(M0930-06-35) Harnessing Camel Milk-Derived Exosomes to Enhance Oral Bioavailability of Anticancer Agents with Poor Solubility and Permeability (ARV-825 and CBD) and Revealing the Mechanism of Improved Absorption

Purpose: Oral administration is an appealing route of delivering cancer treatments owing to its convenience and patient compliance. However, the challenges associated with the gastrointestinal tract, including poor permeability, often limit the oral bioavailability. This is a significant impediment to the development of oncology drugs, particularly those for long-term treatment schedule. In recent decades, the field of nanotechnology has developed several delivery platforms such as exosomes with high potential for overcoming the numerous barriers associated with oral administration due to their inherent biocompatibility, stability, and ability to traverse biological barriers. In our study, we harness the potential of camel milk-derived exosomes (CME) as a delivery system for anticancer drugs with poor oral bioavailability and also enhance their activity as CME are reported to have anticancer properties per se.

Methods: Exosomes were isolated from camel milk by ultracentrifugation. CME were characterized by fluorescent nanoparticle tracking analysis (NTA), proteomics and western blotting to determine size, zeta potential, concentration in particles/ml, and exosomal protein biomarkers. CME were then used to encapsulate ARV-825 (8 mg/ml) and CBD (6 mg/ml) by sonication. To dissolve the drugs, kolliphor EL was used as an emulsifier. To determine the entrapment efficiency, LC-MS method was developed. For in-vitro studies, MDA-MB-231 doxorubicin resistant breast cancer cells and PDX Osimertinib resistant lung cancer cell lines were seeded in 96-well plates for cytotoxicity study. The plates were treated with various concentrations of CME, ARV-825, CBD, ARV-825-CME and CBD-CME to study cytotoxicity. For release study, Spectra-Por® Float-A-Lyzer® G2, 1mL dialysis devices were used. Furthermore, in-vitro permeability studies were carried out using MDCK cells and transepithelial electrical resistance was measured to monitor the integrity of the monolayer and the establishment of tight junctions. In-vivo experiments were conducted to show uptake of CME into tumor cells. MDA-MB-231 cells (4 million) were injected subcutaneously in athymic nude mice to develop tumor xenografts. Vybrant™ DiO solution was used to make fluorescent CME. For pharmacokinetic studies, group 1 received a drug-CME formulation (ARV-825-5mg/kg or CBD-12mg/kg), while group 2 received control drug solution. Blood samples were collected at specified time intervals to obtain plasma to be analyzed using LC-MS method. In a separate study, after oral administration of CME, intestines of rats were sectioned at different time points from 0.5 to 4 h to understand their distribution in the gut and mechanism of absorption.

Results: CME exhibited a size distribution of 104.4±1.6 nm and zeta potential of -30.75±0.04 mV using fluorescent NTA, meeting exosomal EV criteria. Approximately 79% exosomes showed fluorescence indicating presence of tetraspanins CD9/63/81 in the particles. After sonication, the NTA properties of the CME formulation did not alter significantly. Western blotting revealed the presence of typical EV markers Alix, CD63/81 (Fig 1). Cytotoxicity studies showed 2.1-fold and 2.6-fold enhancement in CBD’s and ARV-825’s IC₅₀ (p < 0.001) in MDA-MB-231 and PDX cells, respectively. Our release studies showed 2.95-fold increase for CBD and 3.4-fold increase in ARV-825 drug release from the CME compared to the ARV solution (p < 0.01). The apparent permeability of CBD-CME was found to be 22.9*10^-6±0.34 cm/sec at 2 h whereas the apparent permeability of CBD solution was found to be 5.8*10^-6±0.65 cm/sec. Similarly for ARV-825-CME, there was 4.65-fold increase in apparent permeability compared to free ARV-825 (Fig 2). The pharmacokinetic studies in rats showed significantly enhanced bioavailability for both CBD-CME and ARV-825-CME with 3 to 4-fold increase in AUC of the drugs (Fig 3). It is evident from the pharmacokinetic data that there was improved Cmax, Tmax, half-life, AUC, volume of distribution and clearance suggesting that CME could notably enhance the drug bioavailability. In-vivo tumor xenograft studies showed that DiO-labeled CME were internalized by tumors which was confirmed by their presence with fluorescence microscopy. In absorption studies, intestinal sections showed high number of fluorescent CME at 0.5 h, 1 h and 2 h time points in duodenum, ileum, and jejunum, respectively. After 4 h, no fluorescence was observed in any of the gut sections suggesting that CME are absorbed intact from the gut with their payloads within 2-4 hours which is also supported by our in vivo pharmacokinetic studies. Ileum, being the longest part of the intestine, was responsible for the absorption which was confirmed by presence of drug in the ileum section lysates measured by LC-MS (Fig 3).

Conclusion: Our study focused on addressing the limitations in oral bioavailability of CBD and ARV-825, which are potent anticancer agents. We explored the potential of CME to enhance the bioavailability of CBD and ARV-825. In-vivo pharmacokinetic study revealed a significant enhancement in bioavailability of drug-CME formulations compared to the control group. In overcoming the challenges of poor bioavailability, our study introduces a novel and effective method for enhancing the absorption of poorly bioavailable and water-insoluble pharmaceuticals, making it a noteworthy contribution to the field. Further studies are ongoing to understand the mechanism of absorption.


Fig 1. Fluorescent Nanoparticle Tracking Analysis (F-NTA) of CME. A) Overlay of size distribution histogram in scatter mode and fluorescence mode of CME B) Overlay of zeta potential of CME in scatter mode and fluorescence mode combined. Size distribution histogram of C) CME D) CBD-CME and E) ARV-825-CME. F) Western blots of CME expressing CD63, CD81, Alix and HSP70. The values are expressed as mean ± SD (n = 3).


Fig 2. In vitro release and permeability profiles of drug-CME formulations. A) Release profile of ARV-825 and ARV-825-CME B) Release profile of CBD and CBD-CME C) Bar graph representing apparent permeability of ARV-825 and ARV-825-CME D) Bar graph representing apparent permeability values of CBD and CBD-CME. The values are expressed as mean ± SD (n = 3), ****p < 0.0001.


Fig 3. In vivo plasma concentration vs time profiles of drug-CME formulations. A) Plasma conc. Vs time graph of ARV-825 and ARV-825-CME, Dose of ARV-825 = 5 mg/kg B) Plasma conc. Vs time graph of CBD and CBD-CME, Dose of CBD = 12 mg/kg C) Table demonstrating pharmacokinetic parameters of drug and drug-exosome formulations. D) Fluorescence microscopy image showing DiO-labeled CME in the tumor section. E) Brightfield image of the tumor section. F) Fluorescence microscopy images showing DiO-labeled CME in duodenum, ileum and jejunum at 0.5 h. 1 h, 2 h respectively. The values are expressed as mean ± SD (n = 5).