Patent Application
20080167272
The present invention concerns certain hydrolytic thalidomide metabolites, combinations thereof with other anti-neoplastic compounds and their use in the treatment of solid tumours.
Thalidomide (α-phthalimidoglutarimide) has a number of biological activities that, despite its teratogenicity (Mc Bride, 1961; Lenz, 1962) and removal from widespread clinical use as a sedative in the 1950s, has led to its re-emergence for the treatment of a number of inflammatory conditions, and more recently for the treatment of cancer.
Thalidomide's anti-tumour activities have been mainly attributed to its anti-angiogenic properties (D'Amato et al., 1994; Kenyon et al., 1997; Kruse et al., 1998; Joussen et al., 1999), although there is increasing evidence of the involvement of cytokine modulation (Kenyon et al., 1997; Hideshima et al., 2001a; Hideshima et al., 2001b; Dmoszynska et al., 2002; Tosi et al., 2002; Li et al., 2003). Metabolites have been suggested to be responsible for the anti-angiogenic effects of thalidomide (Gordon et al., 1981; Ng et al., 2003), and hydroxylated metabolites have been found to be effective in inhibiting angiogenesis in a number of different assays (Marks et al., 2002; Price et al., 2002; Ng et al., 2003). On the other hand, modulation of cytokine biosynthesis by thalidomide occurs efficiently without metabolic enzymes (Moreira et al, 1993) indicating that either thalidomide itself or a hydrolysis product is responsible for that activity.
Despite the recent successful applications of thalidomide in anti-tumour therapy, its use is still associated with significant side-effects. There is therefore a need for novel therapeutic agents and compositions with more desirable and advantageous properties.
The object of the present invention is to ameliorate one or more disadvantages of current anti-tumour therapies or to provide a useful alternative.
According to a first aspect there is provided a composition comprising one or more thalidomide metabolites, wherein the one or more metabolites exhibit anti-tumour activity and optionally a pharmaceutically acceptable carrier.
According to a second aspect there is provided a composition comprising one or more thalidomide metabolites, wherein the one or more metabolites exhibit anti-angiogenic or cytokine modulatory activity and optionally a pharmaceutically acceptable carrier.
According to a third aspect there is provided a composition comprising one or more thalidomide metabolites, wherein the one or more metabolites exhibit anti-tumour, anti-angiogenic, or cytokine inhibitory activity, and at least one other anti-neoplastic compound, optionally in conjunction with a pharmaceutically acceptable carrier.
Preferably the thalidomide metabolites are hydrolysis products and more preferably the hydrolysis product is CG (N-(o-carboxybenzoyl)glutamic acid imide). The anti-tumour activity of a thalidomide metabolite is preferably activity against any solid vascularised tumours such as for example breast, lung, brain, colon, ovarian, prostate, testicular, uterine, liver and the like.
Preferably the anti-neoplastic compound is selected from the group consisting of DMXAA, cyclophosphamide or variants and analogues thereof. It will be understood however that other commonly used anti-neoplastic agents, such as the taxanes, platinum compounds, tubulin binders, top( )-isomerase poisons, anti-metabolites and vascular targeting agents can also be advantageously used in this combination.
According to a fourth aspect there is provided a method of treating a solid tumour comprising the administration to a subject requiring such treatment a composition of any one of the previous aspects.
Preferably the solid tumour to be treated can be colon cancers, breast cancers, liver cancers, lung cancers, brain tumours, ovarian tumours, prostate cancers, testicular cancers, uterine tumours and the like.
DMXAA 25 mg/kg
DMXAA 25 mg/kg+PG 100 mg/kg
DMXAA 25 mg/kg+PiG 100 mg/kg
DMXAA 25 mg/kg+CG 100 mg/kg
DMXAA 25 mg/kg+Thal 100 mg/kg
DMXAA 25 mg/kg+CG 20 mg/kg
DMXAA 25 mg/kg+Thal 20 mg/kg
DMXAA 25 mg/kg+CG 4 mg/kg
DMXAA 25 mg/kg+Thal 4 mg/kg
Our earlier studies on multiple myeloma patients treated with thalidomide could not detect the presence of hydroxylated thalidomide metabolites but only three hydrolysis metabolites, PG, PiG and CG. (see below for definition).
For the thalidomide metabolites defined above, which contain a chiral center, the structures and definitions provided are intended to define the R-enantiomer and the S-enantiomer or a mixture of these two enantiomers.
The present invention is based in part on further studies which demonstrate the ability of certain thalidomide hydrolysis metabolites, in particular metabolite CG, to inhibit angiogenesis and to modulate cytokine production. It is further based on observations that certain hydrolysis metabolites can enhance the anti-tumour action of the anti-cancer drugs such as DMXAA and cyclophosphamide in an animal model. DMXAA and its analogues, variants and derivatives are described in U.S. Pat. No. 5,281,620, incorporated in its entirety herein by reference. Cyclophosphamide is a well known anti-neoplastic agent. Thus, synergistic anti-tumour combinations comprising CG and other anti-neoplastic compounds are an integral part of the invention described herein.
In one part the present invention employs an accepted animal model to demonstrate the use and efficacy of the compositions, including synergistic combinations, and methods in the treatment of a typical vascularised solid tumour. In the context of the present invention, the term “vascularised solid tumour” is used to describe any solid tumour, whether benign or cancerous, which relies on ample blood supply for its establishment and growth. Thus, the tissue or organ location, or tissue origin, of such tumours is not material to the compositions and methods of the present invention as long as there is reliance by the tumour on its blood supply. For example the compositions and methods of the present invention may be used to treat colon cancers, breast cancers, liver cancers, lung cancers, brain tumours, ovarian tumours, prostate cancers, testicular cancers, uterine tumours and the like.
In another part the present invention employs well recognized and accepted cell culture systems to demonstrate the anti-angiogenic and cytokine modulatory activities of the thalidomide metabolites and in particular metabolite CG.
The inventive concept will now be described in more detail with reference to non-limiting examples.
Mice (C57B1/6 bred at the Animal Resources Unit, University of Auckland, New Zealand) were implanted with Colon 38 tumours, obtained originally from the Mason Research Institute (Auckland, New Zewaland) by transferring 1 mm3 pieces of the tumour into a subcutaneous opening in the left flank of anaesthetized mice. The mice with tumours were used for growth delay experiments when tumours had reached approximately 3-5 mm in diameter. Mice were divided into 10 groups, an untreated control group, a group treated intraperitoneally (i.p.) with DMXAA (5,6-dimethylxanthenone-4-acetic acid, synthesized at the Auckland Cancer Society Research Centre—ACSRC (Auckland, New Zealand) at 25 mg/kg only, and 8 groups treated with DMXAA together with the hydrolytic thalidomide metabolites PG, PiG, CG also synthesized at the ACSRC as previously described (Lu et al, 2003), or thalidomide at 100 mg/kg, or together with CG or thalidomide at 20 mg/kg or 4 mg/kg. DMXAA was dissolved in saline at 2.5 mg/ml and injected i.p. into mice in a volume of 10 μl/g body weight. Thalidomide, PG, PiG and CG were dissolved in dimethylsulphoxide at 40, 8 or 1.6 mg/ml (for dosages of 100, 20, 4 mg/kg respectively) and injected i.p. in a volume of 2.5 μl/g body weight. Tumours were measured 2-3 times weekly after treatment and tumour volumes were calculated as 0.52×a2×b, where a and b are the minor and major axes of the tumour. The arithmetic means and standard errors were calculated for each point, counting cured animals as having zero tumour volume, and expressed as fractions of the pre-treatment volume. Growth delay was determined as the difference in the number of days required for the tumour to reach four times the pre-treatment volume. Mice in which the tumour had completely disappeared were kept for 3 months to ensure that the tumours did not re-grow.
Mice with Colon 38 tumours, approximately 5-7 mm in diameter, were divided into groups of 5. One group was untreated. Other groups were treated with DMXAA (25 mg/kg) alone; DMXAA (25 mg/kg)+thalidomide (100 mg/kg), DMXAA (25 mg/kg)+PG (100 mg/kg), DMXAA (25 mg/kg)+PiG (100 mg/kg), DMXAA (25 mg/kg)+CG (100 mg/kg), DMXAA (25 mg/kg)+thalidomide (20 mg/kg), DMXAA (25 mg/kg)+CG (20 mg/kg), and thalidomide, PG, PiG or CG only at 100 and 200 mg/kg. Three hours after treatment, mice were anaesthetized with halothane (NZ Pharmacology Ltd., Christchurch, New Zealand), and blood was collected from the ocular sinus and tumours were excised. Blood was coagulated overnight at 4° C., and then centrifuged for 30 min at 2,000×g and 4° C., and the serum was removed and stored at −80° C. until use. Tumour tissues were weighed and homogenized in 1.5 ml α-minimal essential medium (Gibco, Grand Island, N.Y., USA). The homogenates were centrifuged (2,000×g, 30 min, 4° C.) and supernatant was removed and re-centrifuged (14,000×g, 30 min, 4° C.). The final supernatants were removed and kept at −80° C. until use. TNF levels in serum and supernatants derived from tumour homogenates were assayed for TNF as described in section 4 (below).
Blood (20 ml) each from seven healthy volunteers was collected into heparinized tubes, and was overlayed on top of 10 ml of Ficoll-Paque Plus (Amersham Biosciences AB, Uppsala, Sweden) in 50 ml Falcon polypropylene conical tubes (Becton Dickson Labware, Franklin Lakes, N.J.). The tubes were centrifuged for 30 min at 300×g and 4° C., and the layer of cells was removed and re-suspended at 1×106 cells/ml in A-MEM medium supplemented with foetal bovine serum (Moregate BioTech Ltd, Hamilton, New Zealand) (10% v/v), streptomycin sulphate (100 μg/ml) and penicillin-G (100 units/ml) (Sigma, St Louis, Mo., USA). Cells were added to a 24-well plate (Nunc, Kamstrup, Roskilde, Denmark) with LPS (Escherichia coli serotype 055:B5; Sigma, St. Louis, Mo., USA) plus thalidomide or CG at 0.04, 0.2, 1, 5, 25 & 100 μg/ml; or PG or PiG at 1, 5 & 25 μg/ml in a final volume of 1 ml. Wells containing cells only and cells plus LPS only were included as controls. After 12 hours of incubation, supernatants were removed and assayed immediately for TNF as described in section 4.
TNF was assayed using a commercially available ELISA kit (OpEIA human or murine TNF kit, PharMingen, San Diego, Calif., USA), according to the manufacturer's instructions. Briefly, samples (100 μl/well) in duplicate together with a serial dilution of TNF (31.25-2000 pg/ml) for the standard curve, were added to flat-bottom 96-well plates pre-coated with immobilised monoclonal anti-TNF antibody and incubated at room temperature for 2 h. The wells were then washed, biotinylated polyclonal antibody to TNF was added, followed by peroxidase-labelled streptavidin, and incubated at room temperature for 1 h. The wells were washed again, and the substrate, tetramethylbenzidine and hydrogen peroxide was added and after 10 min the reaction was stopped with 2 N sulphuric acid. The absorbance at 450 nm was determined using a microtitre plate reader.
Each well in a 24-well plate was coated with 300 μl of Matrigel (Becton Dickinson, San Jose, Calif., USA) which was allowed to solidified at 37° C. for 1 h. ECV304 cells (4×104) (from American Type Culture Collection, CRL-1998, also known as T24) in a final volume of 1 ml of M-199 medium supplemented with 10% FBS, containing thalidomide, CG, PG and PiG at varying concentrations (2-25 μg/ml), were plated onto the Matrigel. After 15-18 h of incubation at 37° C. at 5% CO2, the culture supernatant was aspirated. The cells were fixed and stained using Diff-Quick staining kit (Dade AG, Dudingen, Switzerland) according to the manufacturer's instructions. After removal of the staining solution the stained cells in each well were photographed along with a measuring ruler placed at the bottom edge of the photographing field (Olympus Camedia C-5050 professional digital camera, Olympus Optical, Tokyo, Japan). The total length of the tubes formed in the photographs was measured and divided by the area of the field (mm/mm2).
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, which measures mitochondrial metabolic activity, was used to determine cell viability (Mosmann, 1983). Cells (4×103/well) were cultured with various concentrations of thalidomide, CG, PG and PiG in a final volume of 100 μl M199 medium (Gibco, Grand Island, N.Y., USA) supplemented with 10% foetal bovine serum (1,100 μg/ml streptomycin sulphate and 100 units/ml penicillin-G (Sigma, St Louis, Calif., USA) in flat-bottomed 96-well plates at 37° C. at 5% CO2 for 18 h. At the end of the incubation period, 10 μl of MTT (Sigma, St. Louis, Mo., 5 mg/ml in PBS) was added to each well, and the cells were further incubated for 2 h. The supernatants were then removed and the formazan crystals in the cells were dissolved by the addition of 100 μl of DMSO per well, and the absorbance at 590 nm was read using a plate reader. Each concentration of drug was tested in triplicate wells, and the mean ±standard error of the mean (SEM) was calculated.
CG (50 μM) with 60 μM of phenacetin (as an internal standard) at pH 3.5, 6.8, 7.1, 7.4, 7.7 and 8 in 8 mm clear polyethylene starburst conical snap vials (Alltech, Deerfield, Ill., USA), was incubated in the autosampler (Waters Associates, Milford, Mass., USA) at 37° C. Aliquots (50 μl) of the solutions were then automatically loaded at 0, 6, 12, 18 and 24 hours after incubation onto a Waters Breeze chromatograph (Waters Associates, Milford, Mass., USA) which consisted of a Model 717plus autosampler, Model 1525 binary pump and Model 2487 dual wavelength absorbance detector.
Compounds were separated using a previously described (Chung et al, 2004) HPLC assay for thalidomide using a 100×4.6 mm stainless steel Luna 5 μm Phenylhexyl column (Phenomenex, Torrance, Calif., USA) and a mobile phase (Milli Q water:acetonitrile 10:1.3 v/v) that was pumped at a flow rate of 2 ml/min. Thalidomide and phenacetin were detected at ultraviolet wavelengths of 220 and 248 nm, respectively. The retention times of phenacetin and thalidomide and were 13 and 11 min, respectively. Data acquisition and integration was achieved using Breeze™ Software, Milford, Calif., USA. Each HPLC run included standard curves of thalidomide (0.6-100 μM) or CG (0.2-50 μM) with phenacetin (60 μM) as the internal standard in PBS. Thalidomide in plasma at 0.6, 5, and 25 μM and CG in plasma at 0.2, 5, and 25 μM were stored at −80° C. and were included where appropriate as quality controls, and were found to be stable over a period of 14 days within 10% and 5% of the validated value respectively. The r2 of calibration curves of thalidomide was 0.9995, and for CG calibration curves was 0.9999. The intra-assay recovery was 90-110% (n=8) and the CV was 5-9% (n=4), while the inter-assay recovery was 96-104% (n=15) and the CV was 2-4% (n=6).
To construct the calibration curve, the peak-area ratios to the internal standard were plotted against thalidomide concentration and the best-fit straight line was obtained by linear regression analysis. The correlation coefficient of the calibration curves had r2 values of greater than 0.9995. The thalidomide/phenacetin peak ratio was determined for all samples and the concentration of thalidomide in each sample was determined from the calibration curve.
The intraassay accuracy and precision were acceptable with relative recoveries and coefficient of variation (CV) of 90-110% and 5-9% (n=8), respectively. Similar results were achieved for interassay accuracy and precision with relative recoveries and CV's of 96-104% and 2-4% (n=15). Quality control plasma and tissue homogenates with added three nominal thalidomide concentrations (0.6, 40, and 80 μM) were stored at −80° C. These were included in each analysis and were found to be stable over a period of 14 days and within 10% of the validated value (n=15).
The ratio of the area of the thalidomide peak or the CG peak to that of the phenacetin peak in the HPLC chromatographs then calculated and the concentration of thalidomide and CG was determined using the calibration curve. The percentage degradation of CG was calculated as: [1-(CG concentration after incubation/starting concentration)]×100.
To investigate whether the hydrolysis metabolites of thalidomide had anti-tumour activity and whether or not they could also potentiate the anti-tumour activity of DMXAA, mice with Colon 38 tumours were treated with CG, PG, PiG and thalidomide at 100 mg/kg together with DMXAA (25 mg/kg), and the growth of the tumours was followed. Co-administration of PiG or PG with DMXAA did not inhibit tumour growth above that of DMXAA alone (
CG was then compared with thalidomide at the lower doses of 20 mg/kg and 4 mg/kg for potentiation of DMXAA activity against Colon 38 tumours. At 20 mg/kg, CG plus DMXAA was more effective than thalidomide plus DMXAA or DMXAA alone (
Tumour-bearing mice were administered thalidomide, PG, PiG, CG or DMXAA alone, or administered DMXAA (25 mg/kg) plus PG (100 mg/kg), PiG (100 mg/kg), thalidomide (100 mg/kg or 20 mg/kg) or CG (100 mg/kg or 20 mg/kg). Serum and tumour samples of mice were assayed for TNF production 3 h after drug administration. TNF levels in serum and tumours from mice treated with PG, PiG, CG or thalidomide alone were similar to that of untreated mice (student's t-test). Mice treated with DMXAA alone (25 mg/kg) had a 20-fold (
Significant TNF production was obtained when HPBL were cultured with 100 pg/ml of LPS (
Tube formation by ECV304 cells on matrigel layers was used as an in vitro model for angiogenesis, and the ability of thalidomide or its hydrolysis metabolites to inhibit tube formation was tested. Near complete inhibition of tube formation was observed with thalidomide and CG at 250 μg/ml (
As a control that inhibition of tube formation was not as result of cytotoxicity of the drugs, ECV304 cells were cultured with thalidomide, PG, PiG and CG at the same the concentrations used for tube inhibition (2-250/g/ml), and cell viability after 18 h was measured using the MTT cytotoxicity assay (see above). No toxicity was observed with any of the compounds compared with untreated or DMSO-treated cultures.
To examine its stability, CG was incubated at different pHs and at 0, 6, 12, 18 and 24 h after incubation, the concentrations of CG and thalidomide in solution was measured using HPLC assay which resolved CG, phenacetin and thalidomide completely with the retention times of 8.5, 22.8 and 23.6 min respectively. At pH 3.5, 7.7 and 8, 21.5%, 8.6% and 0% of CG was remaining in the solution after 24 h (
The present studies have shown that CG was as active as thalidomide in the in vitro assays of angiogenesis inhibition (
The stability studies showed that CG was stable in solution at pHs between 6.8-7.4 (
In example 1, CG was shown to potentiate the activity of the DMXAA. Studies were performed to examine if CG potentiated the anti-tumour response to the anti-neoplastic drug, cyclophosphamide. Studies performed with cyclophosphamide were conducted in accordance with details provided in examples 1 and 2 except that cyclophosphamide (from Sigma Aldrich, St Louis, Mo., dissolved in water) was used in place of DMXAA. The results of these studies are presented in
Mice with Colon 38 tumours were administered cyclophosphamide alone (220 mg/kg), CG alone (100 mg/kg), or the combination. The growth of tumours in each treatment group was compared with that of untreated tumours. The results (
The results of these studies indicate that CG would be an effective anti-tumour agent for clinical use, since it is more soluble, more stable at physiological conditions, and appears to be at least as active as thalidomide in the panel of biological assays described herein. A major advantage of using CG over thalidomide would be that CG appears to be non-teratogenic. CG is able to cross the placenta, but is unable to cause teratogenicity.
Synergistic combinations of, for example, CG and DMXAA or CG and cyclophosphamide provide further novel compositions for effective treatment of tumours in humans and animals. It is clear from studies described herein that other anti-neoplastic agents can be used in combination with CG to achieve the advantageous compositions for the treatment of solid tumours.
Although the present invention has been described with reference to certain preferred embodiments, it will be understood that variations and alternatives which are in keeping with the spirit of the invention are also within its scope.
This applications claims benefit of U.S. provisional application 60/691,385 filed Jun. 16, 2005, the entire contents of which is herein incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60691385 | Jun 2005 | US |
