Optimized 5-Fluorouridine Prodrug for Co-Loading with Doxorubicin in Clinically Relevant Liposomes

Abstract

Liposome-based drug delivery systems have allowed for better drug tolerability and longer circulation times but are often optimized for a single agent due to the inherent difficulty of co-encapsulating two drugs with differing chemical profiles. Here, we design and test a prodrug based on a ribosylated nucleoside form of 5-fluorouracil, 5-fluorouridine (5FUR), with the final purpose of co-encapsulation with doxorubicin (DOX) in liposomes. To improve the loading of 5FUR, we developed two 5FUR prodrugs that involved the conjugation of either one or three moieties of tryptophan (W) known respectively as, 5FUR-W and 5FUR-W₃. 5FUR-W demonstrated greater chemical stability than 5FUR-W3 and allowed for improved loading with fewer possible byproducts from tryptophan hydrolysis. Varied drug ratios of 5FUR-W: DOX were encapsulated for in vivo testing in the highly aggressive 4T1 murine breast cancer model. A liposomal molar ratio of 2.5 5FUR-W: DOX achieved a 62.6% reduction in tumor size compared to the untreated control group and a 33% reduction compared to clinical doxorubicin liposomes in a proof-of-concept study to demonstrate the viability of the co-encapsulated liposomes. We believe that the new prodrug 5FUR-W demonstrates a prodrug design with clinical translatability by reducing the number of byproducts produced by the hydrolysis of tryptophan, while also allowing for loading flexibility.

Keywords: drug combination; liposome; nanoparticle; targeting.

Figure 1

Prodrugs used were designed with the conjugation of tryptophan (W) to 5-fluorouridine to make the compound weakly basic and favorable to ammonium sulfate gradient loading in liposomes. (A) The monoester 5FUR−W has tryptophan conjugation to the primary alcohol of 5FUR. (B) The triester 5FUR−W₃ has tryptophan conjugated to every alcohol of 5FUR.

Figure 2

5FUR−W was determined to be the more stable prodrug for proceeding with liposomal loading. (A) Hydrolysis of 5FUR−W and 5FUR−W₃ showed that 5FUR−W is significantly more stable in heated solution (p < 0.01). (B) 5FUR−W achieved a large range of liposomal ratios when loaded in tandem with DOX. 5FUR−W₃ was not as efficient in loading.

Figure 3

In vitro comparison of 5FUR−W and 5FUR−W₃ dose-response cellular fractional inhibition on 4T1 breast cancer cells showed a high degree of similarity. All points represent n = 6. (A) Comparison of free single drugs DOX, 5FUR−W, and 5FUR−W₃. (B) Comparison of 1:5 ratio of 5FUR prodrugs to DOX. (C) Comparison of 1:1 ratio of 5FUR prodrugs to DOX. (D) Comparison of 5:1 ratio of 5FUR prodrugs to DOX.

Figure 4

Drug pharmacokinetics of liposomal formulations showed sustained release. All formulations were injected inTable 1. 5 mg/kg DOX (n = 3) (A) Drug plasma concentrations of 5FUR−W and DOX from DAFODIL_R=2.5 and DOX from DLI. (B) DAFODIL_R=2.5 drug plasma ratio over time.

Figure 5

DAFODIL was compared to DLI in the 4T1 tumor model using different 5FURW:DOX ratios. (A) DAFODIL_R=10 significantly reduced tumor growth. All formulations began dosing on day 6, with four administrations spaced every two days. (B) DAFODIL_R=10 caused significant weight loss in treated mice. (C) All formulations were administered on day 6. DAFODIL_R=2.5 (four injections, every two days, n = 6) was similar to DAFODIL_R=6 (4 injections, every 4 days, n = 5) and both significantly reduced tumor volume compared to DLI (four injections, every two days, n = 6) (D) While DAFODIL_R=6 caused significantly different weight loss, DAFODIL_R=2.5 expressed no toxicity associated with weight loss. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 6

Storage stability of 5FUR−W in DAFODIL_R=2.5 (n = 3). (A) Hydrolysis of 5FUR−W at 25 °C. (B) Hydrolysis of 5FUR−W at 4 °C. (C) Ratio of 5FUR−W to all 5FU containing species at both 4 °C and 25 °C.