Improving the distribution of Doxil® in the tumor matrix by depletion of tumor hyaluronan

Abstract

Liposomes improve the pharmacokinetics and safety of rapidly cleared drugs, but have not yet improved the clinical efficacy compared to the non-encapsulated drug. This inability to improve efficacy may be partially due to the non-uniform distribution of liposomes in solid tumors. The tumor extra-cellular matrix is a barrier to distribution and includes the high molecular weight glycosaminoglycan, hyaluronan (HA). Strategies to remove HA or block its synthesis may improve drug delivery into solid tumors. Orally administered methylumbelliferone (MU) is an inhibitor of HA synthesis, but it is limited by low potency and limited solubility. In this study, we encapsulate a water-soluble phosphorylated prodrug of MU (MU-P) in a liposome (L-MU-P). We demonstrate that L-MU-P is a more potent inhibitor of HA synthesis than oral MU in the 4T1 murine mammary carcinoma model using both a quantitative ELISA and histochemistry. We show that HA depletion improves the tumor distribution of liposomes computed using Mander's colocalization analysis of liposomes with the tumor vasculature. Hyaluronan depletion also increases the fraction of the tumor area positive for liposomes. This improved distribution extends the overall survival of mice treated with Doxil®.

Keywords: Breast cancer; Extra-cellular matrix; Liposomes; Tumor penetration.

Figure 1. MU-P is rapidly converted to MU by phosphatases and inhibits HA synthesis

(A) MU-P is encapsulated in liposomes and rapidly converted to MU upon release; MU inhibits HA synthesis. (B) MU-P is rapidly converted to MU in serum and (C) both MU and MU-P deplete HA in culture media (n=3). Addition of a phosphatase inhibitor (PI) abolishes the activity of MU-P.

Figure 2. Characterization of MU-P liposomes

(A) TEM images of POPC:Cholesterol and HSPC:Cholesterol liposomes encapsulating MU-P (top) and summary of liposome characteristics including polydispersity index (PDI), diameter, and surface potential (table, n=3). Scale bar is 50 μm. (B) MU-P is passively encapsulated in both liposomes to a similar extent (n=3), but (C) leaks more quickly from POPC liposomes in the presence of serum at 37°C. (D) Both HSPC and POPC MU-P liposomes have extended circulation times compared to free MU-P. Inset shows early timepoints of HSPC and POPC liposome leakage.

Figure 3. L-MU-P depletes HA in 4T1 tumors

(A) Mice with orthotopic 4T1 tumors were given 1–4 doses of L-MU-P according to the given schedule or oral MU daily. A single dose of doxorubicin was administered to measure liposome uptake into tumors (n=10). (B) L-MU-P significantly reduced HA levels in 4T1 tumors as compared to oral gavage of MU. This reduction occurred in a dose dependent manner as measured by an HA-specific ELISA assay or (C) Alcian Blue staining. (D) MU-P and MU accumulate in the tumor in a dose dependent manner, while (E) Doxil® accumulation is not affected by HA levels. Statistical analyses were performed with (D) Student’s t test (B) or ANOVA and are relative to oral MU. Error bars denote mean ± standard deviation.

Figure 4. Liposome localization in endogenous 4T1 tumor macrophages

(A) Liposomes colocalize with 4T1 tumor macrophages. White arrows highlight areas of colocalization. (B) HA depletion with L-MU-P does not alter tumor macrophage content or (C) liposome colocalization with tumor macrophages. (D) 4T1 tumor sections show macrophage staining. (E) Primary bone marrow macrophages in culture are positive for CD68 while (F) 4T1 tumor cells are not.

Figure 5. L-MU-P modulates gene expression in vitro, but not in vivo.

Gene expression levels were examined by qRT-PCR. (A) HAS2 is the most abundant HA-synthase in cell culture. (B) MU down regulates expression of HAS2 in vitro (n=3). (C) HAS2 is the most abundant HA-synthase in 4T1 orthotopic tumors, but (D) L-MU-P has no effect on its expression in vivo (n=10).

Figure 6. HA depletion improves liposome distribution in 4T1 tumors

(A) Orthotopic 4T1 tumors were treated with 4 doses of L-MU-P followed by a single dose of fluorescently labeled liposomes (n=4). (B) Liposome penetration in control tumors is less pronounced than in (C) tumors with reduced HA. (D) Liposome distribution quantified by colocalization analysis across the entire tumor as well as by (E) percent area analysis across the entire tumor. Statistical analyses were performed with Student’s t-test. Error bars denote mean ± standard deviation.

Figure 7. Improved liposome distribution following HA depletion improves efficacy of Doxil®

(A) 4T1 orthotopic tumors were treated with 4 doses of L-MU-P followed by a single dose of Doxil® at 6 mg/kg (n=6). (B) When combined with Doxil®, HA depletion by L-MU-P statistically reduced primary tumor growth and (C) extended overall survival. Statistical analyses were performed with (B) Student’s t-test (C) or Log-rank (Mantel-Cox) test. Error bars denote mean ± standard deviation.