Complete regression of xenografted human carcinomas by a paclitaxel–carboxymethyl dextran conjugate (AZ10992)

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Abstract

Clinically available taxanes, such as paclitaxel and docetaxel, represent one of the most promising classes of anticancer agents, despite their toxicity. To improve their pharmacological profiles, AZ10992 was synthesized based on the concept that a rational design of a polymer–drug conjugate would increase the efficacy of the parent drug. This prodrug is a paclitaxel–carboxymethyl dextran conjugate (molecular weight 150,000 g/mol) via a gly–gly–phe–gly linker. The in vivo antitumor study using AZ10992 against colon26 carcinoma cells, resistant to paclitaxel, supported this concept. Additionally, the comparative efficacy studies of AZ10992 and paclitaxel using a panel of human tumor xenografts in nude mice showed the advantages of drug–polymer conjugation. The maximum tolerated dose of AZ10992 was more than twice as high as the MTD of paclitaxel. A repeated intravenous administration of AZ10992 at 30 mg/kg/day (five injections for 4-days) showed complete regression of MX-1 mammary carcinoma xenografts. Also, HT-29 colorectal tumor xenografts, which are highly refractory to paclitaxel, showed complete regression after AZ10992 administered at 30 mg/kg/day (seven injections for 4-days). Pharmacokinetic studies showed that there were significant increases in the amount and the exposure time of total paclitaxel in the tumors after intravenous administration of AZ10992, which explains the enhanced efficacy of AZ10992.

Introduction

The anticancer agents paclitaxel (Taxol®) and docetaxel (Taxotere®) are currently the only two US Food and Drug Administration (FDA)-approved representatives of the taxane family. Paclitaxel, an anti-microtubule agent isolated from the trunk bark of the Pacific Yew tree, Taxus brevifolia [1], shows great promise as an anti-neoplastic agent for a variety of human cancers, including breast, ovarian, non-small-cell lung, head and neck [2], [3], [4], [5], [6]. Its unique mechanism of action is related to its ability to promote microtubule assembly and inhibit cell replication in the late G2 or M phase of the cell cycle [7].

A major problem that is associated with the administration of paclitaxel is its low solubility in water (less than 0.01 mg/ml), as well as in most pharmaceutically acceptable solvents. The clinically used formulation of Taxol® contains polyoxyethylated castor oil (Cremophor® EL) and ethanol as excipients. Cremophor® EL has long been considered to be the cause of the hypersensitivity reactions that are observed with paclitaxel infusions [8]. Side effects of Taxol® include nausea, vomiting, diarrhea, mucositis, myelosuppression, cardiotoxicity and neurotoxicity [9], [10]. In an attempt to alleviate the systemic toxicity of paclitaxel, several alternative formulations have been evaluated. Many such forms have entered Phase I or II trials as intravenous injectable anticancer agents – for example, polyglutamate–paclitaxel (XYOTAX, CT-2103; many Phase II/III studies are ongoing); liposome entrapped paclitaxel easy to use (LEP-ETU, PNU-93914; Phase I); N-(2-hydroxypropyl) methacrylamide (HPMA)-copolymer–paclitaxel (PNU-166945; failed at Phase I); polymeric micelle-formulated paclitaxel (Genexol-PM; Phase I); paclitaxel-incorporating micellar nanoparticle formulation (NK105; Phase I). Recently, an albumin-stabilized nanoparticle formulation of paclitaxel (Abraxane, ABI-007) has been approved by the FDA for the treatment of metastatic breast cancer [11].

Polymer–drug conjugates are a subclass of the family of novel drugs and drug-delivery systems that have been defined as ‘polymer therapeutics’ [12]. Polymer therapeutics include polymeric drugs, polymer–drug conjugates, polymer–protein conjugates, polymeric micelles to which the drug is covalently bound, and polymeric non-viral vectors for gene delivery. A number of the first-generation polymer–anticancer-drug conjugates are being tested clinically: HPMA-copolymer–doxorubicin (PK1, FCE28068; Phase II); HPMA-copolymer–doxorubicin–galactosamine (PK2, FCE28069; Phase I/II); HPMA-copolymer–platinate (AP5280; Phase II); HPMA-copolymer–platinate (AP5346; Phase I); and oxidized dextran–doxorubicin (DOX-OXD, AD-70; failed at Phase I). More recently, an increasing number of conjugates containing other anticancer agents – particularly, paclitaxel and camptothecin – are being transferred into the clinic: HPMA-copolymer–camptothecin (MAG-CPT, PNU-166148; failed at Phase I); PEG–camptothecin (PROTECAN; Phase II); polyglutamate–camptothecin (CT-2106; Phase I); and CM dextran–camptothecin derivative (MEN4901/T-0128; Phase I) [13].

These prodrugs are made up of a minimum of three components, i.e. the polymeric polymeric carrier, a biodegradable linker and a bioactive antitumor agent. These macromolecular prodrugs, which are conjugates with prolonged circulation times, target tumors by the ‘enhanced permeability and retention effect’ (EPR). This radically changes the pharmacokinetics of the bound drug. Maeda called the passive targeting phenomenon that he observed, the EPR effect, and attributed it to two factors: the disorganized pathology of tumor vasculature, with its discontinuous endothelium, leading to hyperpermeability to circulating macromolecules, and the lack of effective tumor lymphatic drainage, which leads to subsequent macromolecular accumulation [14], [15]. It is well established that long-circulating macromolecules – including albumin, polymer conjugates, polymeric micelles and liposomes – accumulate passively in solid tumors by the EPR effect, and intravenously administered drug-delivery systems can increase the tumor concentration of antitumor drugs. The polymer–protein conjugate styrene maleic anhydride (SMA)–neocarzinostatin (NCS) – which is called SMANCS – was originally synthesized by Maeda and colleagues, and was subsequently approved in Japan as a treatment for hepatocellular carcinoma. Several conjugates have peptidyl polymer–drug linkers that are amenable to cleavage by lysosomal thiol-dependent proteases. In this case, prodrug activation occurs intracellularly. In contrast, other conjugates that contain an ester link between drug and polymer can release the drug by chemical hydrolysis or esterase degradation extracellularly. Some satisfactory results that have been obtained with the use of macromolecules for the targeted delivery of anticancer agents have generated considerable interest regarding the enhancement of therapeutic efficacy and the reduction of systemic side effects. However, some of the dosage forms have failed, such as oxidized dextran–doxorubicin, probably due to toxicity of the oxidized dextran carrier; and HMPA-copolymer–paclitaxel and HMPA-copolymer–camptothecin, probably due to linkage and/or molecular weight being inadequate.

The concept of a polymer–drug conjugate, combination of suitable polymeric carrier, biodegradable linker and a bioactive antitumor agent, is attractive and could well form the basis of a new generation of anticancer agents. In an attempt to overcome the side effects and to improve the pharmacological profile of paclitaxel, AZ10992 is designed as a polymer–drug conjugate that consists of carboxymethyl (CM) dextran and paclitaxel, linked with the gly–gly–phe–gly (GGFG) linker. CM dextran was chosen as a candidate of the paclitaxel carrier for the following reasons: Dextran is generally recognized to be safe; CM dextran contains a sufficient number of carboxyl groups for drug attachment, which provides sufficient carrying capacity for the drug; and the resulting CM dextran–drug conjugate has a high probability of being water-soluble. We previously reported the preparation of CM dextran and doxorubicin conjugates using a peptide linker. Retention of the conjugate in blood circulation and accumulation of doxorubicin in tumors were both increased by CM dextran with a suitable anionic nature and a molecular weight of 150,000 g/mol [16]. Furthermore, CM dextran–peptide–doxorubicin conjugates containing a GGFG linker were more efficacious in a Walker-256 carcinoma rat model than free doxorubicin or a conjugate with no linker. We also reported the synthesis of CM dextran–paclitaxel conjugates with various amino-acid linkers and the effect of linkers on the in vivo activity [17]. In a tumor distribution study and a tumor growth regression study using intravenous administration of these conjugates in colon26-carcinoma-bearing mice, the amounts of paclitaxel in the tumor and antitumor efficacy were found to correlate. These studies seem to support the use of a CM dextran carrier with a GGFG linker as a way to form an improved drug conjugate.

The purpose of the present study was to test the concept that the rational design of a paclitaxel–polymer conjugate would achieve tumor targeting of the active drug, resulting in improved therapeutic efficacy. To this end, we examined the preclinical profile of the antitumor activity of AZ10992 against colon26 carcinoma in BALB/c mice and a panel of human tumor xenografts in nude mice. The efficacy of AZ10992 was compared with that of non-polymer-bound paclitaxel. In addition, we investigated the plasma and tissue (tumor and non-tumor) pharmacokinetics of both released and polymer-bound paclitaxel after intravenous administration of AZ10992 to mice bearing colon26 carcinoma. These data were also compared with those obtained after dosing of non-polymer-bound paclitaxel to tumor-bearing mice using the same dosage. The results showed potential advantages of AZ10992 that were attributable to the passive tumor targeting.

Section snippets

Chemicals

Paclitaxel (Taxol®) was purchased from Hauser Chemical Research, Inc. (Boulder, CO); Dextran T110 was purchased from Pharmacia Biotech (Uppsala, Sweden); Cremophor® EL and Chymotrypsin were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were of reagent-grade purity or better.

Characterization of AZ10992

The relative molecular weight of AZ10992 was determined using gel permeation chromatography (GPC). A TSK-gel G4000PWXL 300 × 7.8 mm column (TOSOH, Tokyo, Japan) was used, and the column temperature was

In vitro drug release

The in vitro release profiles of paclitaxel from AZ10992 in various conditions – in a buffer, and in plasma and tissue homogenates – are shown in Fig. 2. More than 80% of paclitaxel was liberated from AZ10992 after 24–48 h incubation in plasma or serum. In the buffer, the amount of paclitaxel, as well as 7-epi-paclitaxel released from AZ10992, increased as pH increased. In the buffer (pH 7.4), the amount of released paclitaxel after 24 h incubation was lower than that in plasma or serum. After

Discussion

The objective of this study was to test the concept that the rational design of a paclitaxel–polymer conjugate would achieve the targeting of the tumor with the active drug, resulting in improved therapeutic efficacy. AZ10992 is comprised of paclitaxel that is covalently bound to CM dextran by a GGFG linker. It contains 5.5–6.5 wt.% paclitaxel, molecular weight 150,000 g/mol, linked through the 2′ position – that is, via an ester bond, to the carboxylic acid of polysaccharide. We compared the

Conclusions

AZ10992 was chosen for a paclitaxel tumor-targeting system, with a peptidyl linker GGFG with moderate stability in plasma, and a suitable polysaccharide carrier CM dextran with a molecular weight of 150,000 g/mol. AZ10992 consistently produced regression of tumor xenografts, which are highly refractory to paclitaxel. Altered pharmacokinetic properties of AZ10992 in terms of plasma half-life, tumor targeting and kinetics of paclitaxel release may account for the effectiveness. Results

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