Taxol-oligoarginine conjugates overcome drug resistance in-vitro in human ovarian carcinoma

Abstract

Objective

Multidrug resistance is the major cause of failure of many chemotherapeutic agents. While resistance can arise from several factors, it is often dominated by drug efflux mediated by P-glycoprotein (P-gp), a membrane-bound polysubstrate export pump expressed at high levels in resistant cells. While co-administration of pump inhibitors and a drug could suppress efflux, this two-drug strategy has not yet advanced to therapy. We recently demonstrated that the reversible attachment of a guanidinium-rich molecular transporter, polyarginine, to a drug provides a conjugate that overcomes efflux-based resistance in cells and animals. This study is to determine whether this strategy for overcoming resistance is effective against human disease.

Methods

Tumor samples from ovarian cancer patients, both malignant ascites cells and dissociated solid tumor cells, were exposed to Taxol-oligoarginine conjugates designed to release free drug only after cell entry. Cell viability was determined via propidium-iodide uptake by flow cytometry. To analyze bystander effect, toxicity of the drug conjugates was also tested on peripheral blood leucocytes.

Results

Human ovarian carcinoma specimens resistant to Taxol in vitro demonstrated increased sensitivity to killing by all Taxol-transporter conjugates tested. These studies also show that the drug conjugates were not significantly more toxic to normal human peripheral blood leukocytes than Taxol.

Conclusions

These studies with human tumor indicate that oligoarginine conjugates of known drugs can be used to overcome the efflux-based resistance to the drug, providing a strategy that could improve the treatment outcomes of patients with efflux-based drug-resistance.

Introduction

Ovarian carcinoma (OVCA) is commonly diagnosed at a late stage, two thirds of patients eventually relapse and a subpopulation develops resistance to Taxol drugs [1], [2], [3], [4], [5]. A significant problem in the treatment of OVCA is multidrug resistance (MDR). Chemotherapeutic agents initially produce a cytoreductive response, but the development of MDR leads to failure of the drug and frequently cross-resistance to other therapeutics. While many factors contribute to MDR, including drug target mutation, drug metabolism and sequestration [6], a major contributor is the active export of drugs by transmembrane polysubstrate efflux pumps that prevent drugs from reaching their intracellular targets. The recently reported X-ray crystal structure of the mouse P-gp efflux pump, which has 87% sequence identity to human P-gp [7], along with earlier binding studies on the interactions of P-gp with its substrates [8], [9], provides a rationale for how this protein recognizes and mediates the unidirectional efflux of numerous drugs. Hydrophobic drugs with high membrane solubility can enter the internal cavity of the pump through two portals positioned on the inner leaflet of the membrane after which an energy-dependent conformational change in the pump results in drug efflux into the extracellular milieu. Drug efflux by P-gp represents one of the best-studied mechanisms of resistance to hydrophobic anticancer drugs [10].

Much effort has been invested in the development of new drug candidates that are less susceptible to efflux, often requiring synthesis and evaluation of numerous analogs to identify a candidate that maintains target efficacy but is not a P-gp substrate. An alternative approach has focused on using a second agent, to suppress pump expression, accelerate the post-translational degradation of the pump, or inhibit pump efflux by the co-administered drug. However, because export pumps are ubiquitously expressed and required for normal function and because the co-administration of two agents can produce highly variable and often undesired pharmacokinetic responses, this approach has not advanced beyond clinical trials.

We have designed a method that involves the attachment of a drug to oligoarginine, a guanidinum-rich molecular transporter through a releasable linker [11]. The guanidinum-rich molecular transporters, which include oligoarginine and other cationic peptides, have been shown in recent years to facilitate the delivery of attached cargo into cells by rendering them water-soluble [12], [13]. Significantly, because the drug-transporter conjugate is not a substrate for P-gp export and passes rapidly through the cell membrane, it evades efflux-mediated resistance. After cell entry the conjugate is cleaved and the free drug is released at a rate controlled by the linker design.

From a clinical perspective, this approach offers a number of advantages. Taxol, is water-insoluble and must be administered with solubilizing agents like Cremephor-EL that often elicit an acute hypersensitive reaction [14], [15], [16], [17]. In contrast, Taxol conjugated to the transporter can be formulated in minimal volumes (as little as 1–2 ml) of saline. Furthermore, the conjugate is designed to be inactive until it enters cells, thereby minimizing off-target toxicity. Upon entering cells, the conjugate releases free drug over time at a pre-determined rate controlled by linker design, thus avoiding peak-trough effects often associated with a bolus administration. Additionally, the conjugates can be given intraperitoneally (IP), taking advantage of a mode of administration recommended in a National Cancer Institute announcement indicating that IP-administered paclitaxel, when used with intravenously (IV)-administered paclitaxel and cisplatin, provides a 16-month extension to median survival in ovarian cancer patients [18]. Previous biodistribution studies in transgenic mice where the bioluminescent probe luciferin is conjugated to an oligoarginine transporter provide evidence that transporter conjugates injected IP remain localized within the cavity [11]. IP administration of the Taxol-oligoarginine conjugates in larger volumes of saline will minimize localized toxicity associated with the transporter and maintain uniform distribution confined to the IP region. Thus, the balance between drug delivery/maintenance in cancer cells and the drug toxicity profile can be regulated in this drug-conjugate linker system to achieve maximum efficacy and manageable side effects.

To determine if the drug-conjugate linker system is applicable to the complexity and heterogeneity of human disease, we examined the effectiveness of our approach in an in-vitro study using human specimens' from ovarian cancer patients. By testing the cancer cells without selecting for growth advantages or expanding particular subpopulations, the assay emulates the heterogeneity of the clinical disease. The growth rate of primary tumor samples (ascites cells and dissociated solid tumor cells) in vitro replicates the physiological environment where cancer cells grow more slowly than established cell lines [19], [20]. The diverse set of transporter-drug conjugates evaluated includes prodrugs that exhibit extended stabilities under assay conditions, allowing them to be administered and remain intact until cell entry, after which they are rapidly cleaved in the intracellular reducing environment, releasing the free drug at tunable rates ranging from minutes to hours and controlled by linker design.