(T1130-02-11) Cannabinoid Combination Therapy for Doxorubicin Resistant MDA-MB231: Oral Exosome Formulation Versus Parenteral Approaches

Purpose: Combination therapy is the most commonly used approach to overcome resistance developed by single drug therapy in cancer. Cannabinoids were proven to be potential anticancer agents. Previous studies from our laboratory have demonstrated that phytocannabinoids such as cannabichromene, cannabigerol not only possess anticancer activity but also sensitizes resistant tumors to standard chemotherapeutic drugs. Also, these minor phytocannabinoids show high specificity towards tumors and do not exhibit severe side effects. Hence, we hypothesized that testing different combinations of cannabinoids could be an approach to overcome resistance in cancer therapy. However, the oral bioavailability of cannabinoids is very low limiting their potential in cancer therapy. The objective of this study was to develop an effective combination of cannabinoids that can overcome resistance in Triple Negative Breast Cancer (TNBC) as well as to deliver cannabinoids through oral route using camel milk exosomes as drug delivery vehicles.

Methods: Our laboratory had previously reported that Cannabidiol can be used to sensitize resistance cancer cells to standard chemotherapy drugs. In this study, combinations of cannabinoids were studied to overcome resistance in TNBC. Also, exosomes were used to address the low oral bioavailability of cannabidiol (CBD). Briefly, several cannabinoids such as cannabichromene (CBC), cannabidiol (CBD), cannabigerol (CBG), cannabinol (CBN), cannabidiolic acid (CBDA), cannabidivarin (CBDV) were screened against MDA-MB-231 Doxorubicin (DOX) Resistant (RT) and PDX lung cancer cell lines by In-vitro cytotoxicity MTT assays. Combination of cannabinoids were also evaluated and isobolographic analysis was done to evaluate the ideal synergistic combination. In-vitro assays such as wound healing assay, live/dead staining, cell cycle analysis were performed to understand the mechanism of anticancer activity. In-vivo studies were conducted in BALB/s athymic nude mice using MDA-MB-231 xenografts. The animals were euthanized, tumors were collected and processed for further studies. Western blot analysis was done to investigate the molecular mechanisms. Furthermore, CBD was loaded into camel milk exosomes (CBD-CMDE) to improve the oral bioavailability. In-vitro cell permeability studies were conducted using MDCK cell line to assess the apparent permeability of CBD-CMDE. In-vivo Pharmacokinetic studies were also performed using Sprague Dawley rats and plasma concentrations were determined using a well-established LC-MS method. Oral CBD-CMDE was evaluated in-vivo using MDA-MB-231 DOX RT tumor xenografts.

Results: The IC50 values of CBD and CBC were found to be 3.88±0.54µM and 7.06±0.1µM respectively against MDA-MB-231 DOX RT cell line whereas other cannabinoids such as CBG, CBN, CBDV were found to have IC 50 values of around 8±1 µM. The IC50 values of CBC was significantly reduced by 17 folds from 7.06±0.1µM to 0.42±0.22µM when combined with CBD at a concentration of 2µM. Likewise CBD when combined with CBG demonstrated a 10 folds drop in IC50 value from 8.08±0.17µM to 0.84±0.14µM. Other combinations like CBD+CBN and CBD+CBDA showed a 2.6 folds reduction in IC50 value. Isobolographic analysis showed combination index values of 0.1-0.4 suggesting strong synergism for CBC+CBD combination at lower concentrations (0.078-1.25µM) and mild synergism (0.6-0.8) at 2.5, 5µM concentration and antagonism at 10µM concentrations (Fig 1). Similar pattern was observed for other combinations as well. From these screening studies, CBC+CBD was found to be the most potent combination and hence was investigated further. Migration and cell cycle analysis assays had shown that combination of CBC+CBD exhibited better efficacy. In-vivo pharmacodynamic studies showed that the tumor volume of combination group was maintained at 1200mm³ (P < 0.0001) over a period of two weeks whereas the tumor volume for control group was around 2500mm³ on 10^th day. Western blot analysis of tumor samples revealed downregulation of apoptotic markers such as PARP (P < 0.0001), NF-KB (P < 0.001), mTOR (P < 0.01), survivin (P < 0.0001) and metastatic marker such as vimentin (P < 0.001). Also, significant upregulation of proteases like caspase 3 (P < 0.000) and caspase 9 (P < 0.001) was observed Fig 2). Furthermore, CBD-CMDE exhibited improved apparent permeability (Papp) and was found to be enhanced by 3.95-fold with Papp of 22.9*10^-6 ± 0.34 cm/sec as compared to control CBD formulation (Papp of 5.8*10^-6 ± 0.65 cm/sec) in MDCK cells. In-vitro release studies showed release of 78.27 ± 5.37 % and 46.42 ± 4.75 % CBD from exosomes and control CBD formulation respectively at 24 hr. In-vivo PK studies showed enhanced bioavailability of CBD from CBD-CMDE with AUC_(0-24h) of 1350.56±187.50 ng/mL*h as compared to CBD control formulation with AUC_(0-24h) of 351.95±39.10ng/mL*h. Our initial studies with MDA-MB-231 tumor xenografts demonstrated that tumor volume was reduced by 1.8 folds when CBD-CMDE (20mg/kg CBD) were administered orally Fig 3).

Conclusion: CBC+CBD was observed to be the most potential combination against TNBC from our screening studies. The mechanism of synergism of combination was found to be mediated through apoptosis and metastatic pathways as well as cell cycle arrest. Furthermore, the oral bioavailability of CBD was significantly improved by loading them into camel milk exosomes. Our initial studies also demonstrated significant tumor reduction of MDA-MB231 tumor xenografts after administration of CBD-CMDE.


Figure 1 A) In-vitro cytotoxicity studies of individual cannabinoids and combination of cannabinoids against MDA-MB-231 DOX RT cells. Results were expressed as µM in Mean ± SD (n=4). B) Isobolographic analysis. C) Table showing combination index values which suggest that the combination of CBC(1.25um) and CBD (2um) has the most synergism.


Figure 2 A) Represents cell cycle analysis of CBC, CBD and CBC+CBD on MDA-MB-231 DOX RT cells. B) Tumor volume of control, CBC (15mg/kg, i.p.), CBD (10mg/kg, i.p.), and CBC+CBD (15mg/kg+10mg/kg, i.p. respectively) animal groups. Results were expressed as Mean ± SD (n=5). C) Representative western blots and quantification of respective Survivin, Vimentin, mTOR, PARP, NF-KB, caspase 3, caspase 9 in tumor tissue lysate.


Figure 3 Pharmacokinetic study of CBD milk exosomes. A) In-vitro permeability study. B) In-vitro release study. C) In-vivo pharmacokinetic study. D) Brightfield image of tumor section E) Fluorescent microscopy image of tumor section demonstrating that DiO labelled camel milk exosomes can reach tumor after oral administration. F) In-vivo efficacy study of oral CBD-CMDE (20mg/kg, oral) in MDA-MB-231 DOX RT mouse models.