealed, as manifested by decidualization of the endometrium and a decrease in folliculogenesis in the ovaries. Since no toxic effect was observed in doses of 2.5 and 25 mg/kg (which exceeded the therapeutic dose 10 and 100 times, respectively), die equivalent dose for humans can be 0.064 mg/kg/day, according to the animal-to-human dose conversion formula. © 2018 Izdatel'stvo Meditsina. All rights reserved

ВЭЖХ-МС методика одновременного количественного определения инновационного отечественного гестагена и его метаболитов в сыворотке крови крыс и кроликов

A method for simultaneous quantitative determination of a new gestagenic pharmacological agent and its two metabolites in the blood serum of rats and rabbits by HPLC-MS has been developed and validated. The method is selective and specific. The detection limits of all analytes were 5 ng/mL and the lower limits of quantification were 10 ng/mL. The calibration curves for all analytes had the form of y = ax + b with correlation coefficients > 0.99. The matrix effect and degree of extraction were studied, as well as the stability of analytes in standard solutions, ready-for-analysis samples, and biomatrix during repeated freezing and thawing cycles.Разработана и валидирована ВЭЖХ-МС методика одновременного количественного определения нового гестагенного фармакологического средства и 2 его метаболитов в сыворотке крови крыс и кроликов. Метод селективен и специфичен. Предел обнаружения для всех аналитов составил 5 нг/мл, нижний предел количественного определения - 10 нг/мл. Для всех аналитов калибровочные кривые имели вид: y = ax + b. Коэффициенты корреляции > 0,99. Изучены эффект влияния матрицы и степень извлечения, а также стабильность аналитов в стандартных растворах, готовых пробах и биоматрице при многократном замораживании и размораживании

HPLC-MS Method for Simultaneous Quantitative Determination of an Innovative Russian Gestagen and its Metabolites in Rat and Rabbit Blood Sera

An HPLC-MS method for simultaneous quantitative determination of a novel gestagenic pharmaceutical and two of its metabolites in rat and rabbit blood sera was developed and validated. The method was selective and specific. The detection limit for all analytes was 5 ng/mL; lower limit of quantitation, 10 ng/mL. Calibration curves for all analytes had the form y = ax + b. The correlation coefficients were >0.99. Matrix effects, degree of extraction, and stability of analytes in standard solutions, analytical samples, and the biomatrix with multiple freeze—thaw cycles were studied. © 2019, Springer Science+Business Media, LLC, part of Springer Nature