Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

Abstract

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its therapeutic benefit. Nanoparticle-based drug combination has been shown to mitigate the problems encountered by free drug combination therapy. Our previous studies have shown that the combination of two anticancer drugs, doxorubicin (DOX) and mitomycin C (MMC), produced a synergistic effect against both murine and human breast cancer cells in vitro. DOX and MMC co-loaded polymer-lipid hybrid nanoparticles (DMPLN) bypassed various efflux transporter pumps that confer multidrug resistance and demonstrated enhanced efficacy in breast tumor models. Compared to conventional solution forms, such superior efficacy of DMPLN was attributed to the synchronized pharmacokinetics of DOX and MMC and increased intracellular drug bioavailability within tumor cells enabled by the nanocarrier PLN. To evaluate the pharmacokinetics and bio-distribution of co-administered DOX and MMC in both free solution and nanoparticle forms, a simple and efficient multi-drug analysis method using reverse-phase high performance liquid chromatography (HPLC) was developed. In contrast to previously reported methods that analyzed DOX or MMC individually in the plasma, this new HPLC method is able to simultaneously quantitate DOX, MMC and a major cardio-toxic DOX metabolite, doxorubicinol (DOXol), in various biological matrices (e.g., whole blood, breast tumor, and heart). A dual fluorescent and ultraviolet absorbent probe 4-methylumbelliferone (4-MU) was used as an internal standard (I.S.) for one-step detection of multiple drug analysis with different detection wavelengths. This method was successfully applied to determine the concentrations of DOX and MMC delivered by both nanoparticle and solution approaches in whole blood and various tissues in an orthotopic breast tumor murine model. The analytical method presented is a useful tool for pre-clinical analysis of nanoparticle-based delivery of drug combinations.

Importance of integrating nanotechnology with pharmacology and physiology for innovative drug delivery and therapy – an illustration with firsthand examples

“…However, the clinical combination of DOX and MMC (DOX-MMC) was halted owing to its severe cardiotoxicity to cancer patients [62,63] . We perceived that this off-target adverse effect could be reduced with optimal scheduling and dosing of the two drugs by rationally designing a drug nanocarrier system [64][65][66][67][68] .…”

Advances in Nanomedicine Design: Multidisciplinary Strategies for Unmet Medical Needs

“…78,79 To maximize the benefit of such drug combinations, nanocarrier systems were designed that circumvented off-target toxicity through tumor-specific release of drugs. 49,50,56,57,80 The synergistic anticancer effect of DOX− MMC combination therapy has been investigated in multiple murine and human breast cancer cell lines and tumor models. 49,50 circulation of both DOX and MMC (Figure 2a) and enabled ratio-metric codelivery of DOX and MMC in breast tumors (Figure 2b).…”

“…This effect, together with glutathione depletion by MMC and formaldehyde formation from MMC enhanced DOX-induced DNA DSBs, demonstrating the synergistic interactions of DOX–MMC combination . However, severe cardiotoxicity has limited the clinical translation of DOX and MMC (DOX–MMC) combinations to cancer patients. , To maximize the benefit of such drug combinations, nanocarrier systems were designed that circumvented off-target toxicity through tumor-specific release of drugs. ,,,, The synergistic anticancer effect of DOX–MMC combination therapy has been investigated in multiple murine and human breast cancer cell lines and tumor models. , A DOX to MMC molar ratio of 2:1 was used in a PLN (DMPLN) formulation. DMPLN prolonged plasma circulation of both DOX and MMC (Figure a) and enabled ratio-metric codelivery of DOX and MMC in breast tumors (Figure b). , The DMPLN also exhibited enhanced tumor apoptosis and extended host-survival in both human and murine MDR tumor models compared to free DOX–MMC solution and liposomal DOX (DOXIL) (Figure c,d). , Importantly, DMPLN formulations did not cause detectable cardiotoxicity in the animals, in contrast to free DOX–MMC (Figure e).…”

Anti‐cancer combination therapy by co‐delivery of hydrophilic and hydrophobic using dual temperature and pH‐responsive liposomes

“…Doxorubicin (DOX) is commonly the first‐line chemotherapeutic agent for many solid tumours and is highly effective in oxygenated regions of the tumour and mitomycin C (MC) is another chemotherapic compound which reveals cytotoxicity against non‐proliferating cells in hypoxic tumour regions via enhanced bioreductive activation. Considering systematic in vitro studies on free drug combinations, it was revealed that DOX and MC can produce synergistic cytotoxicity in breast cancer cells when applying together [15].…”