The purpose of this paper is an analysis of technology for obtaining liposomal cytostatics from the point of view of the Quality by Design approach. Liposomes were manufactured by a lipid film with further high-pressure extrusion method. Number of critical stages of technology that are necessary for the study have been circled during the pharmaceutical development. Optimization of the intraliposomal pH was demonstrated by the example of obtaining the liposomal doxorubicin hydrochloride. The study of the high pressure extrusion parameters was carried out in the development of the liposomal oxaliplatin technology. An optimization of lipid bilayer composition is shown for liposomal irinotecan. It was shown that the optimal pH value for the liposomal form of doxorubicin hydrochloride was at 3 ± 0.5. The optimal composition of the lipid bilayer for the liposomal form of irinotecan is the Egg phosphatidylcholine/Cholesterol ratio, 80/20% (wt.). Which provides a liposome size of 108 ± 3.1, and the degree of encapsulation of irinotecan hydrochloride into liposomes was 87 ± 0.8%. The oxidation index was studied on model samples of liposomal forms of irinotecan hydrochloride (Io = 0.372) and oxaliplatin (Io = 0.323).

An integral part of the original medicines development in pharmaceutical industry is a thorough, coordinated procedure for pharmaceutical technologies reproduction at various stages of development and production – technology transfer.Aim. To study and to analyze the frequent transitions in the pharmaceutical industry, to investigate the problems of technology scaling observed during the transfer, in the manufacture of nanosized dosage forms, and to propose ways to solve them.Materials and methods. For liposomes preparation it was purchased egg phosphatidylcholine from Lipoid, Germany. Lyophilization was carried out in the Quarco model, P.R.C. The encapsulation degree was determined by HPLC on a Shimadzu LC-20 instrument manufactured in Japan, according to a method developed earlier.Results. Liposomal drugs related to complex structured drugs, and the transfer process during their development is one of the stages of development and requires an additional adaptation experiment. We were tasked with make an experience gained in the transfer and scaling of the production of liposomal cytostatics. Additional experiments were carried out, allowing the results of primary pharmaceutical development to be transferred to industrial equipment without loss of quality. Features of the scale of such technological processes as a lipid film obtaining, high pressure extrusion, ultrafiltration, sterilizing filtration and freeze drying was analyzed.Conclusions. As a result of the conducted researches the basic variants of technology transfer during a life cycle of the finished medical form were analyzed. The peculiarities of the transfer and scaling of nanosized liposomal formulation technology, in particular the production of a lipid film, estrusion at high pressure, ultrafiltration, sterilizing filtration and lyophilic drying are considered.

In this article the results from studies of liposomes with the inclusion of active pharmacological substances (irinotecan, oxaliplatin, cytochrome C, doxorubicin, quercetine, etc.) in lipid nanoparticles are introduced. The key techniques of adding substances to liposomes are observed: the lipid-film method and the gradient and chemical-bond methods. The main conditions for the inclusion of drugs in liposomes are presented. Questions of determining the inclusion in liposomes and the synthesis and control of the liposomal drugs are discussed. The synthesized drugs are currently undergoing preclinical and clinical trials. Some of the proposed drugs have already been registered and have been used in medical practice for more than 25 years.

В статье приведены результаты исследования включения в липидные наночастицы — липосомы фармацевтически активных субстанций (иринотекана, оксалиплатина, цитохрома С, доксорубицина, кверцетина и др.). Рассмотрены основные методы включения субстанций в липосомы: метод липидной пленки, методы градиента и химической связи. Приведены основные условия включения лекарственных веществ в липосомы. Обсуждаются вопросы определения включения в липосомы, получения и контроля готовых лекарственных липосомальных препаратов. Полученные препараты находятся на различных стадиях доклинических и клинических испытаний. Ряд предложенных препаратов зарегистрирован и используются в медицинской практике более 25 лет.

Purpose. For creation of liposomal irinotecan form, the influence of lipid concentration on the encapsulation of the active substance and nanoparticle size was necessary to investigate. Materials and methods. We used «chemical gradient" method for liposomes formulation, in the variety of “pH gradient”. Ammonium citrate at pH 2.5 was used as internal buffer. Lipid film was obtained, by evaporation technics with further high pressure homogenization with Microfluidics Microfluidizer M-110P apparatus. “Chemical gradient” was created by ultrafiltration, with “Minim 2” apparatus. Ultrafiltration cartridge with an upper cut-off 30 kDa was used. Encapsulation was measured using HPLC methods developed in variant of gel chromatography, with Shimadzu LC-20 instrument. The particle size was measured by laser diffraction method withe “Zetasizer Nano ZS” instrument. Results. For the preparation of liposomes was applied a constant lipid ratio with varying of total lipid concentration. The ratio of lipids in the experiment was a phosphatidylcholine / cholesterol 80/20 % by weight. A total concentration was investigated in range from 10 mg/ml to 30 mg/ml. The number of extrusion cycles consisted from 3 cycles at 1500 bar, in case of 10 mg/ml concentration, to 17 cycles at 1500 bar, in case of 30 mg/ml concentration. Conclusions. It was shown that the lipid concentration from 25 mg/ml led to particles formation with size more than 5000 nm, and it was not possible to reduce them by high pressure homogenization method. It was proven that the most optimal, in terms of technology and the final characteristics of liposomes, was lipids in concentration 20 mg/ml. The degree of encapsulation in this case was 82 ± 0.98 %. The size of the liposomes was 106 nm, 5000 nm particles were absent.

The articl is dedicated to development of anticancer drug oxaliplatin in liposomal form. The drug is prospective from point of view of use in clinical practice as a form of low-toxic cytostatic drug for anticancer therapy. The principles for development of the lipid particles and impotence of their size for the formulation with high level of encapsulation is discussed. Parameters of high pressure homogenization techniques were researched. Method was standardized in field of the process parameters. Finished product was characterized by following parameters: size of the liposomes, encapsulation degree and zeta potential.

Today, the development of drug delivery systems is focused on creating of products with improved pharmacological efficacy and safety of action for the patient. Therefore, the development of systems such as liposomes, emulsions and polymer nanoparticles is a promising direction of development of modern pharmacy. The objective of the work: To study the optimal extrusion parameters during liposomal irinotecan creation. For the experiment: egg phosphatidylcholine (Lipoid, Germany); cholesterol (Sigma-Aldrich, USA) were using. Liposomes were obtained by «chemical gradient» method. During the experiment, two homogenization techniques were tested. Sonication and extrusion at high pressure methods were applied to cholesterol modified lipid membranes. It is proved that the sonication method is not applicable because of the particles with diameters greater than 1 000 nm formation. Two different extruders were used. It was determined, that Microfluidics Microfluidiser M-110P more appropriate for preparation of current liposomal emulsion. Chosen extruder can be applicable for preparation on the homogenous liposomal emulsion without the presence of particles larger than 1 000 nm. It was tested extrusion mode for liposomes with the following composition of the lipid bilayer: egg phosphatidylcholine/cholesterol 80/20 by weight. For achieving liposomes with diameter 107 nm it is necessary 7 extrusion cycles at 1 500 bar at 20 °C.

Liposomes with irinotecan by “pH gradient” method were obtained. The method of citrate ion in ultra filtrate by the HPLC was used for the determination of internal volume of Liposomes. It was deter-mined that the internal volume of Liposomes is 10.15 % of the total emulsion volume. The method can be used in the development of liposomes by “pH gradient” using citric acid as an internal buffer.

Methods for creations of oxaliplatin liposomal form in laboratory scale are discussed in this mini-review. Analysis of existed methods has been carried out, and the estimation of their usage in pharmaceutical industry view has been given. Oxaliplatin is one of the modern anticancer medicines, which is used both in monotherapy and in combination with other anticancer agents. One of the disadvantages of oxaliplatin as an anticancer drug is high neuro- and cardiotoxicity, which can be reduced by the creation of its liposomal form. In most methods, PEG-conjugated lipids were used as a part of phospholipids’ bilayer. Cationic liposomes modified with DSPE (distearoyl phosphatidyl ethanolamine)-PEG2000, with composition PC (phosphatidyl choline)/ Chol (cholesterol)/DSPE-PEG2000 (2/1/0.2 molar ratio) showed in vivo higher efficiency against human carcinoma SW480 line on mice both in comparison with a control group and with a free oxaliplatin treated group. Moreover, the absence of cachexia, in case of liposomal oxaliplatin was noted. Also, the influence of Chol on liposomes stability was studied. It was discovered that addition of 40% mol of Chol to liposomes with HSPC (hydrogenated soybean PC)/DSPCPEG 2000 increased encapsulation by 8% within 24 h at 37°C. Comparison of trehalose and L-arginine for liposomes HSPC/Chol/PEG2000 was carried out. Both cryoprotectors showed appropriate stability results in a ratio of 1 : 4 to lipids. As a conclusion, liposomal oxaliplatin is a prospective medicine with less toxicity and higher efficiency against tumors in comparison with a free oxaliplatin.

Methods for creations of oxaliplatin liposomal form in laboratory scale are discussed in this mini-review. Analysis of existed methods has been carried out, and the estimation of their usage in pharmaceutical industry view has been given. Oxaliplatin is one of the modern anticancer medicines, which is used both in monotherapy and in combination with other anticancer agents. One of the disadvantages of oxaliplatin as an anticancer drug is high neuro- and cardiotoxicity, which can be reduced by the creation of its liposomal form. In most methods, PEG-conjugated lipids were used as a part of phospholipids’ bilayer. Cationic liposomes modified with DSPE (distearoyl phosphatidyl ethanolamine)-PEG2000, with composition PC (phosphatidyl choline)/ Chol (cholesterol)/DSPE-PEG2000 (2/1/0.2 molar ratio) showed in vivo higher efficiency against human carcinoma SW480 line on mice both in comparison with a control group and with a free oxaliplatin treated group. Moreover, the absence of cachexia, in case of liposomal oxaliplatin was noted. Also, the influence of Chol on liposomes stability was studied. It was discovered that addition of 40% mol of Chol to liposomes with HSPC (hydrogenated soybean PC)/DSPCPEG 2000 increased encapsulation by 8% within 24 h at 37°C. Comparison of trehalose and L-arginine for liposomes HSPC/Chol/PEG2000 was carried out. Both cryoprotectors showed appropriate stability results in a ratio of 1 : 4 to lipids. As a conclusion, liposomal oxaliplatin is a prospective medicine with less toxicity and higher efficiency against tumors in comparison with a free oxaliplatin.

The article is dedicated to creation and validation of fast analytical method for irinotecan hydrochlorid incapsulation degree into liposomes determination. Such parameters, as Specificity, Linearity, Limit of detection were validated.