In vitro synergistic action of geldanamycin- and docetaxel-containing HPMA copolymer-RGDfK conjugates against ovarian cancer

Abstract

HPMA copolymer-RGDfK (HPMA-RGDfK) conjugates bearing either aminohexylgeldanamycin (AHGDM) or docetaxel (DOC) were synthesized and characterized. In vitro stability and binding were evaluated. Cytotoxicity toward ovarian cancer cells was evaluated and the ability of the conjugates to induce cell death was assessed by combination index analysis. Conjugates bearing AHGDM were more stable and exhibited slower drug release than those bearing DOC. Both conjugates demonstrated the ability to bind to avb3 integrins. In combination, HPMA-RGDfK conjugates demonstrated marked synergism as compared to their non-targeted counterparts and free drug controls. HPMA-RGDfK conjugates bearing AHGDM and DOC induce synergistic cytotoxicity in vitro, suggesting their potential as a promising combination therapy.

Keywords: docetaxel; geldanamycin; ovarian cancer; targeted drug delivery.

Figure 1. Representative structure of α_vβ₃ targeted HPMA copolymer-drug conjugates

Random copolymers are comprised of four functional monomers: 1) HPMA, which makes up the primary backbone and increases hydrophilicity and aqueous solubility, 2) MA-GFLG-X, where X is either aminohexylgeldanamycin or docetaxel bound to the lysosomally degradable drug linker GFLG via an amide or ester linkage respectively, 3) MA-Tyr-CONH₂, a modified tyrosine to facilitate radiolabeling in future studies, and 4) MA-GG-cRGDfK, which contains the cyclic peptide RGDfK which binds to α_vβ₃ integrins expressed on the surface of ovarian tumor cells and neovasculature.

Figure 2. Stability of HPMA copolymer-drug conjugates in PBS and serum

Release of free AHGDM and DOC from HPMA copolymer-drug conjugates as a function of time was assessed in: a) PBS pH 7.4 and b) mouse serum:PBS [1:1]. Release rate was significantly higher for HPMA copolymer-DOC conjugates. Data expressed as mean ± SD of three independent experiments, with 3 samples per experiment.

Figure 3. Competitive binding assay of HPMA copolymer conjugates to cell surface α_vβ₃ integrins of A2780 human ovarian cancer cells

All α_vβ₃ targeted conjugates demonstrated the ability to bind α_vβ₃ integrins and all non-targeted conjugates showed no evidence of competitive binding. The relative binding affinity of RGDfK was decreased following conjugation to HPMA copolymers, with drug containing (AHGDM and DOC) copolymers showing a greater reduction in binding affinity as compared to control HPMA copolymers (no drug). Data expressed as mean ± SD of three independent experiments, with 3 samples per experiment.

Figure 4. In vitro growth inhibition IC₅₀ values of HPMA copolymer conjugates

The ability of HPMA copolymer conjugates to inhibit the growth of A2780 and OVCAR-3 human ovarian cancer cells was evaluated in vitro. DOC conjugates exhibited high potency with IC₅₀ values ranging from 1–4 nM, whereas AHGDM conjugates exhibited moderate potency with IC₅₀ values ranging from 3–7 µM. Free RGDfK peptide and control HPMA copolymer-RGDfK demonstrated some potency with IC₅₀ values of 4–7 µM and 16–24 µM respectively. Data expressed as mean ± SD of three independent experiments, with 3 samples per experiment.

Figure 5. In vitro combination index analysis

The combination index (z-axis) was evaluated as a function of drug ratio (x-axis) and effect (y-axis). Three combination treatments were evaluated: 1) Free drug combination (AHGDM + DOC), 2) Non-targeted conjugates combination (p-AHGDM + p-DOC), and 3) α_vβ₃ targeted conjugates combination (p-AHGDM-RGDfK + p-DOC-RGDfK). Various (5) drug ratios were evaluated and effect was defined as (1- [% relative viability / 100]). Combination treatment with α_vβ₃ targeted conjugates demonstrated marked synergism at drug ratios ranging from 50% IC₅₀ AHGDM + 50% IC₅₀ DOC to 83% IC₅₀ AHGDM + 16% IC₅₀ DOC at effect levels above 0.5. A combination treatment of α_vβ₃ targeted conjugates at the optimum drug ratio of 67% IC₅₀ AHGDM + 33% IC₅₀ DOC demonstrated significantly higher synergism as compared to combinations of either non-targeted conjugates or free drugs. Data expressed as mean ± SD of three independent experiments, with 3 samples per experiment.