Abstract
Aplidin was tested in vitro at concentrations ranging from from 0.1 nM to 1.0 μM and in vivo at a dose of 0.6 mg/kg administered intraperitoneally on an every 4 days × 3 schedule that was repeated at day 21. In vitro, Aplidin was most active against acute lymphoblastic leukemia (ALL) cell lines. In vivo, Aplidin induced significant differences in EFS distribution in 12 of 28 (43%) solid tumor models and 2 of 6 evaluable ALL models. Aplidin showed potent in vitro activity and induced significant in vivo tumor growth inhibition in some xenografts, but did not induce tumor regressions.
Keywords: Preclinical Testing, Developmental Therapeutics, Aplidin
INTRODUCTION
Aplidin (Plitidepsin), a potent antitumor agent, was first isolated from the marine tunicate Aplidium albacans and currently is obtained by total chemical synthesis. The mechanism of action of Aplidin is not clearly defined, although apoptosis induction has been described as occurring through a strong, sustained activation of c-Jun NH2-terminal kinase (JNK) [1]. Cells resistant to Aplidin show lesser extent of JNK activation [2]. JNK activation in human breast cancer cells was reported to be dependent upon induction of oxidative stress associated with activation of the Rac1 small GTPase [3]. Aplidin is currently in Phase II clinical evaluation as a single agent for solid and hematologic malignant neoplasias including multiple myeloma, non-Hodgkin lymphoma, and acute lymphoblastic leukemia (ALL). Phase I studies in pediatric acute leukemias and solid tumors as well as combination studies with other chemotherapeutic drugs are currently ongoing. Given the potential novel mechanism of action of Aplidin, the PPTP evaluated this agent to gain insight into its utility against pediatric tumors.
MATERIALS AND METHODS
In vitro testing
In vitro testing was performed using DIMSCAN, a semiautomatic fluorescence-based digital image microscopy system that quantifies viable cell numbers in tissue culture multiwell plates [4]. Cells were incubated in the presence of Aplidin for 96 hours at concentrations from 0.1 nM to 1.0 μM and analyzed as previously described [5].
In vivo tumor growth inhibition studies
CB17SC-M scid−/− female mice (Taconic Farms, Germantown NY), were used to propagate subcutaneously implanted kidney/rhabdoid tumors, sarcomas (Ewing, osteosarcoma, rhabdomyosarcoma), neuroblastoma, and non-glioblastoma brain tumors, while BALB/c nu/nu mice were used for glioma models, as previously described [6–8]. Human leukemia cells were propagated by intravenous inoculation in female non-obese diabetic (NOD)/scid−/− mice as described previously [9]. All mice were maintained under barrier conditions and experiments were conducted using protocols and conditions approved by the institutional animal care and use committee of the appropriate consortium member. Tumor volumes (cm3) [solid tumor xenografts] or percentages of human CD45-positive [hCD45] cells [ALL xenografts] were determined as previously described [10]. Responses were determined using three activity measures as previously described [10]. An in-depth description of the analysis methods is included in the Supplemental Response Definitions section.
Statistical Methods
The exact log-rank test, as implemented using Proc StatXact for SAS®, was used to compare event-free survival distributions between treatment and control groups. P-values were two-sided and were not adjusted for multiple comparisons given the exploratory nature of the studies.
Drugs and Formulation
Aplidin was provided to the PPTP by PharmaMar, through the Cancer Therapy Evaluation Program (NCI). Aplidin was reconstituted in a solution of Cremophor: Ethanol: Water (15:15:70) and further diluted in saline. Aplidin was administered i.p., every 4 days times 3, repeated at day 21, at a dose of 0.6 mg/kg. Aplidin was provided to each consortium investigator in coded vials for blinded testing.
RESULTS
Aplidin in vitro testing
Aplidin demonstrated a high level of cytotoxic activity against the PPTP’s cell lines, with 16 of 23 cell lines showing T/C values < 50% at the lowest Aplidin concentration tested (0.1 nM)(Table I). Therefore, Aplidin cytotoxic activity at 0.1 nM (T/C0.1nM) was used to compare the relative responsiveness of the PPTP cell lines to Aplidin. The median T/C0.1nM was 25.3% (range 0.5% to 96.3%). The cell lines of the ALL panel were the most sensitive PPTP cell lines to Aplidin (median T/C0.1nM = 8.6%), whereas the neuroblastoma (T/C0.1nM = 80.8%) and rhabdomyosarcoma (T/C0.1nM = 57.4%) cell lines were less responsive to Aplidin (Table I and Supplemental Figure 1).
Table I.
Activity of Aplidin against Cell Lines in the PPTP in Vitro Panel
| Cell Line | Status | Histology | T/C0.1nM (%) | IC50 (nM) |
|---|---|---|---|---|
| RD | Rhabdomyosarcoma | 46.4 | <0.1 | |
| Rh41 | Post-Therapy | Rhabdomyosarcoma | 57.5 | 0.19 |
| Rh18 | Diagnosis | Rhabdomyosarcoma | 96.3 | 5.50 |
| Rh30 | Diagnosis | Rhabdomyosarcoma | 57.3 | 0.23 |
| BT-12 | Diagnosis | Rhabdoid | 24.8 | <0.1 |
| CHLA-266 | Diagnosis | Rhabdoid | 35.7 | <0.1 |
| TC-71 | Post-Therapy | Ewing | 5.5 | <0.1 |
| CHLA-9 | Diagnosis | Ewing | 32.9 | <0.1 |
| CHLA-10 | Post-Therapy | Ewing | 25.3 | <0.1 |
| CHLA-258 | Post-Bone Marrow Transplant | Ewing | 46.7 | <0.1 |
| SJ-GBM2 | Post-Therapy | Glioblastoma | 15.5 | <0.1 |
| NB-1643 | Diagnosis | Neuroblastoma | 79.5 | 0.67 |
| NB-EBc1 | Post-Therapy | Neuroblastoma | 95.4 | 5.70 |
| CHLA-90 | Post-Bone Marrow Transplant | Neuroblastoma | 35.0 | <0.1 |
| CHLA-136 | Post-Bone Marrow Transplant | Neuroblastoma | 82.1 | 6.00 |
| COG-LL-317 | Post-Therapy | ALL T-cell | 3.5 | <0.1 |
| NALM-6 | Post-Therapy | ALL B-precursor | 0.5 | <0.1 |
| RS4;11 | Post-Therapy | ALL B-precursor | 21.6 | <0.1 |
| MOLT-4 | Post-Therapy | ALL T-cell | 8.6 | <0.1 |
| CCRF-CEM | ALL T-cell | 11.0 | <0.1 | |
| Kasumi-1 | Post-Bone Marrow Transplant | AML | 11.4 | <0.1 |
| Karpas-299 | Post-Therapy | ALCL | 8.8 | <0.1 |
| Ramos-RA1 | NHL | 3.7 | <0.1 | |
| Median | 25.3 | <0.1 | ||
| Minimum | 0.5 | <0.1 | ||
| Maximum | 96.3 | 6.00 |
Activity of Aplidin against the PPTP in vivo panel
Aplidin was evaluated in 44 xenograft models. Fifty-nine of 830 mice died during the study (7.1%), with 3 of 411 in the control arms (0.7%) and 56 of 419 in the Aplidin treatment arms (13.4%). Ten lines were excluded from analysis due to toxicity greater than 25 percent. A complete summary of results is provided in Supplemental Table I, including total numbers of mice, number of mice that died (or were otherwise excluded), numbers of mice with events and average times to event, tumor growth delay, as well as numbers of responses and T/C values.
Antitumor effects were evaluated using the PPTP activity measures for time to event (EFS T/C), tumor growth delay (tumor volume T/C), and objective response. Aplidin induced significant differences in EFS distributions compared to controls in 12/28 (43%) of the evaluable solid tumor models and 2 of 6 (33%) for the evaluable ALL xenografts (Table II). Only two lines (ALL-3, ALL-8) met the criteria for intermediate activity with EFS T/C values of 11.2 and 4.7, respectively (Table II). No other models met criteria for intermediate activity for the EFS T/C activity measure by having EFS T/C values exceeding 2.0.
Table II.
Activity for Aplidin against the PPTP in Vivo Panel
| Xenograft Line | Histology | P-value | EFS T/C | Median Final RTV | Tumor Volume T/C | P-value | T/C Activity | EFS Activity | Response Activity |
|---|---|---|---|---|---|---|---|---|---|
| BT-29 | Rhabdoid | 0.009 | 1.4 | >4 | 0.52 | 0.002 | Low | Low | Low |
| KT-14 | Rhabdoid | 0.33 | 1.5 | >4 | 0.9 | 0.36 | Low | Low | Low |
| KT-12 | Rhabdoid | 0.406 | 1.1 | >4 | 0.82 | 0.274 | Low | Low | Low |
| KT-10 | Wilms | 0.259 | 1.2 | >4 | 0.64 | 0.043 | Low | Low | Low |
| KT-11 | Wilms | 0.104 | 1.3 | >4 | 0.71 | 0.105 | Low | Low | Low |
| KT-13 | Wilms | 0.048 | 1.2 | >4 | 0.83 | 0.113 | Low | Low | Low |
| SK-NEP-1 | Ewing | 0.224 | 1.2 | >4 | 0.82 | 0.19 | Low | Low | Low |
| CHLA258 | Ewing | 0.825 | 1 | >4 | 0.79 | 0.631 | Low | Low | Low |
| Rh28 | ALV RMS | 0.831 | 1.3 | >4 | 0.75 | 0.573 | Low | Low | Low |
| Rh30 | ALV RMS | 0.19 | 0.8 | >4 | 1.03 | 0.971 | Low | Low | Low |
| Rh30R | ALV RMS | 0.26 | 1.1 | >4 | 0.9 | 0.258 | Low | Low | Low |
| Rh41 | ALV RMS | 0.025 | 1.1 | >4 | 0.79 | 0.094 | Low | Low | Low |
| Rh65 | ALV RMS | 0.16 | 1.4 | >4 | 0.86 | 0.631 | Low | Low | Low |
| Rh18 | EMB RMS | 0.002 | 1.5 | >4 | 0.58 | 0.001 | Low | Low | Int |
| BT-45 | Medulloblastoma | 0.033 | 1.4 | >4 | 0.76 | 0.237 | Low | Low | Low |
| BT-41 | Ependymoma | 0.549 | 0.9 | >4 | 0.97 | 0.867 | Low | Low | Low |
| GBM2 | Glioblastoma | <0.001 | 1.5 | >4 | 0.67 | 0.019 | Low | Low | Low |
| D456 | Glioblastoma | 0.106 | 1.2 | >4 | 0.79 | 0.093 | Low | Low | Low |
| NB-SD | Neuroblastoma | 0.007 | 1.4 | >4 | 0.53 | <0.001 | Low | Low | Low |
| NB-1771 | Neuroblastoma | 0.004 | 1.4 | >4 | 0.75 | 0.075 | Low | Low | Low |
| NB-EBc1 | Neuroblastoma | 0.016 | 1.4 | >4 | 0.72 | 0.579 | Low | Low | Low |
| NB-1643 | Neuroblastoma | 0.152 | 1.1 | >4 | 0.74 | 0.075 | Low | Low | Low |
| OS-1 | Osteosarcoma | <0.001 | 1.2 | >4 | 0.65 | <0.001 | Low | Low | Low |
| OS-2 | Osteosarcoma | 0.698 | 1.1 | >4 | 0.9 | 0.529 | Low | Low | Low |
| OS-17 | Osteosarcoma | 0.029 | 1.2 | >4 | 0.71 | 0.105 | Low | Low | Low |
| OS-9 | Osteosarcoma | 0.201 | 1.3 | >4 | 0.71 | 0.028 | Low | Low | Low |
| OS-33 | Osteosarcoma | 0.127 | 1 | >4 | 0.74 | 0.247 | Low | Low | Low |
| OS-31 | Osteosarcoma | <0.001 | 1.2 | >4 | 0.63 | 0.003 | Low | Low | Low |
| ALL-2 | ALL B-precursor | 0.818 | 1.5 | >25 | . | Low | Int | ||
| ALL-3 | ALL B-precursor | 0.041 | 11.2 | >25 | . | Int | Int | ||
| ALL-4 | ALL B-precursor | 0.371 | 2.1 | >25 | . | Low | Int | ||
| ALL-7 | ALL B-precursor | 0.707 | 1.4 | >25 | . | Low | Low | ||
| ALL-8 | ALL T-cell | 0.001 | 4.7 | >25 | . | Int | Int | ||
| ALL-19 | ALL B-precursor | 0.703 | 3.4 | >25 | . | Low | Int |
The in vivo testing results for the objective response measure of activity are presented in Supplemental Figure 2 in a ‘heat-map’ format as well as a ‘COMPARE’-like format, based on the scoring criteria described in the Material and Methods and the Supplemental Response Definitions section. The latter analysis demonstrates relative tumor sensitivities around the midpoint score of 5 (stable disease). Objective responses were seen in 0 of 34 tumor models.
DISCUSSION
In vitro the ALL cell lines appeared more sensitive to Aplidin compared to other lines, in agreement with previous reports of Aplidin activity against ALL cell lines and primary ALL cells [11,12]. By contrast, the neuroblastoma and rhabdomyosarcoma cell lines appeared less responsive to Aplidin than the remaining cell lines. However, relative sensitivities using IC50 concentrations cannot be determined from our analysis.
Aplidin demonstrated limited activity against the in vivo solid tumor panels. While there were clearly treatment effects as demonstrated by significant differences in EFS distribution between treated and control animals, these effects were modest and in no case did the time to event for a treated group exceed that for a control group by a factor of two or greater. Aplidin showed greater activity against the PPTP’s ALL panel compared to the solid tumor panels, although the level of activity observed for Aplidin was less than that previously noted for standard agents (e.g., vincristine and cyclophosphamide) [10]. Two xenografts (ALL-3 and ALL-8) had significant extensions in time to event, but they did not meet criteria for objective response.
One important issue in assessing the PPTP in vivo data is whether the systemic exposures achieved in test animals are comparable to systemic exposures achieved in humans at tolerable doses. The estimated plasma systemic exposure in mice for the planned six week treatment course is approximately 1000 ng*hr/mL (personal communication1). For humans, the recommended phase II dose for Aplidin using an every two week schedule is 5–7 mg/m2 [13]. The estimated plasma systemic exposure for a six week treatment course at the 5 mg/m2 dose level is approximately 1050 ng*hr/mL, which is similar to the estimated exposure in mice.
The PPTP Stage 1 testing results point to ALL as the diagnosis for which Aplidin has the highest level of activity, though this activity does not equal that of standard agents previously studied. Given the PPTP results as well as prior published results describing anti-leukemia activity for Aplidin, consideration should be given to focusing future research efforts on optimizing the activity of Aplidin (either used alone or in combination) against ALL.
Supplementary Material
Supplemental Figure 1. Aplidin in vitro activity: Graphs illustrate typical cytotoxicity/growth inhibition dose-response curves for cell lines with maximal effects approaching 100% (Nalm-6 and COG-LL-317). Error bars represent standard deviations for each concentration tested.
Supplemental Figure 2. Aplidin in vivo objective response activity: Representation of tumor sensitivity based on the difference of individual tumor lines from the midpoint response (stable disease). Bars to the right of the median represent lines that are more sensitive, and to the left are tumor models that are less sensitive. Red bars indicate lines with a significant difference in EFS distribution between treatment and control groups, while blue bars indicate lines for which the EFS distributions were not significantly different.
Acknowledgments
This work was supported by NO1-CM-42216, CA21765, and CA108786 from the National Cancer Institute and used Aplidin supplied by PharmaMar. In addition to the authors represents work contributed by the following: Sherry Ansher, Ingrid Boehm, Joshua Courtright, Mila Dolotin, Edward Favours, Henry S. Friedman, Min Kang, Debbie Payne-Turner, Charles Stopford, Chandra Tucker, Amy E. Watkins, Jianrong Wu, Joe Zeidner, Ellen Zhang, and Jian Zhang. Children’s Cancer Institute Australia for Medical Research is affiliated with the University of New South Wales and Sydney Children’s Hospital.
Footnotes
Doreen LePage, PharmaMar.
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Associated Data
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Supplementary Materials
Supplemental Figure 1. Aplidin in vitro activity: Graphs illustrate typical cytotoxicity/growth inhibition dose-response curves for cell lines with maximal effects approaching 100% (Nalm-6 and COG-LL-317). Error bars represent standard deviations for each concentration tested.
Supplemental Figure 2. Aplidin in vivo objective response activity: Representation of tumor sensitivity based on the difference of individual tumor lines from the midpoint response (stable disease). Bars to the right of the median represent lines that are more sensitive, and to the left are tumor models that are less sensitive. Red bars indicate lines with a significant difference in EFS distribution between treatment and control groups, while blue bars indicate lines for which the EFS distributions were not significantly different.