Scaling of the technology of obtaining the hydroxyaluminium tetra-3-phenylthiophthalocyanine liposomal form and studying its mutagenic and immunotoxic properties

The use of liposomes as drug delivery systems today is a recognized approach to improving the effectiveness of treatment. Compared with other nanosized carriers, liposomes are highly biocompatible. However, in the literature, there are a number of data on the immunogenicity of some liposomal drugs. Hypersensitivity reactions of moderate to severe severity were noted in some patients with intravenous administration of liposomes Doxil®, Ambisome®, DaunoХome®. The technology of production of liposomes is a laborious process and obtaining liposomal forms on an industrial scale is problematic for researchers and manufacturers around the world. In connection with this, studies on the scaling up of technology for obtaining liposomes and studying the specific toxicity of liposomal preparations are relevant. Objective – evaluation of the quality of prototypes of liposomal dosage form based on tetra-3-phenylthiophthalocyanine hydroxyaluminum obtained by scaled technology in comparison with samples produced in laboratory conditions, as well as studying their mutagenic and immunotoxic properties. Object of the study – tetra-3-phenylthiophthalocyanine hydroxyaluminum liposomal, lyophilizate for the preparation of injection for injection of 1,5 mg, in bottles with a capacity of 20 ml (LLF). Materials and methods. Bengem’s method, extrusion, sterilizing filtration, lyophilization, intercellular contact integrity test (for promoter activity), Ames bacterial test, cytogenetic study of chromosomal aberrations, DNA damage test (DNA comet test), Erne method; hypersensitivity reaction of delayed type, determination of mass and cellness of central and peripheral immunity organs, evaluation of phagocytic activity of peritoneal macrophages. Results. Based on the results of the research, the LLF production technology is scaled, which allows obtaining up to 400 LLF flasks per production cycle. It has been established that LLF at doses of 6 and 24 mg/kg does not cause an increase in the number of chromosomal abnormalities in mice and does not cause DNA damage, in doses of 1,1–22,0 μg/dish LLF does not show mutagenic activity in the Ames bacterial test, in the range concentrations of 0,062–3,1 μg in the test for promoter activity does not separate intercellular contacts. It is determined that in doses of 6 and 12 mg/kg, LLF does not affect humoral and cellular immunity, does not change the mass and cellularity of the central and peripheral organs of the immune system. LLF, 24 hours after administration at doses of 6 and 12 mg/kg, dose-dependently decreases the phagocytic activity of peritoneal macrophages, which is restored after 7 days. Conclusion. The technology of LLF production is scaled. In the doses studied, it was found that LLF does not have the ability to promote the process of carcinogenesis and the lack of influence on the humoral and cellular immune response.

1. Maeda H.,WuJ.,Sawa T.et al.Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J Control Rel 2000; 65:271–84. doi:10.1016/S0168-3659(99)00248-5.

2. Wick P.,Manser P.,Limbach L.K.et al. The degree and kind of agglomeration affect carbon nanotube cytotoxicity. Toxicol Lett 2007; 168(2):121–31. doi:10.1016/j.toxlet.2006.08.019.

3. Ziemba B.,Matuszko G., Bryszewska M.,Klajnert B. Influence of dendrimers on red blood cells. Cell Moll Biol Lett 2012; 17(1):21–35. doi:10.2478/s11658-011-0033-9.

4. Аляутдин Р.Н.,Романов Б.К. Рекомендации по оценке безопасности лекарственных средств,содержащих наночастицы. Безопасность и риск фармакотерапии 2015; 4:10–22.

5. Szebeni J.,Muggia F.,Gabizon A., Barenholz Y.Activation of complement by therapeutic liposomes and other lipid excipient-based therapeutic products: prediction and prevention. Adv Drug Deliv Rev 2011;63(12):1020–30. doi:10.1016/j.addr.2011.06.017.

6. Brissette J.L.,Kumar N.M.,Gilula N.B., Dotto G.P. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the ras oncogene modulate expression and phosphorylation of gap junction proteins. Mol Cell Biol 1991; 11:5364–71.

7. Abbaci M.,Barberi-Heyob M.,Blondel W. et al. Advantages and limitations of commonly used methods to assay the molecular permeability of gap junctional intercellular communication .BioTechniques 2008; 45:33–46. doi:10.1128/MCB.11.10.5364.

8. Na M.R.,Koo S.K.,Kimd.Y. et al.In vitro inhibition of gap junctional intercellular communication by chemical carcinogens. Toxicology 1995; 98:199–206. doi:10.1016/0300-483X(94)02931-J.

9. Maron D.M.,Ames B.N. Revised methods for the Salmonella mutagenicity test. Mutat Res 1983;113(3–4):173–215. doi:10.1016/0165-1161(83)90010-9.

10. Руководство по проведению доклинических исследований лекарственных средств. Методические указания по оценке мутагенных свойств фармакологических препаратов. М.: ГрифиК, 2012. Ч.1. 944с.

11. Белицкий Г.А.,Ревазова Ю.А., Абилев С.К. и др. Методические рекомендации по исследованию канцерогенных свойств фармакологических и лекарственных средств. Ведомости Фармакологического комитета 1998; 1:21–4.

12. Фонштейн Л.М.,Абелев С.К., Бобринев Е.В. и др. Методы первичного выявления генетической активности загрязнителей среды спомощью бактeриальных тест-систем (Методические указания). М.,1983.34с.

13. Ford C.E.,Hamerton J .L. Chromosomes of five rodent species. Nature 1956; 31:247–54.

14. Оценка генотоксических свойств методом ДНК-комет in vitro. Методические рекомендации. М.: Федеральный центр гигиены и эпидемиологии Роспотребнадзора,2010.

15. Olive P.L.,Banath J.P. The comet assay: a method to measure DNA damage in individual cells. Nat Protoc 2006; 1(1):23–9. doi:10.1038/nprot.2006.5.

16. Budunova I.V., Mittelman L.A., Belitsky G.A. Identification of tumor promoters by their inhibitory effect on intercellular transfer of lucifer yellow. Cell Biol Toxico l1989;5(1):77–89. doi:10.1007/BF00141066.

17. Ostling O.,Johanson K.J. Microelectrophoretic study of radiation-induced DNA damage in individual mammalian cells. Biochem Biophys Res Commun 1984;123:291–8. doi:10.1016/0006-291X(84)90411-X.

18. Иванова А.С.,Мастернак Т.Б., Мартынов А.И. Принципы изучения иммунотоксического действия фарма кологических препаратов. Токсикологический вестник 2010;5:26–31.

19. Иванова А.С.,Мастернак Т.Б.,Малкина Е.Ю., Мартынов А.И. Принципы оценки иммунологической безопасности фармацевтических продуктов. Биомедицина 2011;(3):94–7.

20. Jerne N.K., Nordin A.A. Plaque formation in agar by single antibody-producing cells. Science 1963;140:405.

21. Подоплелов И.И.,Крылов О.Р., Медуницын Н.В. Эванс синий как адъювант для получения повышенной чувствительности замедленного типа в эксперименте. Бюллетень экспериментальной биологии и медицины 1985;6:729.

22. Raz A.,Bucana C.,Fogler W.E.et al. Biochemical,Morphological,and Ultrastructural Studies on the Uptake of Liposomes by Murine Macrophages. Cancer Res 1981;41:487–94.PMID:7448797.