Preclinical studies for a cationic liposome formulation containing Il-2 Intended for the treatment of Human Tumors

Human cervical cancer tumours expressing the IL-2 receptor (IL-2R) were induced in the peritoneal cavity of nude mice. The tumours were signifi cantly reduced by the i.p. administration of either free IL-2 or liposomes containing this growth factor. No toxicity was observed in the mice even at the highest doses of IL-2 in liposomes. We did not detect any IL-2 in the blood plasma pointing to a strong retention of the liposomes on the cavity. We concluded that this preclinical study for the treatment of tumours expressing IL-2R in the peritoneal cavity is effective and safe. The liposomes were stable and their IL-2 active for up to one year when kept at -14oC in a cryopreservation media approved by the FDA for human use. Research Article Preclinical studies for a cationic liposome formulation containing Il-2 Intended for the treatment of Human Tumors Maria Teresa Corona-Ortega1*, Arturo Valle-Mendiola1, Leonor Aguilar-Santelises2, Araceli Garcia del Valle2, Rosalva RangelCorona1 and Benny Weiss-Steider1 1FES-Zaragoza, National Autonomous University of Mexico, Mexico City, Lab. 4 P.B. UMIEZ Campo II. Apartado Postal 9-020 Mexico, 15000, D.F. Mexico City, México 2FES-Zaragoza, National Autonomous University of Mexico, Mexico City, Pharmaceutical Manufacturing Plant, Campo II. Apartado Postal 9-020 México, 15000, D.F., Mexico City, Mexico *Address for Correspondence: María Teresa Corona-Ortega, FES-Zaragoza, National Autonomous University of Mexico, Mexico City, Lab. 4 P.B. UMIEZ Campo II. Apartado Postal 9-020 México, 15000, D.F., Mexico City, Mexico, Tel / Fax: (52) 555773-4108; Email: tcvaldes@unam.mx Submitted: 10 October 2018 Approved: 26 October 2018 Published: 29 October 2018 Copyright: © 2018 Corona-Ortega MT, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited How to cite this article: Corona-Ortega MT, Valle-Mendiola A, Aguilar-Santelises L, del Valle AG, Rangel-Corona R, et al. Preclinical studies for a cationic liposome formulation containing Il-2 Intended for the treatment of Human Tumors. Arch Pharm Pharma Sci. 2018; 2: 051-059. https://doi.org/10.29328/journal.apps.1001010 Introduction Tumors can occur in the peritoneal cavity either as primary or metastatic growths [1-7]. Besides the lymphatic and hematologic routes of dissemination the transcoelomic spread of tumor cells is an acknowledged phenomenon that ultimately leads to peritoneal carcinomatosis [8,9]. The administration of anticancer drugs in the peritoneal cavity suffers from a rapid clearance by the lymphatic system into blood circulation and by its absorption in the large surface of the peritoneum [10,11]. Liposomes have been widely studied as drug carriers [12-16] and shown that when injected i.p. their retention in the cavity is signi icantly increased [17-19]. IL-2 has been used to treat human tumors with relative success. Nevertheless the high doses needed for an effective antitumor effect is rather toxic thus limiting the use of this molecule in cancer therapy [20]. The high toxicity associated with IL-2 has been shown to be due to the cytokines released by T lymphocytes activated by this growth factor [21]. The i.p. administration of IL-2 has been shown to be mostly conserved in the cavity with only slight increase in plasma or organs [22]. Lower doses of IL-2 cannot be used in cancer treatment because on one hand they do not activate cytotoxic lymphocytes and on the other it has been shown that they promote tumor growth [23]. We have previously shown that high doses of IL-2 can be included in liposomes with signi icant antitumor activity without the high toxicity produced by free IL-2 [24]. We used a chemical immune depressed mouse model to Preclinical studies for a cationic liposome formulation containing Il-2 Intended for the treatment of Human Tumors Published: October 29, 2018 052 facilitate the formation of human cervical cancer tumors that express the IL-2R. The cationic liposome developed by our group contains the IL-2 molecules presented on its external surface thus facilitating their recognition by cells expressing the IL-2 receptor [25]. Several cancer cells have been shown to express IL-2R [26-29], thus susceptible to be treated by these liposomes. This work was undertaken to evaluate the effectiveness and safety of our liposomes as an anticancer drug in vivo by using a nude mouse model. The advantage of this model is that human tumors can be produced without immune rejection and in our particular case because IL-2 being a lymphocyte growth factor could not excerpt indirectly its antitumor activity by activating cytotoxic T cells. An additional advantage being that the animals were not chemically immune depressed thus avoiding the inherent systemic toxicity. In our previous model the existence of a partially functional immune system generated signi icant organ damage when free IL-2 was used. We expected that in the nude model the absence of thymic lymphocytes this toxicity could be avoided. We reasoned that if liposomes increase the permanence of associated drugs in the peritoneal cavity and if IL-2 by itself is known to remain in that cavity a liposome containing this growth factor could have an important cavity stay. On the other hand if tumors bearing the IL-2R are known to strongly deplete IL-2 from the surrounding media then the administration a liposomes presenting external IL-2 by this via can further avoid its entrance into blood circulation augmenting its antitumor activity in the cavity itself and eliminating the systemic tumor promoting effect associated with low doses of IL-2. Our results showed a strong anticancer effect of these liposomes without any mice toxicity and the absence of plasma IL-2 even at very high doses. We consider this work to be a successful preclinical study by showing the effectiveness and safety of our liposome for treating peritoneal tumors expressing the IL-2R. Materials and Methods Reagents Human recombinant Interleukin 2 (rhIL-2) was acquired from R&D systems (Minneapolis, MN, USA) and was reconstituted according to the supplier’s instructions. Egg yolk phosphatidyl-choline (PC) and cholesteryl-spermidine (SpeCho), synthesized by direct reaction of cholesteryl chloroformate with spermidine base, which were acquired from Sigma (Sigma Chem, St Louis, MO, USA). Cryopreservation agents were acquired too from Sigma (Sigma Chem, St Louis, MO, USA). Preparation and storage of liposomes Cationic liposomes were formed as published by our group [25] using the cationic lipid Cholesteryl-spermidine (SpeCho) (synthesized in our laboratory) and egg yolk phosphatidylcholine (PC), (Sigma Chem, Mo. USA) in a 1:1 molar ratio. The mixture of lipids (10 μmol) was dissolved in chloroform dried under nitrogen at reduced pressure and liposomes produced by hydration of the thin lipid ilm in phosphate buffer solution (PBS) with or without 100IU/mL of IL-2 (2.4 X 103 IU of IL-2 is equivalent to 1 μG of IL-2) in PBS using three 5 second sonication cycles followed by 30 seconds resting period in an Avanti G112SO20_B sonicator. The liposomes were inally sedimented at 200,000g for 40 minutes in PBS and suspended in a solution that containing a mixture of cryopreservation agents (trehalose/glycerol) authorized by the FDA for human use. Samples with 300 μL were used for our experiments or kept frozen at -14oC for stability evaluation. Transmission electron microscopy The liposome suspensions with or without IL-2 were diluted 1:1 with 4% aqueous Preclinical studies for a cationic liposome formulation containing Il-2 Intended for the treatment of Human Tumors Published: October 29, 2018 053 uranyl acetate on carbon-Formvar-coated grids and incubated for 1 min. The grids were drained on absorbent tissue and allowed to air-dry. A JEOL-JEM-1010 transmission electron microscope (JEOL USA, Inc., USA) was used. Flow cytometry Liposomes in 100 μL of PBS with or without IL-2 were analyzed in a FACS Aria low cytometer (Becton Dickinson, CA, USA). Results were reported as laser beam diffraction (FSC) taken to be proportional to liposomes surface area (size) or refraction (SSC) taken as their complexity (roughness). Cell culture A human solid tumor cell line INBL established in our laboratory from a cervical carcinoma was kept in tissue culture and routinely sub-cultured at 37oC with 5% CO2 in RPMI 1640 medium (Microlab, México) supplemented with 10% fetal calf serum (FBS) (Hyclone, USA). Animals Athymic nu/nu female mice between 8 and 12 weeks old were used. Mice were kept in groups of three on sterile environment in cages at room temperature with food and water ad libitum as per the protocols approved by the UNAM’s Animal Experimentation and Ethics Committee which complies with the code of practice regarding the care and use of animals for scienti ic purposes. Tumor induction Mice were inoculated i.p. with 8x106 INBL cells in 300 μl of PBS. The animals were kept for 20 more days for the different antitumor protocols to be applied. Subsequently sacri iced by cervical dislocation and the tumor masses collected. The volumes were calculated assuming they had a spherical shape by using the formula 4/3 πr3 where r was half of their longest diameter. Toxicology assay The toxicology assay was done according to Lorke Model [30], for increasing concentrations of IL-2. Liposomes were administered i.p. in a single dose with 100, 500 and 5000 IU of IL-2. Mice were observed for 15 days and blood samples collected in Microtainer® microtubes (BD Diagnostics, Franklin Lakes, NJ, USA) and the serum separated by 15 min of centrifugation at 350 g at 4oC. The amount of urea, creatinine, transaminases AST and ALT were then evaluated. Pharmacodynamic assay For the pharmacodynamic assay we induced tumor formation, as described in Materials and Methods, in three groups of three mice