Therapeutic effect against human xenograft tumors in nude mice by the third generation microtubule stabilizing epothilones

Results

Potency Against Tumor Cell Growth in Vitro.

A comparative evaluation of the in vitro activities of various epothilone derivatives against a range of tumor cell lines reveals some important trends (Table S1). Each compound in the iso-oxazole series (iso-dehydelone and iso-fludelone) is more potent than its parent compound (dehydelone and fludelone). Fortunately, much of the in vitro potency that had been lost in progressing from dehydelone to fludelone was restored through the installation of the isoxazole moiety. Also, iso-fludelone generally displays markedly enhanced potency when compared with the established cancer chemotherapeutic agents vinblastine and taxol. Most importantly, the epothilone analogues retain their activity against the MDR cell lines CCRF-CEM/VBL and CCRF-CEM/taxol (Table S1).

Physico-Chemical and Metabolic Properties.

The physico-chemical and metabolic properties of the various epothilone analogues are summarized in Table S2. As shown, the microtubule stabilization activity of each derivative is within the range of taxol. The metabolic stability of the epothilones in mouse plasma and human liver microsomal S-9 fraction is greatly enhanced through the installation of the second C₉–C₁₀ double bond in the epothilone framework. Remarkably, the synthesized isoxazole-containing compounds iso-fludelone and iso-dehydelone demonstrate greatly enhanced levels of stability over all previously evaluated epothilone derivatives. Furthermore, the enhanced water solubility of the fludelone family offers advantages related to formulation. These newly developed compounds also retain promising levels of lipophilicity that promote in vivo tissue permeation. The methods of analyzing physico-chemical properties and in vitro metabolic studies are described in SI Methods.

In Vivo Evaluations of Epothilone Analogues.

In our extensive investigation of the in vivo properties of the epothilones, conducted over the course of many years (6–12), we have formulated some general conclusions regarding the optimal modes of administration and dosing schedules of these compounds, noting that the epothilones demonstrate a very strong dependency on the mode of their administration. We have found that the use of slow i.v. infusion brings greatly reduced toxicity (Table S3), which results in a significantly broadened therapeutic index. When epothilones are administered over 6-hour i.v. infusion, the tolerated dose is much higher than that observed for other traditional modes of administration, such as bolus injection. In addition, if any significant sign of toxicity occurs (e.g., marked body-weight decreases), intermittent cessation of treatment is required to allow toxicity to subside. This approach is particularly effective in avoiding delayed neurotoxicities, such as peripheral neuropathy. These findings, observed primarily in mouse models, may well translate to human clinical settings. Unless otherwise specified, all of our in vivo studies reported herein for epothilones use 6-h i.v. infusion. For each xenograft study, tumor-size and body-weight changes were monitored simultaneously. The body-weight changes are provided in corresponding figures in the SI Methods (Fig. S2).

Efficacy and toxicity may also be strongly affected by dose, interval, and schedule of treatment, thus requiring preliminary studies before formal experimentation. In the in vivo studies described herein, we report the results achieved at approximate maximum tolerated doses, those doses that do not result in death to the animal or delayed toxicity. The sole exception to this rule is the survival lifespan studies of SK-NAS brain-tumor-bearing mice (see below).

In many of our in vivo evaluations, we have continued to monitor mice for several months after the cessation of treatment. Our objective has been to determine the extent to which the tumor cells have been eradicated. Because of the unusually long-term nature of these studies, we differentiate between CR and therapeutic cure as follows: CR is defined as disappearance of tumor, whereas a de facto cure is defined as CR without relapse for at least 15% of the lifespan (4 months in mouse models). We further attempt to estimate a log-cell kill (LCK) in tumor mass (11), based on the length of time of CR and the time required for the tumor to double in size (see SI Methods). Typically, a tumor cell kill that reaches below one-billionth to one-trillionth [i.e., LCK > (9–12)] is deemed to be a cure.

Therapeutic Cure of Extra Large Mammary Carcinoma MX-1 Xenografts.

Nude mice bearing an extra-large MX-1 xenograft (up to 7.8% body weight) were treated with iso-fludelone (30 mg/kg, Q12Dx4) by 6-hour i.v. infusion, on day 25 after tumor implantation. Complete tumor remission was observed in four of four mice after administration of the third dose, and a fourth dose was given for consolidation. There was no relapse in any of the mice on day 158 (Fig. 2A). It was encouraging to find that the maximal body-weight decreases were 12%, 10%, and 6% after sequential administration of three doses, indicating not only a lack of cumulative toxicity, but also an alleviation of toxicity over time (Graph 2A of Fig. S2). Photographs of the mice, taken on days 25, 39, and 53 are shown in Fig. 2B. The LCK of iso-fludelone against MX-1 is estimated to be ≈10.94. Thus, based on our definition, a therapeutic cure was achieved against the MX-1 xenograft.

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Fig. 2.

Treatment of mammary and ovarian tumors. (A) Therapeutic effect of iso-fludelone against extra-large mammary MX-1 tumor xenografts. Initial tumor size, 2.26 ± 0.17g, is ≈7.8% of mice body weight. (B) Typical photos of mice on day 25, day 39 and day 53 for the experiment shown in A. (C) Oral therapy of MX-1 xenograft tumor with dehydelone, iso-dehydelone, iso-fludelone, taxotere, and capecitabine. Dose, route, and schedule are as shown. (D) Therapy of ovarian SK-OV-3 tumor xenograft tumor. Dose, route, and schedule for dehydelone, iso-fludelone, and taxol are as shown.

As summarized in Table S4 and Fig. S3, our relevant epothilone analogues—dehydelone, fludelone, iso-dehydelone, iso-fludelone, as well as taxol—have been shown to achieve CR against normal-sized MX-1 xenografts under our standard experimental protocols, in which treatment was started when the MX-1 tumors had grown to 100–200 mm³ in size. Fludelone, iso-dehydelone, and iso-fludelone showed long-term no-relapse cure.

Therapeutic Cure of MX-1 Xenografts by Oral Administration.

Nude mice bearing MX-1 xenografts were treated orally beginning on day 11 after tumor implantation with dehydelone, iso-dehydelone, iso-fludelone, capecitabine, and taxotere at the doses and schedules specified in Fig. 2C. In this study, taxotere, which is known to have poor oral bioavailability, served as a negative control. Of these compounds, iso-fludelone (45 mg/kg, Q8Dx7) achieved CR in four of four mice on D35 without relapse up to day 168. The LCK of iso-fludelone is ≈13.3, indicating the achievement of an oral therapeutic cure of the MX-1 xenograft. At the dose and schedule examined (45 mg/kg, Q9Dx6), dehydelone achieved CR on day 32. Although one of four mice relapsed on D71, the remaining three achieved a cure, with no relapse on day 122 (LCK ≥ 9.03). The manageable body-weight loss observed in this course of treatment suggests that optimal conditions can be identified to produce an even better outcome for dehydelone. Treatment with iso-dehydelone (25 mg/kg, Q10Dx3) led to 90.4% suppression on day 20 without further suppression with subsequent doses. In contrast to the remarkable oral efficacy of the epothilone compounds, the established oral anticancer agent, capecitabine (800 mg/kg, QDx7), achieved only 77.6% tumor suppression on day 20 and produced body-weight losses of up to 21%. As predicted, taxotere (30 mg/kg, Q3Dx4) was only able to achieve 26.6% tumor suppression on day 20, without detectable body-weight decreases, presumably because of its poor oral bioavailability.

Therapeutic Cure of Ovarian Carcinoma SK-OV-3 Xenograft.

The clinical efficacy of taxol in treating ovarian cancer has been well documented. Our third-generation epothilone analogues are also able to effectively achieve therapeutic cures against the SK-OV-3 xenograft. As shown in Fig. 2D, iso-fludelone (15 mg/kg, Q6Dx5), dehydelone (20 mg/kg, Q6Dx7), and iso-dehydelone (10 mg/kg, Q14Dx4) were able to achieve therapeutic cures in fewer doses, without relapse up to day 202 and with LCK ≥ 11.7.

Therapeutic Effect Against Lung Carcinoma A549 and A549/Taxol₁₀₀ Xenografts.

We next evaluated iso-fludelone against the refractory lung carcinoma A549 cell line and the taxol-resistant subline, A549/taxol (574-fold resistant in vitro) (see SI Methods for tumor cell line generation). Each mouse was simultaneously inoculated s.c. with both the A549 and A549/taxol xenograft tumors on the left and right sides of the flank, to minimize interanimal variations for the two tumors and to take advantage of the fact that the two xenografts grow at similar rates. Treatment commenced on day 11 when each tumor had grown to ≈100 mm³ in size.

As shown in Fig. 3A, both iso-fludelone (15 mg/kg, Q7Dx4) and taxol (25 mg/kg, Q3Dx9) were able to achieve >99.7% tumor suppression against A549, but without tumor shrinkage. Iso-dehydelone (10 mg/kg, Q12Dx3) and dehydelone (20 mg/kg, Q7Dx4) achieved only 89.5% and 77% tumor suppression, respectively.

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Fig. 3.

Treatment of refractory tumors. (A) Therapy of nude mice bearing both refractory human lung carcinoma A549 (right side flank) and taxol-resistant, A549/taxol (left side flank) with dehydelone, iso-dehydelone, iso-fludelone, and taxol. Dose, route, and schedule are as shown. (B) Therapy of adriamycin-resistant human mammary adenocarcinoma MCF-7/Adr with fludelone, iso-fludelone, and taxol. Dose, route, and schedule are as shown. (C) Therapy against s.c. implanted human neuroblastoma SK-NAS xenograft with dEpoB, dehydelone, fludelone, iso-dehydelone, iso-fludelone, and taxol. Dose, route, and schedule are as shown. (D) Therapy against refractory, intracranially implanted human neuroblastoma SK-NAS xenograft in nude mice with iso-fludelone, BCNU, taxol, and temodar. Dose, route, and schedule are as shown. The time courses of percent survival in treated and untreated control are shown in a Kaplan–Meier plot.

The therapeutic effects of the epothilones and taxol against the A549/taxol₁₀₀ xenograft are presented in Fig. 3A. As expected, the efficacy of taxol was greatly diminished against the taxol-resistant xenograft, accomplishing suppression of tumor growth by 42%. Under the conditions examined, iso-fludelone demonstrated the best efficacy, suppressing tumor growth by 80%. Dehydelone and iso-dehydelone achieved growth suppression of 72% and 57%, respectively.

Effects Against Adriamycin-Resistant Mammary Adenocarcinoma MCF-7/Adr Xenograft.

Nude mice bearing the adriamycin-resistant MCF-7/Adr xenograft were treated with fludelone (25 mg/kg, Q2Dx4), iso-fludelone (15 mg/kg, Q7Dx2), or taxol (25 mg/kg, Q2Dx4) by 6-h i.v. infusion (Fig. 3B). Fludelone and iso-fludelone were able to effectively suppress tumor growth by 98.2% and 100%, respectively (Day 18), whereas taxol was only able to achieve ≈3.7% tumor suppression.

Effects Against Refractory Neuroblastoma SK-NAS Xenograft Inoculated S.C.

The s.c. SK-NAS xenograft doubles in tumor size in<1.7 days, rendering this refractory xenograft the most aggressive used in our studies. When treatment commenced on day 11, the tumor size was ≈200 mm³ (Fig. 3C). By day 17, the control-group tumor size had grown to 4,000 mm³ and the control animals were killed. On day 17, taxol (30 mg/kg, Q2Dx4) and dEpoB (30 mg/kg, Q2Dx5) exhibited tumor suppression of 74.4% and 81.4%, respectively.

Treatment with dehydelone (20 mg/kg, Q6Dx4) induced CR in one of three mice on day 37, with relapse on day 43 (LCK = 1.06). Fludelone (20 mg/kg, Q2Dx9) achieved CR in one of three mice on day 33, which relapsed on day 63 (LCK = 5.31), and iso-dehydelone (10 mg/kg, Q12Dx2) also yielded CR in one of three mice (Day 19), which relapsed on day 43 (LCK = 4.25). By contrast, only iso-fludelone (45 mg/kg, Q12Dx4) was able to induce CR in four of four mice, with no relapse on day 120 (LCK ≥ 14.60). Thus, among the epothilone analogues evaluated herein, iso-fludelone uniquely achieved a therapeutic cure against the refractory, fast-growing SK-NAS xenograft. Further detailed comparisons of epothilones against SK-NAS xenografts are given in Table S5.

Therapeutic Effect Against Intracranially Inoculated SK-NAS Xenograft.

To evaluate the therapeutic effect of iso-fludelone in a difficult-to-treat brain-tumor model, the fast-growing refractory neuroblastoma SK-NAS was implanted intracranially (10⁶ cells per μl) into the right frontal lobe of nude mice brains. As shown in the Kaplan–Meier plot for the time course of survival (Fig. 3D), the median survival time (MST) of the control group of mice was 16.8 ± 0.4 days. Treatment with the brain tumor chemotherapeutic agents BCNU (15 mg/kg, QDx4, i.v.) or Temodar (200 mg/kg, Q2Dx3, oral) yielded increases in MST to 17.8 ± 0.4 days and 19.2 ± 1.3 days, respectively. As expected, taxol (20 mg/kg, Q2Dx7, i.v.) had little therapeutic effect, with an MST of 18.8 ± 1.2 days. By contrast, treatment with iso-fludelone (30 mg/kg, Q10Dx2, 6-h i.v. infusion) yielded a significantly increased MST of 26.8 ± 1.3 days**. These results, against a highly refractory tumor, reveal a marked improvement for iso-fludelone over the established cancer agents evaluated in this study. The findings suggest that iso-fludelone is readily capable of exerting a strong therapeutic effect against brain tumors.

Tissue Distribution and Pharmacokinetic Properties.

Fig. S4A shows the distribution of various epothilones in the MX-1 tumor, after 3-h i.v. infusion of tumor-bearing nude mice (13). At the low dose of 2.5 mg/kg, iso-fludelone has the highest concentration at 6 h, followed by fludelone and dehydelone. dEpoB, at 10 mg/kg, reached similar levels as dehydelone at 6 h. At 21 h after infusion, the tumor epothilone concentrations were as follows: iso-fludelone > fludelone > dehydelone > dEpoB. It is remarkable that, even at this low dose, the tumor drug concentrations for iso-fludelone and fludelone are several hundred times higher than their in vitro IC₅₀ values: up to 21 h after the end of infusion.

The tumor/brain ratios, after 5-min, 1-h, and 3-h infusion, are shown in Fig. S4 B and C for fludelone and iso-fludelone, respectively. The ratio was as high as 14 for fludelone at 1 h and 22 for iso-fludelone at 3 h, after infusion. These high ratios are rarely seen in antitumor agents. The tumor/brain drug ratios are compared in Fig. S4D, after 3-h i.v.-infusion at 2.5 mg/kg each, with the exception of dEpoB, which was evaluated at 10 mg/kg. At 6 h after infusion, the tumor/brain ratio rank was: iso-fludelone ≫ fludelone ≫ dehydelone > dEpoB. At 21 h after infusion, the rank was: fludelone > iso-fludelone ≫ dehydelone > dEpoB.

The results indicate that the epothilones, particularly fludelone and iso-fludelone, are concentrated selectively in the tumor rather than in the brain (or many other organs) (data not shown). In addition, these compounds persist in the tumor for a very long time (e.g., 21 h) and at very high concentrations (e.g., several-hundred-fold concentrations compared with their IC₅₀ in vitro).