Synergistic antitumor activity of camptothecin–doxorubicin combinations and their conjugates with hyaluronic acid

Abstract

Combinations of topoisomerase inhibitors I and II have been found to synergistically inhibit cancer cell growth in vitro, yet clinical studies of these types of combinations have not progressed beyond phase II trials. The results of clinical combinations of topoisomerase (top) I and II inhibitors typically fall within one of two categories: little to no improvement in therapeutic efficacy, or augmented toxicity compared to the single drug counterparts. Hence, despite the promising activity of top I and II inhibitor combinations in vitro, their clinical applicability has not been realized. Here, we report the use of polymer-drug conjugates as a means to co-deliver synergistic doses of top I and II inhibitors camptothecin (CPT) and doxorubicin (DOX) to tumors in vivo in a 4T1 breast cancer model. At specific molar ratios, DOX and CPT were found to be among the most synergistic combinations reported to date, with combination indices between 0.01 and 0.1. The identified optimal ratios were controllably conjugated to hyaluronic acid, and elicited significant tumor reduction of murine 4T1 breast cancer model when administered intravenously. This study elucidates a method to identify synergistic drug combinations and translate them to in vivo by preserving the synergistic ratio via conjugation to a carrier polymer, thus opening a promising approach to translate drug combinations to clinically viable treatment regimens.

Low-Molecular-Weight Polymer–Drug Conjugates for Synergistic Anticancer Activity of Camptothecin and Doxorubicin Combinations

“…The concentrations required to inhibit 50% cell growth (D 50 ) of DOX-PVA and CPT-PVA were 1.5 μM DOX and 14 μM CPT, respectively. In comparison, previous studies in our lab demonstrated D 50 concentrations of free DOX and CPT to be 0.3 μM and 100 μM, respectively [20]; hence, conjugation to 10 kDa PVA reduced activity of DOX, whereas it enhanced activity of CPT. The enhanced anticancer activity of PVA-bound CPT may be attributed to its enhanced water solubility upon conjugation to PVA.…”

“…Conjugation of chemotherapy drugs to polymers typically alters drug activity, as was evident in our previous study of 250 kDa hyaluronic acid conjugated to CPT and DOX [20]. However, drug activity should not be compromised so much as to entirely prevent its anticancer effects.…”

“…Combination interactions were evaluated and quantified utilizing the Combination Index (CI) method [29], where CI < 1 indicates synergy and CI > 1 suggests antagonism. Previous studies conducted by our lab demonstrated that free CPT and DOX synergistically inhibit BT-474 cancer cell growth at ratios (R) greater than 0.1 M:M CPT:DOX, with the greatest synergy occurring at R > 2 [20]. Figure 7B shows that the same synergistic ratios hold true for the 10 kDa PVA-bound drugs; for R > 2.2, CI values were found to be <<1 and significantly more synergistic than combinations at R < 2.2.…”

“…CPT and DOX co-conjugated to 10 kDa HA was synthesized as previously reported [20]. Briefly, HA carboxylic acid groups were activated with EDC and DMAP, both in a molar ratio of 0.75:1 with respect to HA, in a 1:1 mixture of DMSO:water under slight heating and stirring for 1 h. CPT was slowly added to the reaction mixture at a molar ratio of 0.4:1 CPT:HA, and allowed to react for 3 days.…”

“…Therefore, although lowmolecular-weight polymer-drug conjugates In this report, low-molecular-weight polymers and varying linker chemistries are investigated in order to understand their impact on the co-delivery of synergistic chemotherapy combinations. We have previously demonstrated the tumor reduction capability of high-molecular-weight 250 kDa hyaluronic acid (HA) conjugated to synergistic chemotherapy drugs camptothecin (CPT) and doxorubicin (DOX) [20]. CPT and DOX represent a large class of highly synergistic combinations, topoisomerase I and II inhibitors [21][22][23], and thus serve as a model pair for studying the impact of polymeric properties on combination potency.…”