Introduction

Aplidin is a cyclic depsipeptide that was isolated from a Mediterranean marine tunicate, Aplidium albicans.¹ In both animal and human preclinical studies and clinical phase I and II studies this agent has been shown to have cytotoxic potential against a broad spectrum of tumor types including leukemia and lymphoma.^2,3,4,5,6,7 Aplidin was found to induce strongly apoptosis in the human leukemia cell line MOLT-4.^8,9 Although the primary mode of action of aplidin has not been determined, the following mechanisms of action have been confirmed: (1) inhibition of protein synthesis by controlling GTP-dependent elongation factor 1-, (2) inhibition of ornithine decarboxylase at the transcriptional level, an enzyme involved in polyamine biosynthesis, (3) inhibition of palmitoyl thioesterase by controlling a G-protein-dependent signal transduction pathway for cellular proliferation and (4) inhibition of the autocrine loop for vascular endothelial growth factor stimulation thereby having potential antiangiogenic effect.^10,11,12,13 Aplidin-induced cytotoxicity was shown to be independent of the p53 and the MDR status.⁹ In clinical Phase I studies aplidin was not myelotoxic, except for mild lymphopenia.^4,5,6,7 These characteristics make aplidin a potentially useful agent for the treatment of leukemia. Adding aplidin to the current combination chemotherapy for leukemia could improve efficacy without the necessity of dose reductions of drugs with proven antileukemic activity, because of increased myelotoxicity. This seems especially relevant for the treatment of relapsed acute lymphoblastic leukemia (ALL) and newly diagnosed and relapsed acute myeloid leukemia (AML), since these are diseases with a relatively poor prognosis, which are currently being treated with myelotoxic drug combinations. In phase I clinical studies with aplidin, L-carnitine was given as a 24 h pretreatment or coadministered to prevent myotoxicity. Coadministration of L-carnitine was proven to be able to improve the recovery of the drug-induced muscular toxicity and has allowed for dose escalation of aplidin.⁵ Preliminary observations in these clinical studies suggested that the cytotoxic potential of aplidin was increased by L-carnitine pretreatment. We therefore included drug interaction studies with aplidin and L-carnitine.

The in vitro sensitivity or resistance of leukemic cells as determined by the total cell kill methyl-thiazol-tetrazolium (MTT)-based assay has been shown to give clinically relevant information in childhood leukemia. The assay results correlate with clinical and cell biological features, as well as with clinical outcome after single agent and after combination chemotherapy.^{14,15,16,17,18,19,20,21,22,23,24,25,26} To determine the potential of aplidin as a cytotoxic agent in pediatric leukemia, we have tested bone marrow (BM) and peripheral blood (PB) samples of children with different types of leukemia and of children without leukemia in the MTT assay and compared the results with in vitro cytotoxicity of known antileukemic agents.

Materials and methods

Leukemic and normal samples

To determine in vitro sensitivity or resistance to aplidin in childhood leukemia and normal BM and PB mononuclear cells, we used the MTT assay on BM samples from 12 patients with untreated ALL, 11 patients with relapsed ALL, 20 patients with untreated AML and two patients with relapsed AML. One ALL and four AML samples had to be excluded from the analysis because of a low percentage of blasts as explained further on. We also tested 19 normal, nonleukemic BM samples and 13 normal, nonleukemic PB samples. In 12 normal controls both BM and PB samples were available (paired samples). The 'normal, nonleukemic samples' were derived from 10 healthy children who underwent anesthesia for an ophthalmological procedure and from 10 children who were at least 2 years after treatment for ALL with persistent complete remission for at least 4 years, seen and sampled in the VU University Medical Center in Amsterdam. For normal controls, both healthy children and ex-leukemia patients, informed consent was obtained and the study was approved by the Medical Ethical Committee of our hospital as well as by the national Central Committee for Medical Research in humans. The leukemia samples were sent to our laboratory for research purposes and tested with informed consent, and mainly concerned patients diagnosed and treated in our hospital in Amsterdam.

In a first analysis (results not shown), we compared aplidin LC50 and LC 75 values in BM samples of ten healthy controls and nine ex-leukemia patients as well as in PB samples of six healthy controls and seven ex-leukemia patients by Mann–Whitney U-test and found no significant differences between healthy controls and ex-leukemia patients. Therefore, healthy controls and ex-leukemia patients were taken together as one group of normal controls.

Mononuclear cells were isolated from the normal PB samples by density-gradient centrifugation with Ficoll-Isopaque and were tested immediately after isolation. Normal BM samples were also tested the same day. The leukemia samples were either fresh or had been cryopreserved before testing. We have previously shown that cryopreservation does not influence the results obtained by cellular drug resistance testing.¹⁹ Moreover, in leukemia samples the origin, BM or PB, also does not change the MTT assay results.¹⁹

MTT assay

The MTT assay has been described before.^20,21 This assay is based on the principle that after drug exposure only surviving cells (ie drug-resistant cells) can convert MTT salt into formazan, which can be quantified by spectrophotometry. Aplidin was provided by PharmaMar as dry powder and 2 mg was dissolved in 500 l DMSO, which was then diluted at least 1124 times.

Briefly, 80 l aliquots of a leukemic cell suspension of 2 10⁶ cells/ml for ALL, 1 10⁶ cells/ml for AML and 1 10⁶ cells/ml for normal controls were added to microculture plates and 20 l of 6 concentrations of aplidin from 1.6 10^-1 to 1.6 10^-6 M was added in duplicate. We chose this aplidin concentration range after performing a pilot study on 10 samples to determine the optimal concentration range of aplidin to obtain dose–response curves. As controls, culture medium with and without (background) cells, and without aplidin, were tested at least in triplicate. After 4 days of culture 10 l of 5 mg/ml MTT (Sigma) was added to each well. The microculture plates were incubated for another 6 h allowing viable cells to reduce the yellow MTT into dark-colored formazan crystals that were dissolved with 100 l acidified isopropanol. The optical density (OD) was measured at 562 and 720 nm (the so-called dual measurements to allow correction for 'noise'). Cell survival (CS) was calculated as follows: CS=(OD_{drug-exposed well}/mean OD_{drug-free wells}) 100% after correction for the background OD and the OD at 720 nm. The LC50 and LC75 values, which are the drug concentrations lethal to 50 and 75% of the cells, respectively, were calculated from the dose–response curves and used as parameters of in vitro chemosensitivity. Results for leukemia samples were considered eligible for analysis only if the control wells contained 70% or more leukemic cells before as well as after 4 days of culture, and in case of a control OD of >0.050.

Since L-carnitine was pre- and coadministered with aplidin in clinical studies, we also tested L-carnitine in the MTT assay and showed that L-carnitine itself was not cytotoxic in concentrations from 2.05 10^-6 to 100 M (data not shown). We then selected two concentrations of L-carnitine (1 and 100 M) for combination studies. Since dimethyl sulfoxide (DMSO) was used as a solvent for aplidin we also tested the possible cytotoxicity of DMSO. In the aplidin concentration range used no added cytotoxicity for DMSO was found (data not shown). Since no differences in LC50 or LC75 values were found when aplidin alone was compared to aplidin after L-carnitine preincubation, either in the high or low concentration, the LC50 and LC75 values presented in this paper are taken from the assay in which aplidin was tested without L-carnitine preincubation. After we determined LC50 and LC75 values in the different subgroups and compared these groups with regard to sensitivity to Aplidin, we noticed that the shape of the dose–response curves showed more pronounced differences between groups at higher aplidin concentrations and therefore the results with regard to the LC75 values which demonstrate these differences more clearly are shown in more detail.

To establish whether crossresistance exists between Aplidin and other cytotoxic drugs, we have compared LC75 values for Aplidin with LC75 values for other frequently used cytotoxic drugs both in leukemia samples and in normal BM and blood samples. These other drugs have been tested in the MTT assay in the same way as Aplidin and include (range of concentration): dexamethasone (0.0002–6 g/ml), prednisolone (0.008–250 g/ml), 6-thioguanine (1.56–50 g/ml), 6-mercaptopurine (15.6–500 g/ml), cytarabine (0.002–2.5 g/ml), 2',2'-difluorodeoxycytidine (gemcitabine) (0.0122–400 g/ml), 2-chlorodeoxyadenosine (0.0004–40 g/ml), fludarabine (0.0156–16 g/ml), mitoxantrone (0.001–1 g/ml), doxorubicin (0.008–8 g/ml), daunorubicin (0.002–2 g/ml), idarubicin (0.002–2 g/ml), etoposide (0.05–50 g/ml), teniposide (0.003–8 g/ml), 4-HOO-ifosfamide (0.1–100 g/ml, active metabolite of Ifosfamide), busulfan (1.23–300 g/ml), L-asparaginase (0.003–10 IU/ml), 5-azacytidine (2.441–2500 g/ml), amsacrine (0.006–20 g/ml) and vincristine (0.05–50 g/ml). In general, these concentrations include the pharmacologically achievable concentrations. In some samples, the LC75 values exceeded the maximum concentration tested and if so the maximum concentration of the drug was considered as being the LC75 value, whereas in fact the real LC75 value was higher and unknown.

Statistics

We calculated median LC50 and LC75 values and ranges for the different subgroups of leukemia and normal BM and PB samples and these were compared with the Mann–Whitney U-test. Also, the paired BM and PB samples from the normal controls were compared with the Wilcoxon signed-rank test. The initial and relapsed ALL samples were not paired. For comparison of LC50 and LC75 values in the same sample with and without L-carnitine a paired samples T-test was applied.

Crossresistance of aplidin with other frequently used cytotoxic drugs was analyzed with the Spearman rank correlation test. Statistically significant differences were defined at a P0.01, while a P-value between 0.01 and 0.05 was considered to indicate a trend for a significant difference. If more than half of the samples tested for crossresistance had the maximum LC75 value for Aplidin and/or any other drug tested (ie LC75 value higher than maximum concentration tested and no absolute value available) statistical analysis was not considered feasible (not eligible in Table 2.

Table 2 - Crossresistance of Aplidin with other frequently used cytotoxic drugs in 40 leukemia samples and 19 normal bone marrow samples.

Full table

Results

Since the MTT assay results were considered eligible only if control wells contained 70% or more leukemic blasts, five of 45 leukemia samples (one ALL, four AML) were excluded from analysis, because of blast percentages less than 70% after 4 days of culture.

Aplidin cytotoxicity as tested in the MTT assay was dose dependent in the concentration range tested. Aplidin proved to be cytotoxic in vitro at nanomolar concentrations. Dose–response curves were obtained so that LC50 and LC75 values could be calculated.

Aplidin LC50 values with preincubation with either high (100 M) or low (1 M) concentrations of L-carnitine and Aplidin LC50 values without L-carnitine pre-incubation had a median of 0.011 (range 0.0013–0.16), 0.012 (0.0013–0.14) and 0.012 (0.0012–0.16), respectively, and were not significantly different (P=0.222 for high and P=0.797 for low concentrations of L-carnitine as compared to aplidin without L-carnitine). The same was true when LC75 values were compared (data not shown).

The Aplidin LC75 median values and ranges in the different subgroups are shown in Table 1 and Figure 1. Aplidin LC75 values were not significantly different for initial ALL as compared to initial AML or for initial ALL as compared to relapsed ALL, but both normal BM and normal PB samples were significantly more resistant to aplidin than leukemia samples (median two- to seven-fold, P=0.001 and median four- to 11-fold, P<0.0001, respectively). Aplidin LC50 values were not significantly different between the leukemic subgroups either, but were again significantly lower than LC50 values in normal BM and PB samples (data not shown). We also compared aplidin cytotoxicity in paired BM and PB samples from 12 normal controls with the Wilcoxon signed-rank test. LC75 values were significantly higher in the PB samples as compared to the BM samples from normal controls (P=0.003).

Figure 1.

Aplidin LC75 values and median in different subgroups of leukemia and normal BM and PB samples.

Full figure and legend (18K)

Table 1 - In vitro Aplidin sensitivity as expressed by LC75 values in different diagnostic subgroups.

Full table

Since Aplidin is a new drug that might be useful as an additional drug in the current treatment of high-risk leukemias, we analyzed crossresistance with other frequently used cytotoxic drugs by comparing LC75 values for these drugs with aplidin LC75 values in the MTT assay both in leukemia samples and in normal BM and PB samples (Table 2). In leukemia samples (initial and relapsed ALL and AML taken together) no evidence for significant crossresistance with other cytotoxic drugs was found. There was a trend for a significant correlation with gemcitabine (=0.71, P=0.02). The leukemia samples were taken together as one group, because aplidin LC75 values between the subgroups were not different. If the subgroups iALL, rALL, iAML were each analyzed separately for crossresistance, no significant crossresistance of aplidin with other cytotoxic drugs was found, although numbers were low in each group (11, 11 and 16 samples, respectively). When LC50 values in the leukemia samples were compared the only significant correlation was with gemcitabine (=0.78, P=0.003, 11 samples tested with both aplidin and gemcitabine; data not shown). In normal BM samples evidence for significant crossresistance was found between aplidin and etoposide and teniposide, while a trend for a significant correlation was found with amsacrine, 2-chlorodeoxyadenosine, 4-HOO-ifosfamide and vincristine (Table 2). In nine of 13 normal PB samples aplidin LC75 exceeded the maximum concentration (0.16 M) tested and since no absolute LC75 values were available, crossresistance testing with other drugs was not considered feasible. When LC50 values for aplidin and other cytotoxic drugs in normal PB samples were compared no evidence for crossresistance was found, but the number of samples tested with aplidin and any other cytotoxic drug in this subgroup was never more than eight.

Discussion

Aplidin is a novel cytotoxic drug that has been shown in phase I studies to have a positive therapeutic index in adult patients with advanced pretreated solid tumors and is currently under phase II development.^2,3,4,5,6,7 Aplidin has also been shown to induce apoptosis in human leukemia cell lines.⁸ In the clinical studies, a lack of BM toxicity (except for 'asymptomatic' grades 1–3 lymphopenia in 50% of the cases) in patients treated at the recommended dose was established.^4,5,6,7 Since the dose-limiting toxicity with aplidin is myotoxicity rather than myelotoxicity, this drug could be a useful adjuvant in current myelotoxic treatment protocols for high-risk leukemia (and other malignancies) in order to try and improve outcome without undue myelotoxicity. Therefore, we investigated the cytotoxicity of aplidin in vitro in childhood leukemia samples, and normal BM and PB samples by means of the MTT assay. Assays such as the MTT assay are successful in about 80% of cases. This selection of patients leads to an over-representation of leukemia patients with high tumor burden and an under-representation of DNA hyperdiploid cases. However, the far majority of samples is being tested successfully and all subtypes of leukemia are represented in this type of studies. The results of this assay have previously shown a good correlation with clinical results of leukemia treatment.^{15,16,22,23,24,25} We show that aplidin indeed is cytotoxic for leukemic cells in a dose-dependent fashion and in relatively low concentrations (10^-9 M=nanomolar) as compared to other drugs we have previously tested in the MTT assay. These in vitro concentrations correlate with the drug levels reached in patients treated with pharmacologically appropriate doses of Aplidin.²⁷ Since in phase I and II clinical studies with aplidin patients have been pretreated with L-carnitine to prevent myotoxicity, we tested the influence of L-carnitine preincubation on aplidin cytotoxicity in vitro. L-carnitine in two different concentrations did not influence the cytotoxicity of aplidin in vitro towards leukemic cells, as expressed by the LC50 and LC75 values in the MTT assay.

Normal BM and PB cells from children appeared to be significantly more resistant to Aplidin cytotoxicity as compared to pediatric leukemic cells, median two- to 11-fold. This result is consistent with clinical phase I and II studies in adults, which have shown that myelotoxicity with aplidin is rare, and with the study by Albella et al,²⁷ who showed that the maximum concentration of aplidin in patients was 21- to 75-fold lower than the IC50_24 h value observed for the different hematopoietic progenitors.^4,5,6,7

For many drugs it has been shown that either lymphoid or myeloid leukemic cells are more sensitive.¹⁸ Also, leukemic cells at relapse tend to be less sensitive to cytotoxic drugs than at initial presentation of the leukemia.²⁶ Usually however, this is because of acquired resistance after these drugs have been used to treat the initial leukemia. Our study shows that lymphoid and myeloid leukemic cells are equally sensitive to the cytotoxic effect of aplidin in vitro. The mechanism by which aplidin is cytotoxic and induces apoptosis is still not completely understood. Our results suggest that aplidin acts through a mechanism that is shared by lymphoid and myeloid leukemic cells. Aplidin cytotoxicity in vitro was not different when initial and relapsed ALL samples were compared. However, these samples were not derived from the same patients and they had not been exposed to aplidin during their treatment of the initial leukemia. It is as yet unclear if and at what rate aplidin resistance may develop when leukemic cells would have been exposed to this drug. However, our data do suggest that relapsed ALL patients may benefit from aplidin at similar dosages as in newly diagnosed ALL.

Since aplidin is a new drug with an incompletely understood, but possibly very different mechanism of action as compared to the currently known cytotoxic drugs, we were interested if any crossresistance with other cytotoxic drugs already used in the treatment of leukemia existed. Interestingly, in leukemic samples no significant crossresistance was found for Aplidin with any of the currently used cytotoxic drugs, except for a trend for a significant correlation with gemcitabine. This suggests that Aplidin, which was shown by us to be highly cytotoxic in relapsed ALL samples and lacked major myelotoxicity in clinical studies, could be used in combination with other currently used, but myelotoxic, cytotoxic drugs in patients with relapsed or chemoresistant leukemia. Although there is only a trend for a significant correlation of cytotoxicity of Aplidin with Gemcitabine in leukemic samples this could be clinically relevant. Gemcitabine is a nucleoside analogue that inhibits DNA synthesis through chain termination and ribonucleotide reductase inhibition.²⁸ Therefore, the reason for crossresistance with aplidin is so far unexplained. Phase I studies of Gemcitabine in refractory and relapsed leukemia have suggested clinical activity in these patients.^29,30 Mucositis and hepatotoxicity were the dose-limiting toxicities. Crossresistance between gemcitabine and aplidin suggests that combining these drugs in leukemia treatment might be less useful, but more in vitro and in vivo studies are clearly needed to substantiate our in vitro results.

For normal BM samples significant crossresistance between aplidin and the epipodophyllotoxins (etoposide, teniposide) was found, while a trend for a significant correlation was found with cytotoxic drugs from different groups and with different modes of action: amsacrine, 2-chlorodeoxyadenosine, 4-HOO ifosfamide and vincristine. From what is currently known about the mechanisms of action of aplidin no common pathway to explain this crossresistance is apparent. Also, it is unclear why crossresistance of aplidin with the epipodophyllotoxins is found in normal BM samples but not in leukemic samples. In PB samples crossresistance could not be established by the evaluation of LC75 values for aplidin and other cytotoxic drugs, because in nine of 13 PB samples Aplidin LC75's were above the maximum concentration tested and thus no absolute values for correlation testing were available. No crossresistance was found when LC50 values in normal PB samples were compared (data not shown). Since several subgroups of leukemia as well as normal BM and PB samples were tested, the number of samples in each subgroup is relatively small and therefore correlations with regard to crossresistance may not reach statistical significance, but the nonsignificant correlation coefficients were also not high.

In conclusion, our study shows that aplidin has selective cytotoxicity in vitro towards childhood leukemia cells and within leukemic samples significant crossresistance with commonly used antileukemic agents was lacking. Carnitine did not decrease or increase the cytotoxicity of Aplidin. Clinical studies are warranted and in fact a phase I study in pediatric patients is under implementation.

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Acknowledgements

We thank the laboratory technicians of the research laboratory of Pediatric Oncology of the VU University Medical Center for their help with performing the MTT assays.