Innovation in oncology clinical trial design.

Progress in and better understanding of cancer biology causes a shift in cancer drug development: away from the evaluation of drugs in large tumour histology defined patient populations towards targeted agents in increasingly heterogeneous molecularly defined subpopulations. This requires novel approaches in clinical trial design by academia and industry, and development of new assessment tools by regulatory authorities. Pharmaceutical industry is developing new targeted agents generating many clinical studies, including target combinations. This requires improved operational efficiency by development of innovative trial designs, strategies for early-stage decision making and early selection of candidate drugs with a high likelihood of success. In addition, patient awareness and ethical considerations necessitate that agents will be rapidly available to patients. Regulatory Authorities such as the European Medicine Agency and national agencies recognise that these changes require a different attitude towards benefit-risk analysis for drug approval. The gold standard of randomised confirmatory Phase III trials is not always ethical or feasible when developing drugs for treatment of small cancer populations. Alternative strategies comprise accelerated approval via conditional marketing approval, which can be granted in the EU based on small non-randomised Phase II trials. The paper describes innovative trial designs with their pros and cons and efforts of pharmaceutical industry and regulatory authorities to deal with the paradigm shift. Furthermore, all stakeholders should continue to share their experiences and discuss problems in order to understand the position and concerns of the other stakeholders to learn from each other and to progress the field of novel oncology clinical trial design.

Efficacy, pharmacokinetic and pharmacodynamic evaluation of apaziquone in the treatment of non-muscle invasive bladder cancer.

INTRODUCTION: Apaziquone (also known as EO9 and QapzolaTM) is a prodrug that is activated to DNA damaging species by oxidoreductases (particularly NQO1) and has the ability to kill aerobic and/or hypoxic cancer cells. Areas covered: Whilst its poor pharmacokinetic properties contributed to its failure in phase II clinical trials when administered intravenously, these properties were ideal for loco-regional therapies. Apaziquone demonstrated good anti-cancer activity against non-muscle invasive bladder cancer (NMIBC) when administered intravesically to marker lesions and was well tolerated with no systemic side effects. However, phase III clinical trials did not reach statistical significance for the primary endpoint of 2-year recurrence in apaziquone over placebo although improvements were observed. Post-hoc analysis of the combined study data did indicate a significant benefit for patients treated with apaziquone, especially when the instillation of apaziquone was given 30 min or more after surgery. A further phase III study is ongoing to test the hypotheses generated in the unsuccessful phase III studies conducted to date. Expert opinion: Because of its specific pharmacological properties, Apaziquone is excellently suited for local therapy such as NMIBC. Future studies should include proper biomarkers.

Present and future breast cancer management--bench to bedside and back: a positioning paper of academia, regulatory authorities and pharmaceutical industry.

Insights into tumour biology of breast cancer have led the path towards the introduction of targeted treatment approaches; still, breast cancer-related mortality remains relatively high. Efforts in the field of basic research revealed new druggable targets which now await validation within the context of clinical trials. Therefore, questions concerning the optimal design of future studies are becoming even more pertinent. Aspects such as the ideal end point, availability of predictive markers to identify the optimal cohort for drug testing, or potential mechanisms of resistance need to be resolved. An expert panel representing the academic community, the pharmaceutical industry, as well as European Regulatory Authorities met in Vienna, Austria, in November 2012, in order to discuss breast cancer biology, identification of novel biological targets and optimal drug development with the aim of treatment individualization. This article summarizes statements and perspectives provided by the meeting participants.

Antiproliferative activity and mechanism of action of fatty acid derivatives of gemcitabine in leukemia and solid tumor cell lines and in human xenografts.

UNLABELLED: Gemcitabine is a deoxycytidine analog, which can be inactivated by deamination catalyzed by deoxycytidine deaminase (dCDA). Altered transport over the cell membrane is a mechanism of resistance to gemcitabine. To facilitate accumulation, the fatty acid derivative CP-4125 was synthesized. Since, the fatty acid is acylated at the site of action of dCDA, a decreased deamination was expected. CP-4125 was equally active as gemcitabine in a panel of rodent and human cell lines and in human melanoma xenografts bearing mice. In contrast to gemcitabine, CP-4125 was not deaminated but inhibited deamination of deoxycytidine and gemcitabine. Pools of the active triphosphate of gemcitabine increased for over 20 hr after CP-4125 exposure, while these pools decreased directly after removal of gemcitabine. IN CONCLUSION: CP-4125 is an interesting new gemcitabine derivative.

Antiproliferative activity and mechanism of action of fatty acid derivatives of arabinosylcytosine (ara-C) in leukemia and solid tumor cell lines.

Resistance to, the hydrophilic drug ara-C, might be meditated by decreased membrane transport. Lipophilic prodrugs were synthesized to facilitate uptake. These compounds were equally active as ara-C, while the compounds with the shortest fatty-acid group and highest number of double bonds were the more active. These compounds also show a better retention profile, their effect is retained longer than for ara-C.

Antitumor activity of imidazothioxanthones in murine and human tumor models in vitro and in vivo.

BACKGROUND: A new series of imidazothioxanthones has recently been synthesized as potential anticancer agents with the aim of overcoming drug resistance. The route of synthesis and DNA-binding properties of the compounds were reported previously. This paper describes the general structure-activity relationships for the class of imidazothioxanthones in panels of human and murine tumor cell lines in vitro, and the in vivo activity against human and murine solid tumors of the most potent compound, N-[3-(Dimethylamino)propylo]-11-oxo-11H-benzothiopyrano [3',2':2,3]pyrido[1,2-a]imidazo-2-carboxamide (10a). In addition, the interaction between compound 10a and DNA is also considered in terms of molecular mechanics methods and flexible docking techniques. MATERIALS AND METHODS: The cytotoxicity of compounds 10a, 11-oxo-N-[2-(pyrrolidino)ethylo]-11H-benzothiopyrano [3',2':2,3]pyrido[1,2-a]imidazo-2-carboxamide, 11-oxo-N-[2-(piperidino)ethylo]-11H-benzothiopyrano [3',2':2,3]pyrido[1,2-a]imidazo-2-carboxamide and N-[2-(morpholino)ethylo]-11-oxo-11H-benzothiopyrano [3',2':2,3]pyrido[1,2-a]imidazo-2-carboxamide (10c-10e) was assessed in human tumor cell lines and xenografts using the sulforhodamine B assay, MTT assay and the clonogenic assay. The human ovarian xenograft, PXN/109TC, two human breast carcinomas, MT-1 and MCF-7, and the murine colon adenocarcinoma, MAC15A were used for the in vivo testing of compound 10a. In addition, the interaction between compound 10a and DNA is also considered in terms of molecular mechanics methods and flexible docking techniques. RESULTS: Two compounds, 10a and 10c, showed cytotoxic activity below 10 mM in the NCI in vitro screen of 60 human tumor cell lines. The IC50 value of compound 10a was 6.8 mM and that of 10c, 8.3 mM. In addition, both compounds possessed differential activity against leukemia, colon and mammary cancer. The activity pattern was confirmed in two further screens using monolayer and clonogenic, assays. In vivo antitumor studies showed that 10a was active against the human mammary carcinoma MT-1 and murine colon cancer MAC15A. Marginal activity was observed in human ovarian cancer model PXN/109T/C and the compound was inactive in human mammary cancer MCF-7. CONCLUSION: The results warrant further in vivo testing of 10a in additional human solid tumor models. The molecular modeling showed that the planarity of the chromophore and the side-chain conformation could assist the insertion of compound 10a between the base pairs of the double helix. On the other hand, docking to the nucleotide sequence GGAATTGCCTCA suggested that the molecule could also act as a minor groove binder.

Antiproliferative activity and mechanism of action of fatty acid derivatives of arabinofuranosylcytosine in leukemia and solid tumor cell lines.

1-beta-D-arabinofuranosylcytosine (ara-C) is a deoxycytidine analog with activity in leukemia, which requires phosphorylation by deoxycytidine kinase (dCK) to allow formation of its active phosphate 1-beta-D-arabinofuranosylcytosine triphosphate, but can be deaminated by deoxycytidine deaminase. Altered membrane transport is also a mechanism of drug resistance. In order to facilitate ara-C uptake and prolong retention in the cell, lipophilic prodrugs were synthesized. Fatty acid groups with a varying acyl chain length and number of double bonds were esterified at the 5' position on the sugar moiety of ara-C. The compounds were tested in two pairs of ara-C resistant leukemic cell lines (murine L1210 and rat BCLO and their resistant variants L4A6 and Bara-C, respectively) and two pairs of cell lines with a resistance to gemcitabine, another deoxycytidine analog (human ovarian cancer A2780 and murine colon cancer C26-A and their resistant variants AG6000 and C26-G, respectively). L4A6, Bara-C and AG6000 have varying degrees of decreased dCK activity, while the mechanism for C26-G is not yet clear. In the parent cell lines, ara-C was more active, but in the resistant variants several of the analogs were more active, while the degree of cross-resistance varied. In AG6000 with a total dCK deficiency, all compounds were inactive. Structure-activity relation analysis showed that ara-C derivatives with shorter acyl chains and more double bonds were more active in the parental and drug resistant cells. Further mechanistic studies were performed with the elaidic acid derivative of ara-C (CP-4055). CP-4055 inhibited deamination of dCyd partly and induced DNA synthesis inhibition effectively in C26-A and C26-G cells, but the retention of inhibition was much longer for CP-4055 than for ara-C. In contrast to ara-C, CP-4055 inhibited RNA synthesis for 60% after drug exposure. In conclusion, CP-4055 seems to be a promising prodrug, whose effects were different and longer lasting than for the parent drug.

No evidence of tumor growth stimulation in human tumors in vitro following treatment with recombinant human growth hormone.

In a recent study we demonstrated that recombinant human growth hormone (r-hGH; Saizen) delayed tumor-induced cachexia in human tumor xenografts in vivo. Such a therapeutic effect could have a great impact in the supportive care of advanced cancer patients. Before large clinical studies are initiated possible growth stimulation should be excluded. This question was investigated in vitro in 20 human tumor models, which had been established in serial passage in nude mice. The effect of continuous exposure of r-hGH was investigated at dose levels ranging from 0.3 ng/ml up to 0.1 microg/ml in colorectal (n=2), gastric (n=1), non-small cell lung (n=4), small cell lung (n=1), mammary (n=3), ovarian (n=2), prostate (n=2) and renal cancers (n=2), and melanoma (n=3) using a modified Hamburger and Salmon clonogenic assay. The results show that there was neither tumor growth inhibition nor any evidence for tumor growth stimulation in any of the tumors studies. Therefore this preclinical study in 20 human tumor models indicated no direct risk for tumor growth enhancement.

High antitumour activity of ET743 against human tumour xenografts from melanoma, non-small-cell lung and ovarian cancer.

BACKGROUND: Ecteinascidin-743 (ET743) is a novel antitumour agent originating from the Caribbean tunicate Ecteinascidia turbinata. It has potent cytotoxic and antitumour activity and a potential new mechanism of action. The aim of the present study was to further explore the antitumour activity of ET743 in human tumour xenografts from melanoma, non-small-cell lung and ovarian cancer. DESIGN: As the antitumour profile of ET743 was largely unknown a chemo-sensitive and a marginal chemo-resistant human tumour xenograft were selected for each tumour type. ET743 was administered intravenously using two administration schedules (days 0, 4, 8 and 0-2, 13-15). RESULTS: ET743 was very active at the maximum tolerated dose (MTD) in the chemo-sensitive xenograft melanoma MEXF 989, non-small-cell lung cancer LXFL 529, and ovarian cancers HOC22 and (marginally resistant to cisplatin) HOC18. Activity was also seen at 1/2 MTD. Apart from HOC18, ET743 caused complete remissions in the responding xenografts. The compound was inactive in the chemo-resistant xenograft melanoma MEXF 514 and non-small-cell lung cancer LXFA 629. In terms of antitumour activity the days 0, 4, 8 schedule had advantages over the days 0-2, 13-15 schedule. CONCLUSIONS: ET743 is a very effective agent in chemo-sensitive and marginal chemo-resistant xenografts, but inactive in chemo-resistant tumour xenografts. The activity of ET743 in the marginally cisplatin-resistant ovarian cancer HOC18 might indicate absence or incomplete cross-resistance against cisplatin. It is recommended to include melanoma, non-small-cell lung cancer, and ovarian cancer in phase II clinical trials and to use an intermittent schedule.

Superior therapeutic efficacy of N-L-leucyl-doxorubicin versus doxorubicin in human melanoma xenografts correlates with higher tumour concentrations of free drug.

N-L-leucyl-doxorubicin (Leu-DOX), a prodrug of doxorubicin (DOX), has previously shown antitumour activity against human ovarian, breast and lung carcinomas in nude mice. In the present study, the efficacy of Leu-DOX was compared with free DOX in inhibiting the growth of four DOX-sensitive and -resistant malignant melanoma xenografts. In an attempt to elucidate mechanisms underlying any differential effect, a sensitive high-performance liquid chromatography (HPLC) method was established for measuring plasma and tumour concentrations of the two drugs and their main metabolites. Leu-DOX was more effective than free DOX in inhibiting xenograft growth. At equitoxic intravenous doses of Leu-DOX (28 mg/kg) and DOX (8 mg/kg) administered to tumour-bearing nude mice, comparable levels of DOX were found in plasma, whereas differences were seen in tumour tissue concentrations. Thus, in animals carrying highly sensitive (LOX) and resistant (THX) melanomas, higher tumour concentrations of free DOX were observed in the Leu-DOX treated group from 24 up to 240 h after drug injection. Notably, the difference in drug-induced tumour growth inhibition correlated with differences in tumour exposure to free DOX, assessed as area under the curve (AUC) calculated over the first 48 h. In conclusion, the results confirm the prodrug nature of Leu-DOX and provide a possible explanation for its increased antitumour efficacy.

Cell specific cytotoxicity and structure-activity relationship of lipophilic 1-B-D-arabinofuranosylcytosine (ara-C) derivatives.

Lipophilic derivatives of ara-C were developed with the aim to improve drug penetration and retention in solid tumors. Ara-C was esterified at the 5'-position with fatty acids (16-22 C-atoms, 0-3 double bonds). The derivatives were inactive in cell lines with various forms of ara-C and 2',2'-difluorodeoxycytidine (dFdC, gemcitabine) resistance, including deoxycytidine kinase (dCK) deficiency. The activity in the parent cell lines correlated negatively with chain length and positively with double bonds.

The antitumour activity of the prodrug N-L-leucyl-doxorubicin and its parent compound doxorubicin in human tumour xenografts.

The antitumour activity of the investigational agent N-L-leucyl-doxorubicin (Leu-DOX) was compared with that of doxorubicin (DOX) in human tumour xenografts growing subcutaneously in athymic nude mice. Leu-DOX was developed as a prodrug of DOX, and may be converted into the clinically active parent compound by hydrolytic enzymes present in or on tumour cells. It has been suggested that a better therapeutic index with a reduced cardiac toxicity and higher efficacy might be obtained. Both compounds were administered intravenously weekly for 2 weeks, each at maximum tolerated doses of 8 mg/kg and 28 mg/kg for DOX and Leu-DOX, respectively. The panel of xenografts represented three different tumour types. Leu-DOX showed antitumour activity, defined as tumour growth inhibition > 50% and specific growth delay > 1.0, in 10 of the 16 tumours, including two of five breast, five of seven small cell and three of four non-small cell lung carcinomas. In comparison, DOX was active in one breast, four small cell lung and two lung adenocarcinoma xenografts. In all the DOX sensitive lung tumours, Leu-DOX showed higher efficacy than the parent compound. Based on the results of the present study, and since phase I clinical trials with Leu-DOX have already been performed, phase II clinical evaluation of Leu-DOX in patients with breast and lung cancer is recommended.

KW-2149 (7-N-[2-[gamma-L-glutamylamino]ethyldithioethyl]mitomycin C). A new mitomycin C analogue activated by serum.

KW-2149 (7-N-[2-[gamma-L-glutamylamino]ethyldithioethyl]mitomycin C) is a new mitomycin-C analogue in clinical trial. This study demonstrates that KW-2149, unlike mitomycin C, is activated to a cytotoxic species by extracellular metabolism in serum. The metabolising activity differs between batches of serum and species of origin. Human serum had high activity (which resulted in a 150-fold enhancement of cytotoxicity), whereas mouse serum had low activity. In the presence of serum, the rate of uptake of 3H-KW-2149 into cells increased by 8-fold and drug binding to DNA by 32-fold. The metabolising activity of serum can partially be replaced by glutathione. No anticancer drug has previously been described whose toxicity is mediated by metabolism in serum.

Alkyl and alkoxyethyl antineoplastic phospholipids.

Two series of phosphodiester ether lipid analogs with (N-methylmorpholino)ethyl or (N-methylpiperidino)ethyl polar head groups and long aliphatic or alkoxyethyl chains in the nonpolar portion of the molecule were synthesized as potential antineoplastic agents. The cytotoxic activity of these compounds (9-19) was evaluated in vitro against a panel of six human tumor xenografts and in two biochemical, mechanism-based screens (cdc2 kinase and cdc25 phosphatase). Analogs 13, 14, 17, and 19 showed activity in the in vitro tests. Specifically, 14 and 17 were more active than the reference compound hexadecylphosphocholine (Miltefosine, He-PC) while 13 and 19 possessed activity similar to that of the control. Of the analogs tested the one with the highest potency and least toxicity (17) has an N-methylpiperidino head group and a C16 alkyl chain. In the mechanism-based tests 11 showed weak inhibitory activity in the cdc25 phosphatase screen.

Development, pharmacology, role of DT-diaphorase and prospects of the indoloquinone EO9.

1. The indoloquinone EO9 (3-hydroxymethyl-5-aziridinyl-1-methyl-2- (1H-indole-4,7-dione)-propenol; E85/053; NSC 382,459) is a synthetic bioreductive alkylating agent that is structurally related to mitomycin C (MMC). 2. EO9 does, however, show a different mechanism of action and a broader antitumour profile than MMC. It is also a more potent cytotoxic agent in vitro than MMC, probably because of its impressive efficient activation by reductive enzymes, particularly DT-Diaphorase. This enzyme is elevated in several tumours compared to normal tissues. 3. The preferential cytotoxicity of EO9 under hypoxic conditions makes it an interesting compound to combine with radiation. 4. In preclinical and the Phase I clinical studies, no myelosuppression was observed but reversible proteinuria was dose-limiting. Phase II clinical studies were started in the summer of 1994.

Cycloplatam: a novel platinum compound exhibiting a different spectrum of anti-tumour activity to cisplatin.

Cycloplatam is a novel platinum compound which has shown anti-tumour activity in murine tumour models. In this study, cycloplatam was found to have anti-tumour activity in vitro and in vivo in human tumour models. In 15 cell lines (mainly ovarian), cycloplatam showed similar cytotoxicity as cisplatin, using the sulphorhodamine B assay. Determination of the resistance factor (IC50 of cisplatin-resistant divided by IC50 of parental cell line) clearly showed lower values for cycloplatam than for cisplatin. In the parental ovarian cell line CH1 and the cisplatin-resistant CH1 cisR model, we observed no cross-resistance of cycloplatam and cisplatin. The in vitro anti-tumour activity was confirmed in human tumour xenografts using the clonogenic assay. Mean IC70 values of cycloplatam were 0.54 microgram/ml (1.25 microM) and of cisplatin 0.42 microgram/ml (1.4 microM), respectively. In the murine subcutaneously implanted ADJ/PC6 plasmacytoma in vivo cycloplatam showed less activity than cisplatin, with a 2-fold smaller therapeutic index than cisplatin. In ovarian cancer xenografts cycloplatam was less active than cisplatin. However, anti-tumour activity of cycloplatam in lung cancer xenografts was quite different from cisplatin. In LXFS 538, a model moderately sensitive to cisplatin, a partial remission was observed, but in LXFL 529, a cisplatin-sensitive model, cycloplatam was inactive, cycloplatam thus demonstrating a different spectrum of anti-tumour activity. Based on these results, further preclinical investigations with other tumours, such as cisplatin-sensitive and -resistant gastric cancer models, are warranted with cycloplatam.

Preclinical phase II studies in human tumor xenografts: a European multicenter follow-up study.

BACKGROUND: The EORTC New Drug Development Office has initiated a multicenter collaborative program to evaluate the use of human tumor xenografts to predict phase II clinical activity. A first study confirmed the efficacy of doxorubicin and inactivity of amsacrine against human tumor xenografts (Boven et al., Cancer Res: 52, 5940, 1992). In the follow-up study reported here, the activities of cisplatin, AZQ (diaziquone), pazelliptine and retelliptine have been evaluated against a panel of 40 established tumor lines grown subcutaneously in nude mice. DESIGN: The xenografts used represent carcinomas of the breast, colon, head+neck, ovary, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC) and melanoma. Drugs were administered intravenously on days 0 and 7. Doses were for cisplatin 5 mg/kg, AZQ 3-7 mg/kg, pazelliptine 20-80 mg/kg and retelliptine 6-12.5 mg/kg and were selected to give a median loss of about 10%-15% body weight. RESULTS: When activity was defined as a specific growth delay > 1 and a tumor growth inhibition > 50%, then cisplatin demonstrated activity in 15 of 40 xenografts tested (3 of 5 breast, 1 of 6 colon, 0 of 5 head+neck, 2 of 6 NSCLC, 4 of 7 SCLC, 1 of 5 melanoma and 4 of 6 ovarian cancers); AZQ was active in 23 of 38 xenografts (2 of 3 breast, 2 of 7 colon, 4 of 5 head+neck, 3 of 6 NSCLC, 6 of 6 SCLC, 2 of 5 melanoma, 4 of 6 ovarian cancers); pazelliptine was active in 2 of 38 xenografts (1 of 5 breast cancers, 1 of 5 melanoma) while retelliptine was active in 1 of 39 xenografts (a breast cancer xenograft) tested. CONCLUSIONS: These results are reasonably consistent with the clinical activity of cisplatin, but overpredict the clinical efficacy of AZQ. Since pazelliptine and retelliptine are investigational compounds, the clinical phase II studies will provide a prospective test for this model. The results of the present study and the previous one indicate that the human tumor xenograft model could be suitable for predicting the activity of novel compounds to be developed for treatment of cancer patients.

Flunarizine as a modulator of doxorubicin resistance in human colon-adenocarcinoma cells.

The potential of the calcium-entry blocker flunarizine in modulating the cytotoxicity of doxorubicin was investigated in human colon-adenocarcinoma cell lines sensitive to (LoVo) or with experimentally induced resistance (LoVo/DX) to doxorubicin. Exposure to 1 to 2 micrograms/ml flunarizine for intervals of up to 24 hr did not affect cell survival in either line. Simultaneous exposure to flunarizine and doxorubicin for 1 hr selectively enhanced doxorubicin activity in the resistant cell line and not in the sensitive cell line. In particular, the doxorubicin concentration able to reduce cell survival by 50% dropped to one third. Moreover, simultaneous exposure to flunarizine significantly increased intracellular doxorubicin accumulation, as evaluated by fluorescence spectrophotometry. Again, flow-cytometric analysis showed hyperpolarization of the membrane in resistant cells, starting from 15 min of exposure to 2 micrograms/ml flunarizine. Finally, in LoVo/DX cells, which normally express gp170, a 24-hr treatment with flunarizine markedly reduced the immunoreactivity of cells with 2 monoclonal antibodies (MAb57 and MRK16) directed against different external epitopes of the glycoprotein. The results from our study indicate the ability of flunarizine to positively modulate doxorubicin-resistance in human colon-adenocarcinoma cells expressing the multidrug-resistance phenotype.

EO9: a novel bioreductive alkylating indoloquinone with preferential solid tumour activity and lack of bone marrow toxicity in preclinical models.

EO9 is a novel and fully synthetic bioreductive alkylating indoloquinone. Although structurally-related to mitomycin C, EO9 exhibits a distinct preclinical antitumour profile and there are also differences in its biochemical activation. In this study, EO9 was found to demonstrate preferential cytotoxicity against solid tumours in vitro as compared to leukaemia cell lines both in the Corbett two-tumour assay and in the disease-oriented human tumour cell line panel of the U.S. National Cancer Institute. In the latter system activity was particularly apparent in colon, melanoma and central nervous system lines, together with some renal and non-small cell lung lines. Preferential cytotoxicity towards hypoxic versus aerobic EMT6 mouse mammary tumour cells was observed. In vivo, EO9 was inactive against the P388 murine leukaemia, while exerting significant antiproliferative effects against several murine and human solid tumours, including the generally resistant MAC mouse colon tumours and gastric, ovarian and breast xenografts. These results confirmed in vitro observations of preferential solid tumour activity. In animal toxicology studies, EO9 induced vascular congestion in the gastrointestinal tract, but no significant bone marrow toxicity. The LD10 value of EO9 after a single intravenous injection into mice was 9 mg/kg (27 mg/m2). A dose of one-tenth of the mouse equivalent LD10 (2.7 mg/m2), the recommended starting dose for clinical phase I studies, was found to be safe in rats. Considering its distinct mechanism of bioactivation as compared to mitomycin C, its preferential solid tumour activity, its excellent activity against hypoxic cells, and lack of significant bone marrow toxicity in animals studies, EO9 has been selected for clinical evaluation within the framework of the EORTC.

The anti-tumour effects of the prodrugs N-l-leucyl-doxorubicin and vinblastine-isoleucinate in human ovarian cancer xenografts.

N-l-leucyl-doxorubicin and vinblastine-isoleucinate can be considered as relatively non-toxic prodrugs from doxorubicin and vinblastine, respectively. A comparative analysis was carried out of the anti-tumour activity of the four compounds as well as vintriptol in four human ovarian cancer xenografts different in histology, growth rate and chemosensitivity. Injections were given i.v. weekly twice into mice bearing well-established s.c. tumours. At equitoxic doses, the amount of drug administered for N-l-leucyl-doxorubicin and vinblastine-isoleucinate was respectively 3-fold and 2-fold higher than the doses of the parent compound. N-l-leucyl-doxorubicin induced a growth inhibition > 50% in three out of four human ovarian cancer lines. The anti-tumour effects obtained were significantly better (P < 0.01) than in the case of doxorubicin. Vinblastine-isoleucinate studied in two of these lines could induce a growth inhibition of > 50%. This prodrug appeared slightly less effective than vinblastine. Insignificant growth inhibition (< 50%) was obtained by vintriptol.