Introduction
Since chemotherapy was first developed for the treatment of cancer over four decades ago, a wide range of effective agents have been identified. Despite these advances, the therapeutic benefits of chemotherapy have been limited by the ability of tumors to develop drug resistance [
1]. If tumor cells are repeatedly exposed to an antineoplastic agent, cross-resistance to related agents of the same drug class generally is seen. However, the tumor is likely to remain sensitive to drugs from different classes due to their different mechanisms of action [
2]. Even so, in many cases, tumors display multidrug resistance (MDR), where cross-resistance occurs to multiple drugs that are neither structurally nor functionally related, and to which the tumor has never been exposed.
Several different mechanisms exist whereby tumors become drug-resistant. One key mechanism is overexpression of the P-glycoprotein (P-gp) efflux pump (encoded by
MDR1), which can result in subtherapeutic concentrations of cytotoxic agents such as anthracyclines and taxanes being retained in tumor cells [
3,
4]. For drugs that target microtubules, such as taxanes, other key mechanisms of resistance include overexpression of the βIII-tubulin isotype and tubulin mutations [
5,
6]. Multidrug resistance poses a significant challenge to the treatment of cancer and drives the continued search for new compounds without such limitations.
The epothilones are a novel class of microtubule-stabilizing agents produced by the myxobacterium
Sorangium cellulosum. Four main natural epothilones are produced by
S. cellulosum: A and B and, to a lesser extent, C and D [
7]. These 16-membered macrolide antibiotics are highly effective at inducing cell arrest at the G
2/M phase, resulting in apoptosis [
8,
9]. However, although the naturally occurring epothilones demonstrate impressive activity in vitro, it has been difficult to demonstrate antitumor activity in vivo [
10]. This is, at least in part, due to the unfavorable pharmacokinetic characteristics and relatively narrow therapeutic window of the naturally occurring epothilones.
Ixabepilone is a semisynthetic analog of epothilone B, chemically modified to retain the highly favorable in vitro characteristics of natural epothilone B while improving the pharmacokinetic profile [
11]. Specifically, the lactone oxygen is replaced with a lactam. In previous studies, ixabepilone has demonstrated the ability to overcome tumor resistance due to a range of mechanisms in vivo, showing antitumor activity in the following established models: Pat-7 ovarian carcinoma, HCT116/VM46 human colon carcinoma (both resistant due to P-gp overexpression), A2780Tax ovarian carcinoma (resistant due to a tubulin mutation), clinically derived, paclitaxel-resistant Pat-21 breast carcinoma (resistant due to overexpression of βIII-tubulin), and the inherently paclitaxel-refractory murine fibrosarcoma M5076 (unknown mechanism of resistance, non-MDR) [
5].
These promising preclinical findings have since translated to the clinic. Single-agent ixabepilone has shown encouraging antitumor activity in a broad range of tumor types during phase I [
12‐
15] and phase II [
16‐
28] clinical trials.
The results reported herein extend these previous reports regarding the preclinical efficacy of ixabepilone, explore the susceptibility of ixabepilone to tumor resistance mechanisms, and describe the pharmacokinetics of this new antineoplastic agent.
Discussion
Tumor resistance to chemotherapeutic agents is a significant limitation of this treatment modality in many patients. The taxanes, for example, are susceptible to several mechanisms of tumor resistance, including MDR protein-mediated efflux, overexpression of βIII-tubulin isoform, and tubulin mutations. Because resistance to taxanes and other drug classes ultimately limits their efficacy, development of agents able to overcome any of these resistance mechanisms would address a significant clinical need.
Results presented from these in vitro cytotoxicity studies against three different tissue-specific, cancer cell-line panels demonstrate that ixabepilone has potent and broad-spectrum antineoplastic activity. The effectiveness of ixabepilone in vitro is paralleled by equally broad-spectrum activity in vivo, with robust antitumor activity seen against 35 human tumor xenografts representing a wide array of tumor types including breast, colon, NSCLC, pancreatic, ovarian, prostate, SCLC, gastric, and squamous cell carcinomas. Furthermore, in 33 of 35 tumors, 1 LCK or greater efficacy was seen in addition to significant tumor regression rates, including “long-term absence of measurable disease” in ~50% of the tumor types tested. Moreover, in agreement with previous studies [
5,
6], ixabepilone demonstrated greater activity than taxanes in a range of models resistant to other chemotherapeutics due to various mechanisms, including overexpression of βIII-tubulin (e.g., Pat-21), overexpression of P-gp (e.g., Pat-7, HCT116/VM46), and tubulin mutations (e.g., A2780Tax ovarian carcinoma).
The activity of ixabepilone in these drug-resistant models can most likely be explained by the low susceptibility of ixabepilone to several mechanisms of drug resistance. For example, it was previously shown that ixabepilone displays reduced susceptibility to P-gp and other efflux pumps compared with the taxanes, and that ixabepilone does not induce expression of MDR proteins [
5]. The cellular uptake of ixabepilone, but not paclitaxel, in P-gp-overexpressing cells shown in the present study supports this observation, and is consistent with the activity of ixabepilone in MDR models such as HCT116/VM46. Ixabepilone is also able to overcome resistance conferred by overexpression of the βIII-tubulin isoform, as exemplified by its activity in the paclitaxel-resistant Pat-21 model. The precise mechanism of Pat-21 resistance was unknown until quite recently. However, recent studies suggest that a combination of loss of βII-tubulin and overexpression of βIII-tubulin may be responsible for the paclitaxel resistance of Pat-21 [
6]. In the studies reported here, the head-to-head comparison of ixabepilone versus docetaxel in the Pat-21 model showed that ixabepilone was significantly more potent than docetaxel. The activity of ixabepilone against this taxane-resistant model may be explained by the fact that the tubulin-binding mode of ixabepilone affects the microtubule dynamics of multiple tubulin isoforms, including βIII-tubulin. Moreover, ixabepilone preferentially suppresses dynamic instability of αβIII-microtubules compared with αβII-microtubules [
6]. Thus, the ability of ixabepilone to bind βIII-tubulin, and its preferential binding of βIII-tubulin over βII-tubulin, may explain the selective sensitivity of the Pat-21 model to ixabepilone.
An understanding of pharmacokinetic parameters for ixabepilone, along with its antitumor activity, allows for greater maintenance of overall effective drug concentration while minimizing toxicity. The pharmacokinetics of ixabepilone reported herein in mice are characterized by rapid tissue distribution and extensive tissue binding, as shown by the large Vd
SS (107 L/m
2) at a dose of 30 mg/m
2, which corroborates the large Vd
ss (530–840 L/m
2) observed in humans administered the currently approved dosage of 40 mg/m
2 [
33‐
35]. It was also important to establish the in vivo stability of ixabepilone to better understand the dynamics of its metabolism following administration. Importantly, the half-life of ixabepilone (13 h at 6 mg/kg and 16 h at 10 mg/kg) was considerably longer than that of desoxyepothilone B, which has a lactone at position 16 and a half-life in mice of approximately 20 min [
36]. This suggests that replacing the lactone oxygen with a lactam improves the metabolic stability of the molecule through overcoming lactone hydrolysis by esterases in the mouse. Although there are reduced esterase levels in human compared with mouse plasma, P450-bearing microsomes in human liver are able to hydrolyze epothilone B lactone [
37].
Therefore, it is likely that the lactam modification is important in extending the half-life of ixabepilone in humans. This is supported by the favorable half-life of ixabepilone in humans (35 h) seen in a phase I dose-finding study [
33]. The favorable clinical pharmacokinetic characteristics of ixabepilone are further demonstrated by the finding in phase I clinical studies that AUC of ixabepilone at the approved dose of 40 mg/m
2 (1,760–2,560 ng h/ml, 3.5–5.1 μM h) was higher than that of patupilone (204–237 ng h/mL, over a range of administration schedules) [
33‐
35,
38]. It is also worth noting that this exposure level is similar to those produced in mice at therapeutically efficacious doses (Fig.
4). Therefore, the overall pharmacokinetic profile of ixabepilone reported here and previously demonstrates that plasma levels of ixabepilone required for antitumor activity are attainable clinically.
Consistent with its favorable pharmacokinetics and notable preclinical activity in this study, promising clinical activity of ixabepilone has been seen against a wide range of tumor types, including breast [locally advanced (LABC) and metastatic (MBC)], lung, renal, prostate, pancreas, and lymphoma [
16‐
28]. In several instances, tumors had been heavily pretreated or were resistant to current therapies, including anthracycline-pretreated MBC [
25], taxane-resistant MBC [
26], and MBC or LABC resistant to an anthracycline, a taxane, and capecitabine, [
39] suggesting that the reduced susceptibility of ixabepilone to mechanisms of drug resistance seen in these and other preclinical studies can translate to the clinic.
In summary, ixabepilone has broad-spectrum in vitro and in vivo activity across a range of tumor types, accompanied by desirable pharmacokinetics. Consistent with its reduced susceptibility to several mechanisms of drug resistance, ixabepilone is active against a range of resistant tumor xenografts, including those resistant to taxanes. These preclinical findings have translated into clinical activity against a wide range of tumor types, including heavily pretreated and drug-resistant tumors. One phase II neoadjuvant study has correlated response to ixabepilone in breast cancer patients with the expression of specific cellular genes such as
ER [
40]. In addition, a phase III study in patients with anthracycline- and taxane-resistant MBC demonstrated superior efficacy for ixabepilone in combination with capecitabine versus capecitabine alone, with 40% prolongation of PFS and 2.5-fold higher rate of response [
41].