The objective of this study was to investigate the hypothesis that co-encapsulation of two drugs in the same liposome can enhance the efficacy of synergistic agents compared to two drugs delivered in different liposomes. This required us to devise liposome formulations of FOA, IRN, and FOA + IRN combination.
The combination activity of FOA + IRN was tested in C26 cells. FOA + IRN at a 5:1 molar ratio was synergistic in C26 cells in vitro (). It was surprising that IRN showed good activity since it is a prodrug that has to be activated by a carboxylesterase to the active compound SN38. The results from the screen indicated that the synergism exhibited by FOA and IRN is ratio-dependent. This makes it important to control the ratios in vivo in order to achieve maximum therapy.
To prepare L-FOA, a method developed by Heath and coworkers [25
] was initially used which allowed encapsulation of 1–3 mM of FOA. Dissolving FOA in 7M urea, which can disrupt hydrogen bonding between molecules, allowed us to make a more concentrated FOA solution. As a result, liposome that encapsulated higher concentrations (~10mM) of FOA could be prepared. With the L-FOA formulation, one could deliver a 10 mg/kg dose to mice in a 200 μL volume.
The MTD of L-FOA was established because: 1) L-FOA has never been tested in mice and 2) we needed to determine whether L-FOA could be administered safely at a schedule similar to an IRN dosing schedule. The maximum tolerated dose of L-FOA is 10 mg/kg, which is a 10 fold less dose than the MTD of free FOA. This enhanced toxicity is probably due to the longer circulation, sustained release, and enhanced accumulation of FOA to sites of toxicity due to the liposome formulation. This increased toxicity for a water soluble anti-metabolite has been observed in liposomal cytosine arabinoside formulations [37
]. Balb/c mice were able to tolerate a single i.v. dose of both treatments, but could not endure multiple i.v. doses of FOA 100 mg/kg given four days apart (). The mice also could not tolerate multiple i.v. doses of 10 mg/kg L-FOA given four days apart. Therefore, L-FOA could not be administered on the schedule followed in the L-IRN therapy study in HT29 mice. The MTD of the two formulations were also evaluated in the Balb/c mice on a 3×q7d dosing schedule (). This is the schedule that Mayer and coworkers used to evaluate the therapeutic activity of their liposome IRN + floxuridine co-formulations [5
]. The formulations were not toxic to the mice at this schedule; therefore, the weekly schedule was selected for use in animal studies with the FOA and IRN liposome formulations.
The therapeutic efficacy of BTCA loaded L-IRN was tested in a HT29 human xenograft mouse model at a similar dose and schedule used by Drummond and coworkers [20
]. The data in Fig. S2 and Fig. S3 (supplementary information)
demonstrate that L-IRN was efficacious and safe at the dose and schedule administered.
Lastly, a liposome formulation that encapsulated both FOA and IRN was developed. Co-encapsulating these two drugs in one liposome formulation was challenging because of the disparate physico-chemical properties of these two drugs. Weakly acidic drugs like FOA are traditionally passively loaded into liposomes; while amphipathic drugs like IRN can be actively loaded into preformed liposomes. Passive loading of drugs occurs through hydrating a lipid film with an aqueous solution of drug. This method is inefficient, and the resulting encapsulated drug concentration relies on the maximum solubility of the drug in solution [34
]. It is difficult to remote load FOA because it is deprotonated in aqueous solution; therefore, FOA cannot readily cross a lipid bilayer. Whereas, IRN can partition into and diffuse across a bilayer when deprotonated. To co-encapsulate FOA and IRN in liposomes, FOA was passively loaded into vesicles and then was used to remote load IRN (). In this protocol, some of the FOA leaked out during IRN remote loading (, , as well as Table S1 and Fig. S4 in the supplementary information
). Reducing the loading temperature, loading time and IRN drug/lipid ratio enhanced FOA retention but minimized IRN loading. The IRN drug/lipid ratio had the biggest impact on FOA retention and IRN loading, and adjusting the loading conditions allowed us to reproducibly encapsulate FOA + IRN at ratios between 5:1 to 1:1.
We investigated the combination therapy of FOA + IRN when delivered in the same liposome or when delivered together in separate liposomes in C26 tumor-bearing mice. L-FOA (57.4 μmol/kg) was more effective than L-IRN (73.8 μmol/kg). Although IRN + floxuridine, another fluoropyrimidine, co-encapsulated in liposomes had greater anti-tumor activity than the single liposome agents [5
], we did not observe the same results. Although delivering the two drugs at the 5:1 ratio in the same liposome was statistically superior than delivering the two drugs at the 5:1 ratio in separate liposome, none of the combinations were more effective than L-FOA alone at 10 mg/kg (). This might have been due to: 1) lower dose of FOA in the co-encapsulated formulations than in the single liposome formulation or 2) co-delivery of the drugs in separate liposomes. The dose of FOA in the co-encapsulated formulation, L-FOA-IRN 5:1, was 28.7 μmol/kg while that in L-FOA was 57.4 μmol/kg. It was challenging to devise a co-encapsulated formulation with this concentration of FOA because of the leakage of FOA due to IRN remote loading in the liposome. To deliver a co-formulation with FOA at the dose of the single agent formulation, we had to deliver the drugs in separate liposomes. The L-FOA + L-IRN 5:1 (double strength) combination was administered at the same dose of FOA as the L-FOA formulation and had slightly but not significantly less, tumor growth inhibition as L-FOA.
It is possible that this co-formulation did not show enhanced efficacy compared to L-FOA because the drugs were in separate liposomes. Delivering drugs in the same liposome formulation may help to coordinate the release of the drugs in the cell such that the encapsulated drugs leak at similar rates [15
]. Perhaps the L-FOA + L-IRN 5:1 (double dose) mixture did not deliver the 5:1 synergistic ratio into the cell.
In conclusion, we describe the development and evaluation of liposomal formulations for FOA and IRN alone and in combination. An optimized method for encapsulating FOA into liposomes was developed which allowed us to encapsulate up to 10mM of FOA. This method enabled us to co-encapsulate FOA and IRN, which have disparate physico-chemical properties, at different molar ratios. L-FOA as a single agent has anti-tumor efficacy in the C26 tumor mouse model that was superior in tumor growth inhibition and in the increase in survival time compared to L-IRN 73.8 μmol/kg (50 mg/kg). However, the co-delivery of IRN with FOA in either the same or different liposomes failed to improve further the anti-tumor activity in the C26 model.