Patent Application
20080039485
The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings:
It is found in the invention that camptothecin derivatives are unexpectedly effective in the treatment of digestive tract cancers, particularly CRC, when dissolved in an aqueous solvent and administered orally. In this way, camptothecin derivatives become suitable for use in a low-dose-long-term regimen, which is more beneficial to cancer patients due to lower toxicities.
As used herein, the term “camptothecin derivative” comprises camptothecin per se as well as analogs derived from camptothecin, such as the known compounds 10-HCPT, CPT-11, topotecan, rubitecan, lurtotecan and exatecan (see Thomas, C. J. et al., Bioorg Med Chem 12: 1585-604 (2004); and Lorence, A. and Nessler, C. L., Phytochem 65: 2735-49 (2004)). Preferably, the term “camptothecin derivative” refers to a compound of the following formula:
wherein R1 represents H, —OCH2O or —O(CH2)2O; R2 represents H, OH, —OCH2O, —O(CH2)2O, —OR wherein R is —C(O)—Cn alkyl and n is 9, 11, 13, 15, 17 or 19, or —OC(O)-piperidyl-piperidyl; R3 represents H or —CH2N(CH3)2; and R4 represents H, methyl, ethyl, n-butyl, —Si(CH3)3 or —Si(CH3)2-t-butyl.
As used herein, the term “liquid vehicle” refers to a pharmaceutically acceptable liquid solvent able to dissolve or suspend CPT derivatives. Suitable non-aqueous solvents include, but are not limited to propylene glycol, DMSO, PEG and cremophor. It is well known in the art that CPT is water-insoluble. Some CPT derivatives, like topotecan and irinotecan, have better water-solubility than CPT, but their water-solubility is still limited. Therefore, if an aqueous vehicle is desired, it is first necessary to dissolve the derivative in a non-aqueous solubilizing agent, such as one of the above solvents, for use in the aqueous vehicle.
As used herein, the term “digestive tract cancer” refers to cancers in the digestive tract and digestion-related organs, such as esophageal cancer, stomach cancer, gastrointestinal cancer, gallbladder and bile duct cancer, liver cancer, pancreatic cancer, and colorectal cancer.
According to the present invention, the dose of the composition to be administered can vary in accordance with the age, size, and condition of the subject to be treated. Useful dosages of the composition are those which will deliver about 0.05 to about 1 mg/kg body weight/day, and preferably deliver about 0.15 to about 0.5 mg/kg body weight/day of the camptothecin derivative. The amount of the camptothecin derivative needed in the composition should be an amount suitable for oral administration in view of the metabolism of the compound and of the composition.
The pharmaceutical composition of the present invention can be manufactured by conventionally known methods with one or more excipients that are normally employed in oral liquid formulations, such as surfactants, solubilizers, stabilizers, emulsifiers, thickeners, coloring agents, sweetening agents, flavoring agents, and preservatives. Such excipients are known to those skilled in the art.
The pharmaceutical composition of the present invention can be prepared as any liquid formulation suitable for oral administration, such as syrups, elixirs, oral drops, and oral sprays.
The pharmaceutical composition of the present invention may be used in combination with other conventional cancer treatments, such as surgery, radiotherapy and biological therapy. The pharmaceutical composition of the present invention may also be used in combination with other anticancer drugs in chemotherapy.
The effect of the present invention can be well understood from the following experimental examples, which are intended to be a way of illustrating but not limiting the present invention.
Two human colon cancer cell lines, Colo-205 (ATCC accession No. CCL-222) and HT-29 (ATCC accession No. HTB-38), were used in this study. Cells were treated with 0.2, 0.76, 1.52, 7.62, 15.24, 22.87, 30.49, 38.11, 45.73 or 53.35 μM 10-HCPT or CPT-11 for 1-3 days, and the relative number of the viable cells was determined by MTT assay as described below.
Cells were seeded in a 24-well cell culture cluster (Costar, Cambridge, Mass.) at a density of 1×106 cells per mL and cultured overnight prior to drug treatment. After 10-HCPT or CPT-11 treatment for 3 days, the medium was discarded and replaced with an equal volume (0.5 mL) of fresh medium containing 0.456 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT; Sigma Chemical Company) and incubated for 2 hours at 37° C. in the dark. The medium was discarded, and cells were then combined with 300 mL dimethyl sulfoxide (Sigma Chemical Company) to dissolve the formazan produced. Cell viability was determined according to the calorimetric comparison by reading optical density (OD) values from a microplate reader (Spectra Max 250; Spectra Diode Laboratories, CA) at an absorption wavelength of 570 nm. The results are shown in
As shown in
To investigate the effect of 10-HCPT on the growth of Colo-205 cell line, the cells were treated with a series of dosages of 10-HCPT and harvested at different time points. The cell numbers were determined by trypan blue exclusion assay as described below.
Colo-205 cells (5×105) were seeded in 25T flasks overnight and then treated without (control) and with 5, 10, 15, or 20 nM of 10-HCPT, respectively. After treatment for 24 to 120 hr, cells were harvested by trypsin-EDTA and then centrifuged at 1500 rpm for 5 min at 4° C. The cell pellet was resuspended in culture medium containing 0.04% trypan blue and the viable cells were enumerated by a hemocytometer. The results are shown in
As can be seen from
We further investigated if the suppression of cell growth induced by 10-HCPT on Colo-205 was due to either cell cycle arrest or cell death. The Colo-205 cells were treated with various concentrations of 10-HCPT for 48 hrs and analyzed by propidium iodide staining and flow cytometry as described below.
After treatment with 10-HCPT, cells were trypsinized and resuspended in 70% ethanol. The cells were incubated on ice for at least 1 hr and resuspended in 1 mL of cell cycle assay buffer (0.38 mM sodium citrate, 0.5 mg/mL RNase A, and 0.01 mg/mL propidium iodide) at a concentration of 5×105 cells/mL. Samples were stored in the dark at 4° C. until cell cycle analysis, which was carried out by use of a flow cytometer and ModFit LT 2.0 software (Verity Software, Topsham, Me.).
A FACScan flow cytometer (Becton Dickinson, Bedford, Mass.) equipped with a 488-nm argon laser was used for analysis of cellular function by flow cytometry. Forward and side scatters were used to establish size gates and exclude cellular debris from the analysis. The excitation wavelength was set at 488 nm. The observation wavelength of 530 nm was chosen for green fluorescence and 585 nm for red fluorescence and the intensities of emitted fluorescence were collected on FL1 and FL2 channels, respectively. In each measurement, a minimum of 20,000 cells was analyzed. Data were acquired and analyzed using the Cell Quest software (Becton Dickinson). Relative change in the mean fluorescence intensity was calculated as the ratio between mean fluorescence intensity in the channel of the treated cells and that of the control. The results are shown in
As shown in
The progression of the cell cycle is promoted by various CDKs binding with their respective cyclin partners and inhibited by CDK inhibitors such as p21 and p27. In general, CDK2 in association with cyclin E regulates the G1/S transition, and CDK1 in concert with type-A and -B cyclins controls the G2/M transition. Both cell cycle inhibitory proteins p21 and p27 block cell cycle progression at the G1/S as well as the G2/M transitions of cell cycle. Since 10-HCPT led to an accumulation of cells in the G2/M phase as demonstrated in Example 3, we speculated that the underlying molecular mechanism might involve changes in the expression of cell cycle regulatory proteins. To confirm this speculation, we analyzed the expressions of several cell cycle regulatory proteins by means of Western blotting assay as described below.
Colo-205 cells were cultured in Dulbecco's modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37° C. in a 5% CO2 atmosphere. Cells were seeded in 6-well plate and incubated with 3 μM of 10-HCPT. After 48 hr, the treated cells were washed twice with phosphate-buffered saline (PBS), flash frozen in liquid nitrogen, and stored at −80° C. The harvested cells were lysed in ice-cold report lysis buffer (Promega). After cleaning the resulted lysates by centrifugation, protein in the clear lysate was quantified by Dc protein assay kit (Bio-Rad). 15 μg of lysate protein were resolved by 10% SDS-PAGE, transferred onto a polyvinylidene difluoride membrane (PVDF membrane, Amersham Biosciences), and immunoblotted with antibodies against followed cellular proteins: DNA topoisomerase I, p53, p27, phosphorylated p27 (Santa Cruz Biotechnology), cyclin E, CDK2 (BD Pharningen), PCNA, p21 (Upstate), and α-tubulin (Sigma). Protein content was visualized with BM Chemiluminescence Blotting Kit (Roche Molecular Biochemicals). The blots were exposed to X-ray film (Fuji) for various times. The results are shown in
As can be seen from
Because apoptosis always takes place as a consequence of a cell cycle block, it is possible that 10-HCPT is able to cause cell death through apoptosis pathway. To verify this possibility, we first performed DNA fragmentation assay since DNA fragmentation is one characteristic of apoptosis.
The DNA of Colo-205 cells treated with various concentrations of 10-HCPT for 48 hrs was extracted and analyzed by 1.5% native agarose gel electrophoresis. As shown in
In addition, to further determine whether caspases participate in the propagation of apoptosis induced by 10-HCPT in Colo-205 cells, cell lysates from drug-treated cells were analyzed for the cleavage of the p-nitroaniline-tagged peptides N-Acetyl-Asp-Glu-Val-Aps-pNA (DEVD-pNA), which represent preferential substrates for caspase-3. Caspase activation leads to the release of pNA that can be monitored by absorbance measurements at 405 nm using a plate reader. Specifically, DEVD-pNA cleavage activity was measured using Caspase-3 Colorimetric Assay Kit (BioVision) according to the recommended protocols. 3×106 of 10-HCPT treated cells were harvested, resuspended in 50 μl of chilled cell lysis buffer and incubated on ice for 10 min before centrifugation (12,000 rpm for 3 min at 4° C.). The supernatant was collected and the total protein concentration of the supernatant was determined by Dc protein assay kit (Bio-Rad). Each assay reaction containing 100 μg of cell lysates, 50 μl of 2× reaction buffer supplemented with 10 mM DTT, and 5 μl of the 4 mM of DEVD-pNA (200 μM final concentration) was carried out at 37° C. for 1 hr. The formation of p-nitroanilide was detected at 405 nm in a microtiter plate reader.
With the DEVD-pNA substrate, the absorbance measured after 10-HCPT treatment of the cells for 48 hrs was clearly elevated compared to that measured in the control (drug-free) lysates (
We further examined whether 10-HCPT is effective in growth suppression in vivo after tumor formation. Colo-205 cells were transplanted into BALB/c-nu mice, and when the tumors were palpable (3-5 mm), the mice were treated either with vehicle control or 10-HCPT.
The in vivo experiment was carried out with ethical committee approval and met the standards required by the Guiding Principles in the Use of Animals in Toxicology, which were adopted by the Society of Toxicology in 1989. The BALB/c-nu mice were obtained from National Laboratory Animal Center of National Applied Research Laboratories (Taipei, Taiwan) and housed in a laminar flow room under sterilized conditions with the temperature being maintained at 25±2° C. and the light controlled at 12 h light and 12 h dark cycle. The Colo 205 cells were harvested and resuspended in serum free RPMI-1640 medium. Cells were adjusted to 1×107 cells/mL, and transplanted subcutaneously on the flank regions of the mice. Each experimental group included six to seven mice bearing tumors.
To calculate the suitable dosage for mice, we adopted the scaling method. That is, we used the dose for human as reference to calculate the equivalent dose for mice. The dose for mice was calculate through the following formula:
Dose for Mice (mg/kg/day)=Dose for Human (mg/kg/day)×[65 kg (Human Average Body Weight)]/1600 (Human MEC Value)]×[3.7 (Mice MEC Value)/0.02 (Mice Average Body Weight)]
[MEC: minimum energy cost, which equals to the basal metabolism rate (BMR); for more reference, see Ruckebush, Y. et al., Physiology of Small and Large Animals, pp. 387-398. B. C. Decker Inc., USA, 1991]
10-HCPT was dissolved in propylene glycol and treatment began when the tumor size reached 3-5 mm. 10-HCPT was administered via p.o. once per two or four days at doses of 1-7.5 mg/kg (volume of injection: 0.1 mL/20 g of body weight), respectively. The control group received propylene glycol vehicle once per two days. Tumor size and body weight were monitored twice a week throughout the experiment. The tumor size was measured using a vernier caliper. Tumor volume (V) was calculated according to the formula: V (mm3)=0.4AB2, where A and B are the longest diameter and the shortest diameter, respectively. At the end of experiment, all mice were sacrificed by CO2 gas. Tumors, livers, kidneys, and lungs were collected, fixed, embedded and stained with hematoxylin and eosin for pathological analysis.
The challenge of 10-HCPT (1-7.5 mg/kg, p.o., once per two or four days) in nude mice produced no obvious acute toxicity. No significant reduction in body weight was found in 10-HCPT-treated mice, except the 7.5 mg/kg/2 days-treated group (
Previous pharmacokinetic studies indicate that 10-HCPT has a short distribution half-life and a prolonged elimination half-life. Dose-dependent toxicity was observed with i.v. administration of 10-HCPT. It can be best administered as a loading dose followed by a maintenance dose every other day. Since large amounts of drug were excreted within a short period following i.v. administration, the possible side effects on renal function may be observed, especially in the early phase of drug disposition (Zhang, R. et al., Cancer Chemother Pharmacol 41: 257-67 (1998)).
The results in Example 6 demonstrate that following oral administration at doses of 2.5-7.5 mg/kg/2 days, but not 2.5-7.5 mg/kg/4 days, tumor growth suppression by 10-HCPT was obviously observed in nude mouse xenografts. The challenge of 10-HCPT (2.5-7.5 mg/kg, p.o., once per two or four days) in mice produced no obvious acute toxicity. No significant reduction in body weight was found in 10-HCPT-treated mice, except the 7.5 mg/kg/2 days-treated group. These results agree with the studies of R. Zhang, and the low maintenance dose of 10-HCPT is the best oral administration strategy. We concluded that 10-HCPT could be administered orally and the long-term low maintenance dose of 10-HCPT should be the best administration strategy to achieve maximal anticancer effect.
It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.
