(W0930-07-39) Development of Biodegradable Injectable In Situ Forming Implants for Controlled Release of Cannabinoids: In Ovo Assessment

Purpose: Cannabinoids have emerged as potential anticancer agents due to its ability to inhibit tumor growth, tumor angiogenesis, and metastasis. Among all cannabinoids, cannabidiol (CBD) that lacks psychotropic effects is one of the most promising. CBD has demonstrated a relevant anticancer effect in several neoplasms like breast, prostate, and ovarian cancer [1]. In breast cancer, CBD is useful not only as monotherapy but also in combination with conventional antineoplastics as potentiates the effect of doxorubicin and paclitaxel. Nevertheless, its high lipophilicity hampers the development of parenteral formulations. In this context, in situ forming implants (ISFIs) that are minimally invasive and provide a controlled drug delivery [2] could be a promising platform for CBD delivery, allowing a prolonged anticancer effect that could improve conventional chemotherapy. The aim of this work was to develop, optimize and characterize ISFIs for the subcutaneous administration of CBD that could be useful as adjuvant treatment in breast cancer.

Methods: Poly-D, L-lactide (PLA) resomer 202 (PLA-202) and poly-D, L-lactide-co-glycolic acid (PLGA) resomer 502 (PLGA-502) and 502-H (PLGA 502H) were used as polymers due to their biocompatibility, biodegradability, and safety. N-Methyl-2-pyrrolidone (NMP) and Dimethyl sulfoxide (DMSO) were used as water-miscible solvents. Formulations loaded with at a CBD drug: polymer ratio of 2.5:100 (ISFI-2.5%) and 5:100 (ISFI-5%) were elaborated using the direct injection technique. Firstly, injectability studies (using 23G and 25G needles) were carried out to select the most suitable and manageable polymer, and the optimal polymer:solvent ratio. Viscosity of the formulations was also evaluated. Optimized CBD loaded implants were characterized by scanning electron microscopy (SEM) and drug release was determined. Toxicity studies and implant formation were evaluated in ovo using the chick choriollantoic membrane (CAM) model and Irritation scores (IS) were calculated. Finally, the antiangiogenic effect of CBD loaded ISFIs was determined in ovo using both: yolk sack model (YSM) and CAM assays [3].

Results: Injection tests using both DMSO and NMP as solvent demonstrated that all PLGA and PLA solutions showed good injectability properties, with an easy and continuous injection using both 23G and 25G needles. In all cases, the optimal polymer: solvent ratio was 100 mg: 400 µL that was the selected to prepare CBD loaded ISFIs. All the formulations allowed the easy formation of CBD-loaded implants when injected in phosphate buffer solution. A controlled CBD release was observed in all formulations (Figure 1). PLGA based ISFIs showed a faster drug release than PLA based formulations, especially when NMP was used as solvent with more than 70% of the CBD released within one week. In all the ISFIs loaded with CBD 5%, a slightly slower drug release was detected, probably due to their higher loading of CBD. Formulations prepared with PLA-202 plus DMSO or NMP at a CBD:polymer ratio of 5:100 allowed a CBD controlled drug release for at least one month and were selected for the following studies. Morphology of CBD loaded ISFIs was studied using SEM, appreciating a smooth surface with some spherical holes. Selected CBD loaded ISFIs were successfully formed when injected onto the hydrated CAM (Figure 2). While DMSO based formulations did not show any sign of toxicity (IS=0), ISFIs prepared with NMP, showed a slight irritation onto the CAM (IS=7.1 ± 0.6) that were attributed to the solvent. NaOH 0.5M that was used as positive irritation control showed an IS of 18.4± 0.6 (very irritant agent). Therefore, ISFIs prepared with DMSO and PLA-202 at a CBD:polymer ratio of 5:100 were selected for angiogenesis studies. After 48 hours of treatment, CBD ISFIs (administered at a CBD concentration of 100 µM) showed an antiangiogenic effect in both YSM and CAM models. A statistically significant (p value < 0.05) lower vascular density, especially in terms of tertiary and quaternary blood vessels were detected in CBD treated eggs. An inhibition percentage of 35.59 ± 10.62 % and 64.05 ± 7.79% in both tertiary and quaternary blood vessels were observed (Figure 3). Interestingly, CBD implants showed a similar antiangiogenic effect than celecoxib that were used as positive control of angiogenesis inhibition.

Conclusion: ISFIs consisting of PLA-202 and DMSO and loaded with CBD at a drug:polymer ratio of 5:100 showed good injectability properties to be administered subcutaneously, a controlled CBD release for at least one month, no toxicity signs in CAM model and an antiangiogenic effect in both YSM and CAM assays. This formulation could be useful as adjuvant treatment in breast cancer.

References: [1] Future Aspects for Cannabinoids in Breast Cancer Therapy. Int J Mol Sci., 2021.
[2] Drug Release from in Situ Forming Implants and Advances in Release Testing, Advanced Drug Delivery Reviews, 2021.
[3] Establishment of an in ovo chick embryo yolk sac membrane (YSM) assay for pilot screening of potential angiogenic and anti-angiogenic agents. Cell Biology International, 2018.

Acknowledgements: Irene Lozza was granted a postgraduate research grant from the University “La Sapienza” of Rome. Scholarship of the programme
“Perfezionamento all’estero” according to law no. 398 of 1989. This work has been funde by " Fondo Especifico de investigación FEI19/30- 01" of UCM.


CBD release profile of drug loaded ISFIs prepared with the different polymers plus DMSO (left panel) and NMP (right panel).


CBD loaded PLA-202-DMSO ISFI formed onto CAM model


YSM angiogenesis studies