Analogues of etoposide for the treatment of tumours

Abstract

Compounds for treatment of a patient having a tumour that is metastatic and/or that reduces an organ function, wherein the compounds are of the general formula: ##STR00001##
wherein X is O, NH and S, wherein n is 0, 1 or 2, wherein R.sup.1 and R.sup.2 are H, methyl or ethyl, or together form a group CR.sup.3R.sup.4, and wherein R.sup.3 and R.sup.4 are H, methyl or ethyl.

Claims

1. A method of treating tumor in a subject in need thereof, wherein said method comprises administering to the subject a compound of formula (I) ##STR00004## wherein X is selected from the group consisting of O, NH and S, n is 0, 1 or 2, and R.sup.1 and R.sup.2 are independently selected from the group consisting of H, methyl, and ethyl, or together form a group CR.sup.3R.sup.4 wherein R.sup.3 and R.sup.4 are independently selected from the group consisting of H, methyl and ethyl, wherein the tumor is metastatic and/or that reduces an organ function; and, wherein the tumor is resistant to etoposide.

2. The method of claim 1, wherein said method comprises administering to the subject a compound selected from the group consisting of ##STR00005##

3. The method of claim 1, wherein the subject is a human.

4. The method of claim 1, wherein the tumor is resistant to treatment with a therapeutic protein or a chemotherapeutic drug exclusive of the compound of formula (I) ##STR00006## wherein X is selected from the group consisting of O, NH and S, n is 0, 1 or 2, and R.sup.1 and R.sup.2 are independently selected from the group consisting of H, methyl, and ethyl, or together form a group CR.sup.3R.sup.4 wherein R.sup.3 and R.sup.4 are independently selected from the group consisting of H, methyl and ethyl.

5. The method of claim 1, wherein the tumor is a solid tumor.

6. The method of claim 5, wherein the tumor is selected from the group consisting of adenocarcinoma, hypopharynx cancer, lung cancer, diffuse large cell lymphoma, Burkitt's lymphoma, Hodgkin lymphoma, Non-Hodgkin lymphoma, histiocytic lymphoma, lymphatic lymphoma, acute T-cell leukemia, pre-B-acute lymphoblastic leukemia, gall bladder cancer, bile duct carcinoma, thymus carcinoma, urothelium carcinoma, testicular cancer, prostate cancer, bladder cancer, brain tumor, AIDS-related Kaposi's sarcoma, Ewing sarcoma, rhabdomyosarcoma, neuroblastoma, ovarian cancer, breast cancer, and unknown primary origin (CUP) syndrome.

7. The method of claim 1, wherein the treatment leads to a stagnation of tumor growth or a remission of the tumor.

8. The method of claim 1, wherein said tumor reduces an organ function.

9. The method of claim 1, wherein the tumor expresses at least one of CES1 and CES2.

10. The method of claim 9, wherein the fraction of CES1 and/or CES2 expressing cells of the tumor is 0.50% or higher.

11. The method of claim 1, further comprising administering to the subject a second therapeutic drug compound.

12. The method of claim 1, wherein the compound of formula (I) is administered to the subject intravenously.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the time-dependent conversion of CAP7.1 to etoposide by different carboxylesterases and by foetal calf serum.

(2) FIG. 2 shows the inhibition of topoisomerase II proteins in U937 cells by CAP7.1 and by etoposide at the transcriptional level.

(3) FIG. 3 shows the inhibition of MDR-1 activity in Kelly cells by CAP7.1 in a functional MDR-1 assay.

(4) FIG. 4 shows the effect of CAP7.1 and etoposide on the cell cycle in U937 cells.

(5) FIG. 5 shows the cytotoxicity of CAP7.1 and of etoposide on the cell line HT-29.

(6) FIG. 6 shows the expression of CES1 and CES2 in tumour cell lines responsive to CAP7.1.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(7) In various investigations (cellular assays, animal models, human clinical studies), the compounds of formula (I) and in particular CAP7.1 show a unique therapeutic profile within the class of topoisomerase inhibitors: a better efficacy (even at low doses) in comparison to some other chemotherapeutic drugs (especially etoposide), an efficacy also in tumours resistant to other therapies and in particular etoposide-resistant tumours (examples are hypopharynx cancer, gall bladder cancer, ovarian cancer, testis cancer (testicular cancer), thymus carcinoma, cancer of unknown primary origin (CUP) and neuroblastoma), an efficacy in advanced cancer patients at a low dose, a better tolerability/fewer side effects (even at a higher dose) in comparison to some other chemotherapeutic drugs and a prevention of an immediate increase of etoposide concentration through slow conversion of the compounds of formula (I).

EXAMPLES

Example 1: Conversion of CAP7.1 to Etoposide by Carboxylesterases

(8) The time-dependent conversion of CAP7.1 to etoposide by different carboxylesterases and foetal calf serum was analysed by incubating CAP7.1 with CES1, CES2 and CES3, respectively. 5 M CAP7.1 was pre-incubated for 3 min in 999 l 0.1 mol/l sodium phosphate (NaH.sub.2PO.sub.4, Roth, Karlsruhe, Germany) pH 7.4 at 37 C. After that, CES1, CES2 and CES3, respectively, were added. Controls were incubated with 10% foetal calf serum (FCS, positive control) or in buffer (negative control). At different time points, aliquots were subjected to HPLC in order to analyse the formed products.

(9) In FIG. 1, the levels of the etoposide formed are depicted. CES2 (square symbols) cleaves CAP7.1 as efficiently as the positive control FCS (X symbols). CES1 (diamond symbols) also efficiently cleaves CAP7.1, whereas in the case of CES3 (triangle symbols), substantially no cleavage of CAP7.1 is observed. The negative control (cross symbols) shows that CAP7.1 is stable under the chosen conditions.

Example 2: Inhibition of Topoisomerases II at the Transcriptional Level by CAP7.1 and by Etoposide

(10) U937 cells grown in RPMI 1640/DMEM medium supplemented with 10% foetal bovine serum, 2 mmol/l L-glutamine and 100 U/ml/100 g/ml Penicillin/Streptomycin were washed and resuspended at a concentration of 10.sup.6 cells/well into 6-well plates in solutions containing 10.sup.4 mol/l CAP7.1 and 10.sup.4 mol/l etoposide, respectively. One of the controls involved the treatment of the cells with 0.05% (1 l) DMSO. Untreated cells are also shown as a control.

(11) After an incubation time of 24 h, expression was analysed by determining mRNA levels by real-time PCR. In the respective lanes of FIG. 2, the bands for topoisomerase II a (322 bp), topoisomerase II (304 bp) and the GAPDH control (358 bp) are shown. In the H.sub.2O control lane, showing the results of the H.sub.2O control sample, no bands were observed.

(12) Untreated cells expressed both topoisomerase II a and at a strong level. Both CAP7.1 and etoposide lead to a significant decrease of expression of both topoisomerase II a and .

Example 3: Inhibition of MDR-1 Activity by CAP7.1

(13) By means of an assay using JC-1, MDR-1 activity was assessed in Kelly cells, which express MDR-1. JC-1 is a cationic staining dye which is taken up by living cells. JC-1 is recognized by the MDR-1 gene product (P-glycoprotein) and pumped to the outside of the cell (substrate efflux). Thus, MDR-1 activity is indicated by a reduction in intracellular JC-1 signal.

(14) Kelly cells grown in RPMI medium were washed and resuspended at a concentration of 10 cells/ml in solutions containing JC-1 in a final concentration of 50 nmol/l. Cells were incubated for 15 min in the presence of 10 mol/l CAP7.1 and 10.sup.4 mol/l etoposide, respectively. The control contained 1 l (0.05%) DMSO. Cells were analysed for JC-1 staining in a FACScanto II flow cytometer.

(15) In FIG. 3, the top diagram shows an intracellular JC-1 signal in the presence of the negative control (DMSO). The intracellular JC-1 signal in the presence of etoposide is identical (middle diagram). In the presence of CAP7.1, the intracellular JC-1 signal is increased (bottom diagram). This intracellular accumulation of JC-1 shows the inhibition of the MDR-1-mediated (P-glycoprotein-mediated) substrate efflux by CAP7.1, but not by etoposide.

Example 4: Effect of CAP7.1 and Etoposide on the Cell Cycle

(16) Multidrug-resistant U937 cells grown in RPMI medium were washed and resuspended at a concentration of 10.sup.6 cells/ml in PBS containing 10.sup.4 mol/l CAP7.1, 10.sup.4 mol/l etoposide and 1 l (0.05%) DMSO, respectively. A control contained no CAP7.1, no etoposide and no DMSO. The cells were incubated for 48 h. After 18 h, 24 h and 48 h of incubation, respectively, cell cycle analysis was performed as follows:

(17) Cells were washed once with CellWash, fixed with fridge-cold 70% Ethanol for 30 min at 4 C. and washed three times. RNA was digested with RNAse A (100 pg/ml in PBS) for 10 min at room temperature. Cells were washed once with CellWash and stained with propidium iodide (50 pg/ml in PBS) for 5 min at room temperature in the dark. Cells were analyzed immediately by flow cytometry using a FACScanto II apparatus.

(18) The percentage of cells in G2 phase is shown in FIG. 4. With increasing incubation times, etoposide (X symbols) leads to a reduction in the fraction of cells in G2 phase, whereas CAP7.1 (triangle symbols) leads to an increase in the fraction of cells in G2 phase. This is indicative of a delayed induction of apoptosis as compared to etoposide.

Example 5: Cytotoxicity of CAP7.1 and of Etoposide on the Cell Line HT-29

(19) HT-29 cells, which are not multidrug-resistant, were grown in DMEM, 10% foetal calf serum, 2 mM glutamine and 100 U/I Pen/Strep medium and then washed. Cells were resuspended at a concentration of 10.sup.6 cells/ml in medium as described above containing various concentrations of CAP7.1 and etoposide, respectively, ranging from 10.sup.10 mol/l to 10.sup.4 mol/l.

(20) Cells were harvested, counted, and inoculated at the appropriate concentration of 10.sup.4 cells/well (100 l volume) into 96-well microtiter plates. After 24 h, the drugs were applied to triplicate culture wells, and cultures were incubated for given times at 37 C. XTT was prepared at 1 mg/ml in pre-warmed (37 C.) medium (RPMI 1640) without serum. PMS was prepared at 5 mmol/l (1.53 mg/ml) in PBS. Fresh XTT and PMS were mixed together at appropriate concentrations. For a 25 ol/l XTT-PMS solution, 25 l of the 5 mmol/l PMS solution were added per 5 ml of XTT (1 mg/ml). 50 l of this mixture were added to each well on given time points after cell inoculation. After incubation at 37 C., the plates were mixed on a mechanical plate shaker, and absorbance was measured with an ELISA Reader to determine cell viability.

(21) FIG. 5 shows cell viability plotted against the concentrations of CAP7.1 (square symbols) and etoposide (diamond symbols), respectively. The IC.sub.50 value of CAP7.1 is at a 10-fold lower concentration than the IC.sub.50 value of etoposide.

Example 6: Expression of CES1 and CES2 in Tumour Cell Lines

(22) Different cell lines responsive to compounds of formula (I) were tested for endogenous Expression of CES1 and CES2. Expression was quantified by real-time PCR.

(23) Cells were harvested, counted, and inoculated at the appropriate concentration of 10.sup.6 cells/well (2 ml volume) into 6-well plates with drugs for given time points at 37 C. RNA was extracted and cDNA was synthesized with random hexamers and TaqMan reverse transcriptase to be used in real-time PCR in a reaction with PCR buffer mix and specific primers for human CES1 and CES2.

(24) The results for CAP7.1 obtained in the cell lines Raji, U-937 and HT-29 are illustrated in FIG. 6, which shows inverted ACT values.

(25) Raji cells express only CES2. U-937 and HT-29 cells express both CES1 and CES2. Accordingly, these cells respond to treatment with compounds of formula (I), in particular CAP7.1.

Example 7: Effect of CAP7.1 in Animal Studies

(26) The objective of this study was to evaluate the potential modifications of cardiovascular function and cardiac electrophysiology by CAP7.1. The intravenous route was selected, as it is the intended mode of administration in human therapeutic use.

(27) A total of four cynomolgus monkeys (two males and two females) were implanted with telemetric devices and allocated to a single group. They received a single dose of the vehicle (Cremophor/ethanol/NaCl) or CAP7.1, by intravenous 30-minute infusion. Cardiovascular parameters were monitored up to 24 hours following single infusion. The cardiovascular parameters evaluated in this study included heart rate, diastolic, systolic and mean arterial pressure, and duration of the PQ, QRS and QT intervals. CAP7.1 induced no relevant modifications in the cardiac electrophysiological parameters of conscious monkeys. No arrhythmias were observed.

(28) The lack of cross-resistance to CAP7.1 of MDR-mediated etoposide-resistant cell lines in vitro was confirmed in vivo in mice bearing NSX2 tumour xenografts. CAP7.1 administration resulted in a clear inhibition of tumour growth compared to control.

(29) The toxicity profile of CAP7.1 in animals corresponds to that of other topoisomerase II inhibitors and in particular etoposide, i.e. the main toxicity at sublethal dose is bone marrow suppression affecting predominantly neutrophils and red cells. However, animals treated with etoposide experience significant weight loss whereas animals receiving CAP7.1 do not.

(30) Animals receiving CAP7.1 experienced reversible tooth damage in the high dose group.

(31) Animal toxicology studies have shown that the LD.sub.10 value of CAP7.1 in mice is 120 mg/kg (360 mg/m.sup.2) whereas the LD.sub.10 value reported in the literature for etoposide given to mice is 34 mg/kg (102 mg/m.sup.2) after i.v. administration.

Example 8: Clinical Studies

(32) Nine heavily pre-treated patients were recruited to a combined Phase I/I Ia safety/efficacy study. The patients had tumours that were metastatic and/or that reduced the function of at least one organ.

(33) In all patients, the tumours were clinically assessed and the history of their disease was recorded. Tumour markers were determined.

(34) Each of the patients had been treated before with different drugs, which on admission to the study had failed. Thus, the respective tumours were resistant to treatment with the respective drugs (listed under previous treatment for the respective patients).

(35) Patient 1: 57 years old, male, squamous cell hypopharynx carcinoma with pulmonary metastases. Previous treatment: cisplatin, pololobe kinase, cisplatin, 5-fluorouracil, panitumumab.

(36) Patient 2 (non-responder after two cycles of treatment): 49 years old, squamous cell oropharynx carcinoma. Previous treatment: cetuximab, cisplatin.

(37) Patient 3: 62 years old, male, sarcomatoid thymus carcinoma with pulmonary metastases, Previous treatment: tyrosine kinase inhibitor, trofosfamide, etoposide, doxorubicin (adriamycin).

(38) Patient 4: 65 years old, female, adenocarcinoma of the gall bladder with lymph node metastases. Previous treatment: cisplatin, gemcitabine.

(39) Patient 5 (non-responder after two cycles of treatment): 65 years old, urothelium carcinoma of urine bladder with liver metastasis. Previous treatment: cisplatin, gemcitabine.

(40) Patient 6: 50 years old, male, cancer of unknown primary origin with small-cell brain metastases. Previous treatment: cisplatin, etoposide.

(41) Patient 7 (non-responder after two cycles of treatment): 64 years old, squamous tonsillar carcinoma with metastases in lymph nodes, liver and lung.

(42) Patient 8: 32 years old, male, mixed seminoma and non-seminoma germ cell carcinoma of the testis with renal, pulmonary, retroperitoneal, hepatic and cerebral metastases.

(43) Patient 9: 67 years old, female, ovarian adenous carcinoma with abdominal metastases.

(44) A cycle of treatment consisted of CAP7.1 administration by infusion on 5 consecutive days, followed by an intermittent period of 16 days in which no CAP7.1 was administered. After completion of such a cycle, the next cycle of treatment was initiated. The first cohort (patients 1-3, 8 cycles in total) received CAP7.1 at a daily dose of 45 mg/m.sup.2, the second cohort (patients 4-6, 9 cycles in total) at a daily dose of 90 mg/m.sup.2 and the third cohort (patients 7-9) at a daily dose of 150 mg/m.sup.2.

(45) Pharmacokinetic evaluations were performed before administration, at 30 min during infusion, and after infusion at 15 min, 45 min, 90 min, 3 h, 6 h, 10 h on day 1, and before administration and 15 min after end of infusion on the remaining days. Pharmacokinetics showed that CAP7.1 was processed to etoposide in a delayed way. The majority of CAP7.1 was converted to etoposide after 45 min of CAP7.1 infusion.

(46) All patients tolerated the therapy well without major adverse reactions. Patients 1 to 6 did not experience toxicity. Thus, a total dose per cycle of CAP7.1 of 450 mg/m.sup.2 is tolerated without toxicity. This represents a 25% increase over the recommended dose of etoposide in small cell lung cancer.

(47) Patients 1, 3, 4, 6, 8 and 9 showed that treatment with CAP7.1 was successful: a stagnation of the disease after the second cycle of treatment was observed in each case.

Example 9: Clinical Studies for Combination Therapy

(48) CAP7.1 was administered with carboplatin to three children with heavily pre-treated advanced neuroblastoma. Two of these patients had received etoposide previously.

(49) CAP7.1 was administered on day 1 of each cycle starting at 200 mg/m.sup.2 (the usual dose of etoposide in this regimen) and was escalated in successive cycles to 400, 600 and 800 mg/m.sup.2; carboplatin was administered at a dose of 150 mg/m.sup.2 daily on days 2, 3, 4 and 5. Cycles were repeated every 21 days and a total of 4 cycles were administered in each patient. Toxicity was mainly haematological, grade 1-2 at 200 mg/m.sup.2 and increased to grade 3-4 in the rest of the cycles, but cycle delay due to toxicity was not necessary.

(50) In two of the three patients there were evidences of tumour shrinkage. Thus these observations provide indirect support of the lack of cross-resistance in humans between CAP7.1 and etoposide. Additionally the study supported the good tolerability of CAP7.1 in patients. The half life in plasma in this study evaluated in one of the children treated was 12-30 minutes with a C.sub.max of 3.7 pg/ml.

Example 10: Formulation of CAP7.1

(51) CAP7.1 is formulated as a dry white powder. CAP7.1 is presented in 3 mg strengths as a solution for intravenous infusion. The active ingredient is solubilised in 20 ml polyethoxylated castor oil (e.g. Cremophor EL) and ethanol (50:50) in glass vials before dilution into sterile sodium chloride (0.9%) for infusion. 1, 2 and 3 mg/ml final solution strengths are provided.