Разработка и регистрация лекарственных средств (May 2022)
Development of Dihydroquercetin-based Oral tablets and Evalution of the General Toxic Effect
Abstract
Introduction. Dihydroquercetin is a flavonoid from the group of flavanonols with a wide spectrum of pharmacological activity. Its positive therapeutic profile was established for chronic venous insufficiency, chronic obstructive pulmonary disease and bronchial asthma, for the prevention of stroke. Its use leads to an improvement in the course of atherosclerosis and Alzheimer's disease. In addition, it is characterized by radio-protective, antidote, anti-inflammatory, anti-allergic, reparative, hepatoprotective effects. Currently, there are no drugs containing dihydroquercetin on the domestic pharmaceutical market. However, the pronounced therapeutic potential of dihydroquercetin, the economic and technological availability of its substance indicate the relevance of the development of dosage forms based on this molecule.Aim. Development of the optimal composition and technology of tablets based on dihydroquercetin and assessment of their physico-chemical properties, general toxic effect and safety.Materials and methods. The tablets were obtained by direct compression on a laboratory rotary XS press (Robert Bosch GmbH, Germany). The flowability and bulk density of the powders were checked by tester ERWEKA GT (ERWEKA GmbH, Germany). Tablets hardness and geometric dimensions were checked by tester SOTAX HT1 (SOTAX AG, Switzerland), the friability of the tablets was checked by tester SOTAX F2 (SOTAX AG, Switzerland), disintegration time was checked by tester DISI-A touch (CHARLES ISCHI AG, Switzerland). Pharmaceutical substance dihydroquercetin of the company "Khimiya Drevesiny" was used as an active pharmaceutical substance. Silicated cellulose for direct compression, aerosil 200, sodium starch glycolate and magnesium stearate were used as a excipients. The qualitative and quantitative content of dihydroquercetin in tablets was determined using HPLC-UV (Agilent 1260 Infinity II). The tablets were dissolved in a certain volume of the mobile phase, and then the solution was filtered (0.22 microns PTFE syringe filters, VWR, USA). The following reagents were used: acetonitrile for chromatography and concentrated phosphoric acid, "BDA", Russian Pharmacopoeia XIV, OFS.1.3.0001.15 "Reagents. Indicators". Determination of acute, subacute toxicity and local irritant effect was carried out according to the generally accepted method according to Russian Pharmacopoeia XIV on mature male and female white mice and rats in the laboratory of Pathomorphology and Drug toxicology of the Central Research Laboratory (REC) of the RUDN. The study was performed on 36 healthy sexually mature outbred mice with a body weight of 18–20 g and on 30 healthy sexually mature outbred rats with a body weight of 180–200 g. The drug is administered to animals once intragastrically with the help of an atraumatic probe in the form of a suspension prepared from crushed DQV tablets on 1 % starch gel. The comparison preparation was 1 % starch gel. The duration of observation of laboratory animals is 14 days for the study of acute toxicity and 30 days for the assessment of subacute. Daily clinical examination of the animals was performed, physiological parameters were evaluated, and the condition was recorded. The choice of doses was carried out according to the already available data on toxicity requirements described in the OESD regulatory documentation No. 425. A detailed clinical examination of the animal is carried out in the holding cage, in the hands and in the open area. Note the manifestation and expression, where acceptable, of signs of intoxication. The frequency of the examination depends on the severity of the intoxication pattern, but not less often than on the 2nd, 7th and 14th day of the experiment. Body weight is recorded immediately, on the 2nd, 7th day, and immediately before euthanasia. Deviations in the consumption of feed and water by the animals in the group planting cages are recorded visually on a daily basis. Food deprivation is performed the night before euthanasia. Animals are deprived of food, but not water. The animals are subjected to euthanasia by being placed in an ether-containing desiccation chamber 14 days after the administration of the study drug and the reference drug.Results and discussion. The technological characteristics of the substance of dihydroquercetin for the implementation of the technological process of making tablets based on it have been determined. It has been found that the substance dihydroquercetin has very poor flowability. Four types of excipients were used to improve the technological properties of the active substance. In the course of tabletting, the pressing modes were selected. The optimal pressing force was 5 kN. The depth of filling was – 7 kN. The optimal distance between of the punches was 2.5 mm. The rotor speed was constant at 15 rpm. The speed of the feeder is 25 rpm. Chromatography conditions were selected for the determination of dihydroquercetin in tablets by HPLC-UV method. The lethal dose for a single administration was determined, and the toxic effects of repeated repeated administration of the drug dihydroquercentin in the dosage form of an oral tablet were evaluated. The local irritant effect of the drug during oral administration was evaluated in laboratory animals. In the course of the conducted experiments, the absence of a local irritant effect was established when using tablets based on dihydroquercetin, when determining the toxicity class, the drug is assigned to the III class of "Moderately toxic" substances.Conclusion. The formulation and technology of tablets based on dihydroquercetin have been developed. It was found that the optimal composition for the manufacture of tablets with dihydroquercetin by direct compression should contain a filler silicated cellulose, sodium starch glycolate, aerosilglidant and a sliding agent – magnesium stearate. The HPLC-UV technique for identification and determination of the quantitative content of dihydroquercetin in tablets has been developed and validated. It is shown that in addition to the main peak, the peaks of bioflavones of eriodictyol and quercetin are determined on the chromatogram of tablets. This may contribute to the expansion of the spectrum of pharmacological activity of the tablets obtained. On the basis of the developed composition and production technology of tablets based on dihydroquercetin, an assessment of the general toxic effect and safety of the preparation based on bioflavonoids of the Daurian leaflet was carried out. The average lethal dose of the drug (LD50) was established, which, when administered orally to mice, corresponded to 159 mg/kg. During the study, the clinical picture of intoxication was evaluated. With intragastric administration of the drug, at a dose of 159 mg/kg, the death of 50 % of the animals in the experimental group was observed.
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