CarboCell: a novel drug delivery system for the formulation of immunotherapeutics for cancer treatment

Immunotherapy has emerged as a revolutionary cancer treatment and an encouraging alternative to traditional cancer therapies as surgical resection, radiotherapy and chemotherapy. Cancer immunotherapy is focused on generating and enhancing an anti-cancer immune response in the body of the patient that is both effective and durable. However, in the current clinical practice, only a small fraction of patients are respondent to cancer immunotherapy. Moreover, most of the immunotherapies are systemically administered, which is often associated with severe toxic effects particularly for therapies aimed at activating and bridging the innate and adaptive immune responses. Intratumoral administration can be a useful alternative strategy, but low injection reproducibility, rapid drug clearance from tumors and drug leakage into systemic circulation can negatively affect the therapeutic response and compromise patient safety.
We have developed the CarboCell drug delivery system to address the aforementioned challenges for intratumoral immunotherapy. CarboCell is comprised of three main components: (i) a carbohydrate ester, (ii) a co-solvent, and (iii) a solvent, which form a low viscosity fluid. Upon injection, CarboCell self-assembles as a highly viscous depot that acts as both drug reservoir and localization marker. CarboCell provides a sustained drug release that can enable continuous immune stimulation of the tumor microenvironment while minimizing systemic drug exposure, in addition to secure accurate and reproducible intratumoral injections. This project was focused on the formulation and delivery of Toll-like receptors 7/8 agonists, particularly resiquimod (R848), and the transforming growth factor-β (TGF-β) inhibitor RepSox, which are water-insoluble drugs. The present PhD thesis describes the characterization and formulation development of the CarboCell as well as its in vivo therapeutic efficacy in murine models.
In Chapter 2, the drug stability of R848 in the CarboCell was investigated. The chemical stability of R848 was evaluated in multiple CarboCell formulations, at different temperatures and in the presence of benzoic acid (BA) or trimethylamine. It was observed that R848 was susceptible to chemical modifications resulting from interactions with some carbohydrate esters. The type of carbohydrate ester played a key role in R848 stability, where non-reducing carbohydrates esters comprising benzoate groups promoted the stability of R848. Also, R848 was less reactive when adding BA to the formulation and upon storage at low temperatures (≤ 4°C). R848 showed excellent short-term stability in CarboCells comprising sucrose octabenzoate (SuBen), so SuBen was deemed as the main carbohydrate ester for subsequent formulations.
Chapter 3 contains a manuscript to be submitted to the Journal of Controlled Release, presented in its intended submission format. In such manuscript, the characterization, formulation and delivery of five novel R848 prodrugs is described. R848 and R848 prodrugs were formulated in various CarboCells that were tested both in vitro and in vivo. CarboCell was able to provide sustained drug release, which could be tailored based on both the chemical composition of the CarboCell and drug hydrophobicity. Moreover, the incorporation of a novel computed tomography (CT) contrast agent named CLA-8 was evaluated. CLA-8 provided excellent CT contrast to the CarboCell depot thus highlighting the application of CarboCell as localization marker for image-guided injections and subsequent monitoring. Intratumoral injections with R848 and the R848 prodrugs were well tolerated and significantly increased the median survival time of mice bearing CT26 tumors.
Chapter 4 describes the simultaneous formulation and co-delivery of R848 and RepSox from CarboCells. The multi-drug therapy enhanced the therapeutic response in mice compared to the R848 monotherapy. Furthermore, the combination treatment induced both a local and systemic anti-cancer response. Moreover, an improved CarboCell formulation was developed in which the drug-releasing period was doubled. Thus, the interval between injections was extended from 7 to 14 days thus reducing by half the number of injections per treatment. By increasing the SuBen to co-solvent ratio in the CarboCell, the viscosity and hydrophobicity of the self-formed depot was increased resulting in an overall slower drug release. The results of the in vivo efficacy in mice for the extended release formulation were equivalent to those of the original CarboCell.
Chapter 5 comprises multiple characterization studies of numerous CarboCell compositions aimed at gaining further knowledge and understanding of the system. Overall, the data showed a correlation between viscosity, solvent diffusion and drug release. It was observed that the drug burst release was highly dependent on the amount and type of solvent in the CarboCell. In contrast, the long-term release profile was generally influenced by the type of carbohydrate ester and co-solvent as well as the ratio between them. In addition, two sterilization methods were tested: steam sterilization via autoclave and gamma irradiation. The latter was better at maintaining the chemical drug stability in the CarboCells; however, gamma irradiation also caused the modification of 3 to 5% of the CLA-8 in the formulation.
In Chapter 6, hydrophobic ion-pairing (HIP) was investigated as a formulation strategy to enable the solubilization of hydrophilic charged drugs in the CarboCell. The fluorescent dye indocyanine green (ICG), a hydrophilic anionic molecule, was used as a model molecule. HIP complexation of ICG with several co-ions was successfully done using the Bligh-Dyer method. The solubility of ICG in the CarboCell was significantly improved when formulated as a HIP complex. Furthermore, the release of ICG could be tailored depending on the type and concentration of the co-ion as well as the chemical composition of the CarboCell. Lastly, an in vivo test in rats showed that CarboCells containing ICG-HIP complexes might be used as markers for near-infrared (NIR) fluorescence imaging applications.