WO2010095965A1 - The use of sulindac and/or its metabolite as adjuvant treatment for colon cancer in humans - Google Patents

Abstract

The use of sulindac and/or its metabolite in combination with cytostatic drugs as adjuvant treatment for colon cancer in humans. The cytostatic is selected from a group including: 5-f luorouracil, capecitabine, oxaliplatin, leucovorin and irinotecan and/or their combinations.

Description

The use of sulindac and/or its metabolite as adjuvant treatment for colon cancer in humans

The subject of the invention is the use of sulindac and/or its metabolite as adjuvant treatment for colon cancer in humans .

Colon cancer is one of the most common malignancies of the gastro-intestinal tract in humans. In Poland, the incidence is estimated at 11 000, which accounts for approximately 10% of all malignancies in women and men. In Poland, the mortality rate is very high, at 83%, which means that less than 20% of patients are successfully cured (Nowacki MP: Standards of diagnostic and therapeutic management in cases of colon cancer in: Standards of systemic treatment for malignant neoplasms in adults in Poland, ed by: M. Krzakowski and P. Siedlecki, in Polish) . In the USA, the survival rate is 66% compared to approximately 35% in Eastern Europe, including Poland (Parkin et al.: Global cancer statistics, 2002. CA Cancer J Clin 2005; 55:74- 108. ) .

Colon cancer occurs as transformation within polyps. Polyps may develop in the course of familial adenomatous polyposis as well as spontaneous polyposis due to the APC gene mutation. The development of colon cancer, which may last over a few to a few dozen years, consists of excessive proliferation of intestinal epithelial cells (as the result of the mutation) leading to the formation of characteristic thickening, a polyp, which protrudes into the lumen. This lesion is adenomatous and with time as the cells continue to proliferate and become invasive, cancer develops. Molecular basis is related to the genomic instability comprising chromosomal and microsatellite instability in addition to mutations in APC and P53 genes (Markowitz et al.: Molecular basis of colorectal cancer. New Engl J Med 2009; 361 : 2449) .

In advanced stages, when the tumour is no longer a local lesion, but spreads to adjacent or distant tissues, after surgical resection of the tumour, the patient is given adjuvant therapy with cytostatics. Its aim is to destroy potential mictrometastases and to reduce the likelihood of recurrence .

The cytostatics used are a group of natural and synthetic substances which have a toxic effect on rapidly dividing tumour cells. Cytostatic drugs act essentially by affecting the cell cycle and causing cell death or inhibiting cell development and divisions. The efficacy of treatment depends on the degree to which the population of cancer cells is destroyed. Usually, a chemotherapy regimen is multidrug and incorporates several drugs belonging to different groups of cytostatics to increase the efficacy of treatment. The drugs are selected in such a way that they have different mechanisms of action (cause the tumour cell kill in different ways) and have different adverse effects which helps to avoid intensification of the same toxicities.

Unfortunately, chemotherapeutics also damage other rapidly dividing healthy cells (bone marrow, mucous membranes, hair cells) with the commonly observed adverse effects such as anaemia, nausea and vomiting, and hair loss. Their disadvantage is a low therapeutic index, defined as the ratio the dose producing symptoms of toxicity to the dose producing therapeutic effects. The therapeutic index (TI) is different for different drugs and is used to assess their safety. The higher the TI is, the safer is the drug. Regimens incorporating drugs with high therapeutic indices are sought, but that is not always possible. Cytostatics, whose aim is to destroy living cells of the body, have a very low therapeutic index, approximately 0.25 and thus are dangerous drugs.

The use of adjuvant therapy in the treatment for colon cancer was based on the finding of improved survival compared to surgery alone (Andre T et al.: Oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment for colon cancer. N Engl J Med 2004; 350:2342-2351; Chau I and Cunningham D: Adjuvant therapy in colon cancer - what, when and how? Ann Ocol 2006; 17:1347-1359) . The cytostatic drugs employed belong to different groups. 5-fluorouracil is a pyrimidine antagonist, oxaliplatin is an alkylating agent, irinotecan is a derivative of camptothecin, capecitabine is a prodrug given orally, which is converted into fluorouracil in tumour tissue (it acts in the same way as 5-fluorouracil) while leucovorin (folinic acid) is used to enhance the inhibitory effect of 5- fluorouracil (5-FU) on thymidylate synthase (an enzyme involved in the synthesis of DNA) . Several regimens are used, the most commonly: 5-fluorouracil (or more recently, capecitabine, which is converted into 5-fluorouracil in tumour tissue) with leucovorin and oxaliplatin or irinotecan and leucovorin. The results of such treatment are very poor. In advanced cancer (distant metastases are present in nearly 50% of cases with first symptoms), the 5-year survival rate is below 30% in stage III and very rare in stage IV (Nowacki MP: Standards of diagnostic and therapeutic management in cases of colon cancer in: Standards of systemic treatment of malignant neoplasms in adults in Poland, ed: M. Krzakowski and P. Siedlecki, in Polish) . This situation makes oncologists search for other drugs which may be added to adjuvant therapy to improve the efficacy of the chemotherapy. Sulindac first aroused interest when it was found to cause regression of polyps of the colon (Waddell WR and Loughry RW: Sulindac for polyposis of the colon. J Surg Oncol 1983; 24:83-87) . Since then there have been many reports confirming that sulindac reduces the number of polyps of the colon (Ishikawa H: Chemoprevention of carcinogenesis in familial tumors. Int J Clin Oncol 2004; 9:299 - 303) but the drug has not been used in the clinic because it has adverse gastro-intestinal effects and cannot be administered in long- term treatment. Also, the drug does not prevent the development of adenomas in patients with familial adenomatous polyposis (Giardiello FM et al: Primary chemoprevention of familial adenomatous polyposis with sulindac. N Engl J Med 2002; 346:1054-1059) .

Sulindac - as all anti-inflammatory drugs - is an inhibitor of the enzyme cyclooxygenase (COX) . There are two isoforms of COX: COX-I and COX-2. Sulindac acts mostly on COX-I with small effect on COX-2. Overexpression of COX-2 has been found in polyposis of the colon. Celecoxib (Celebrex), however, is a specific COX-2 inhibitor and it slows down the development of adenomas in the colon of humans . Although the findings have been confirmed and the approved therapeutic indications of celecoxib include "to reduce the number of adenomatous colorectal polyps in familial adenomatous polyposis", the drug is not used in long-term treatment because of the risk of cardio-vascular events, including myocardial infarction. However, the fact that celecoxib prevents polyp growth, suggested that it may be effective in adjuvant treatment for colon cancer in humans. A phase II study did not produce positive results. It was found that celecoxib neither enhanced the effects of cytostatic drugs nor decreased their toxicity. As sulindac was not ultimately approved for use in the prevention of polyposis of the colon, there was no interest in its use as adjuvant treatment of colon cancer.

Sulindac is a non-steroidal anti-inflammatory drug used for the treatment of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, acute painful shoulder and acute gouty arthritis (Physician Desk Reference, 56 edition, Medical Economic Company 2002, www. PDR. net) . No indications are approved for treatment of polyposis, prevention of cancer development (although relevant studies were conducted) and adjuvant treatment for colon cancer.

Also the state of the technology established on the basis of the patent literature does not suggest any potential uses of sulindac in combination with cytostatic drugs as adjuvant treatment of colon cancer.

The patent specification No EP 1428528 disclosed that the combination of NSAIDs (non-steroidal anti-inflammatory drugs) , such as sulindac, piroxicam or aspirin, with fermenting dietary fibres such as inulin found in chicory or oligofructose or their combination, had a synergistic anti- tumour effect in humans and in other vertebrates.

The patent specification No US 2008207751 disclosed the use of sulindac and other non-steroidal anti-inflammatory drugs in combination with DFMO (difluoromethylornithine) for both prevention and treatment of tumours of the colon characteristic of the expression of Ki-ras protooncogene .

The use of sulindac in combination with ursodeoxycholic acid (a bile acid) for prevention of the recurrence of colon adenoma is known from the patent specification No Mx 9700190.

The combination of NSAIDs, including sulindac and/or its metabolites, with oxidants, such as hydrogen peroxide or arsenic (III) oxide for the treatment of skin cancer and precancerous states is known from the patent specification No US 2008119559.

The patent specification No PL 3532267 disclosed the effect of NSAIDs, including sulindac with an EFGR kinase inhibitor for the treatment and inhibition of growth of colon polyps and the treatment of colorectal cancer.

The therapeutic and tumour preventing effect of sulindac derivatives used in monotherapy and combination therapy is known from the patent specification No US 2008207751.

However, unexpectedly sulindac proved to act synergistically with cytostatic drugs, which allowed several- fold reduction of their concentrations while maintaining the same "lethal" effect on cancer cell. The study used the active sulfide metabolite which is formed in vivo by reduction of the sulfinyl group to the sulfide group. The reduction is achieved due to, among other factors, the presence of intestinal bacteria and sulindac sulfide (metabolite) is preferentially accumulated in the cells of intestinal epithelium; after oral administration, the ratio of epithelial to blood concentrations is 20:1. Concentrations of sulindac sulfide used in studies on cell lines (up to 200 μM) corresponded to concentrations achieved in vivo after oral administration of sulindac. Sulindac sulfide was found to potently induce apoptosis in the cells of colon cancer, hence the conclusion that sulindac sulfide (and thus sulindac administered in vivo) may synergistically potentiate the action of classical cytostatic drugs used in the treatment of colon cancer.

The studies have demonstrated a synergistic effect of sulindac sulfide used in combination with oxaliplatin and 5- FU on the cancer cell lines Colo-205, HT-29 and SW48. When sulindac sulfide was used in combination with oxalipatin, the concentration of oxalipatin required for 75% reduction of proliferation was decreased 5-fold (Colo-205 line) , 14-fold (SW48 line) or 11-fold (HT-29 line) . The use of sulindac sulfide in combination with 5-FU allowed a several-fold reduction in the concentration of 5-FU.

The invention consists of the use of sulindac and/or is metabolite in combination with cytostatic drugs as adjuvant treatment for colon cancer.

Since, as mentioned earlier, cytostatic drugs (including 5-FU and oxaliplatin) are only partially effective in the treatment of colon cancer, due to, among other factors, their enormous toxicity, and thus very harmful effects on the body, the possibility of several-fold reduction of cytostatic doses used in clinical practice offers an exceptional chance of advances in the treatment of colon cancer.

The cytostatic drugs used belong to the group including 5-fluorouracil and its precursor capecitabine as well as oxaliplatin, leucovorin and irinotecan and/or their combinations. The inclusion of 5-fluorouracil and oxaliplatin was advantageous.

The example describes the study method and illustrates the synergistic effect of sulindac with oxaliplatin or 5- fluorouracil .

Human cell lines of colon cancer: SW48, HT-29 and Colo- 205 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) . The cells were cultured on RPMI- 1640 medium (IITD, Wroclaw, Poland) with the addition of 5% fetal bovine serum - FBS (Gibco) , 2 mM glutamax (Gibco) , 10 mM sodium pyruvate (Polpharma, Poland), and antibiotics: 100 IU/ml penicillin sodium, 100 μg/ml streptomycin and 250 ng/ml amphotericin B (Gibco) . Cell cultures were conducted in sterile, single-use polystyrene culture flasks with the surface area of 80 cm² or 25 cm² kept in an incubator at 37 'C in a moist atmosphere with 5%CO_2. With regular change of the medium and passaging of cells, the culture was maintained in the exponential growth phase and continuously checked for the presence of mycoplasm.

Experiments on cell lines were conducted in 80-cm² culture flasks and 6-, 24- and 96-well culture plates. The choice of single-use culture vessels depended on the type of experiment and method used. All experiments were carried out at least four times in duplicate or triplicate. Two days prior to experiment the cells were inoculated on to fresh medium to obtain the initial culture in the exponential growth phase. The experiment was started with appropriate preparation of the culture vessels into which cell suspensions of the same density were inoculated.

The cells were pre-incubated overnight at 37 °C and next study substances dissolved in DMSO at relevant concentrations were added to the medium and incubated for 24, 48 and 72 hours at 37 °C in 5%CC>₂. After the incubation, the cells were viewed in an inverted light microscope and next trypsinized. To do so, the medium was transferred to a new test tube, the cells adhering to the medium were rinsed with phosphate buffer (PBS) without Ca²⁺ and Mg²⁺ ions (CMF) and trypsin was added (0.25% trypsin solution/0.03% EDTA), and the cells were incubaded at 37 °C until they became detached . After trypsin inactivation by addition of the culture medium, the detached cells were transferred into a test tube with the earlier collected medium and used in the assays.

The following substances were used in the study: oxaliplatin, 5-FU and sulindac sulfide (Sigma, St. Luis, MI, USA) at the concentration range from 3 to 200μM. The substances were dissolved in 100% DMSO (Sigma) and then for the purposes of the experiment diluted in the culture medium to an appropriate concentration. Cells with DMSO served as controls. The final DMSO concentration in the medium did not exceed 0.2% (the value which does not affect cell survival) .

The MTT cytotoxicity assay was used to evaluate the percentage of live cells. In the MTT assay, water-soluble tetrazole salt in live cells is reduced (oxidoreductive activity of the mitochondria) to water-insoluble dark-blue formazan. The assay was performed in 96-well plates.

After the incubation of cells with the study substances, the medium was removed and 50 μl of MTT solution was added to each well (5 mg/ml solution of MTT in PBS was diluted 10-fold in RPMI-1640 medium without phenol red) . The plates were incubated at 37 °C for 4 hours and then aliquots of 150- μl of 10% SDS (sodium dodecyl sulfate) were added to extract formazan crystals. After 24-hour incubation in the dark at room temperature, absorbance was measured using a Power Wave microplate reader (Bio-Tek, USA) at wave length 570 nm.

Apoptosis induction in the cells was assessed by determining the degree of binding of FITC-conjugated annexin to phosphatidylserine in the surface of the cell membrane. The apoptotic index was determined by addition to 100 μl of cell suspension (IxIO⁵) of 5 μl annexin and 5 μl propidium iodide from an Annexin V Kit (Becton Dickinson, USA) . Before the cytometric measurements, cells were incubated for 15 minutes in the dark.

The cell cycle was analysed in cells fixed in 80% ethanol. The cells (at least IxIO⁶) were thawed, rinsed with PBS buffer and stained with the solution of 50 μg/ml PI and 100 μg/ml TNase in 0.1% PBST (phosphate buffer, Triton) . After incubation for 30 minutes at room temperature, fluorescence was measured with a BDFACSCalibure flow cytometer BD Bioscences, San Jose, CA, USA) .

DNA histograms were analysed using the ModFit LT V.3.0 software (BD Biosciences, San Jose, CA, USA) .

Drug interaction study

In the experiment, cells were incubated with the study substances for 72 hours. A single drug and its combinations with other drugs at five different concentrations were investigated, each concentration assessed in triplicate in 8 independent experiments.

After pre-incubation the drugs were added to the cells to achieve an appropriate final concentration. For each drug combination, parallel cell cultures were conducted with single drugs, which served as controls.

At the end of incubation, the percentage of live cells was assessed with the MTT test. Interactions between study substances were analysed by the isobologram method and the Chou-Talalay method based on the median-effect equation using the CalcuSyn ver. 2.0 software (Biosoft, Cambridge, UK) .

The isobolograms were determined based on the IC₅₀ values obtained experimentally for the cytostatic drugs and for sulindac sulfide given alone or in combination. The doses determined for each drug given alone were recorded on the x and y axes respectively and the line connecting the two points formed the so-called "theoretical addition line". The IC₅₀ dose for a drug combination located close to or on the addition line identified an additive drug-drug interaction while the IC₅₀ values located below or above the addition line identified synergism and antagonism respectively. The median effect method was used to determine the Combination Index (CI) depending on the percentage of dead cells (Fa) . CI values < 1 identified a synergistic effect, CI=I identified an additive effect and CI>1 an antagonistic effect.

Also, the so-called Dose Reduction Index (DRI) was determined using the Chou -Talalay method. The value of DRI shows by how many times the concentration (dose) of a drug may be reduced when it is given in a given combination. The higher the DRI is, the more the drug dose may be reduced when a multidrug regimen is used, Fig 1.

The results of the study are given in Fig. 2. The synergism between sulindac sulfide and oxaliplatin and 5- fluorouracil (5-FU) was demonstrated in human colon cancer cell lines (Colo-205 and SW48) . y axis: sulindac sulfide concentration, x axis: oxaliplatin or 5-FU concentration; circles: drug concentrations which cause death of 50% cells. Thus, e.g. Colo-205, y axis: sulindac sulfide at ^'a concentration of approximately 70 μM (oxaliplatin 0 μM) caused death of 50% cells; x axis: oxaliplatin at a concentration of approximately 33 μM (sulindac sulfide 0 μM) caused death of 50% cells. Along the line connecting the two points runs the so-called "addition line", i.e. the concentrations of the two drugs are located which are required to produce the same effect assuming that their actions are addititive. Theoretically, approximately 20 μM oxaliplatin and 30 μM sulindac sulfide are required to produce the same effect (lines plotted from the concentrations of 20 μM for oxaliplatin and 30 μM for sulindac sulfide meet at the "addition line") . Experimentally, however, it has been demonstrated that to produce the same effect of 50% cancer cell "kill" approximately 3 μM oxaliplatin and 38 μM sulindac sulfide should be used. The synergistic effect of sulindac sulfide and the studied cytostatic drugs is accounted for by the fact that sulindac sulfide in combination with cytostatic drugs in all cell lines studies potently induces apoptosis (programmed cell death) of cancer cells, as shown in Figs 3 and 4 (abbreviations: SD; sulfide; 5-FU: 5-fluorouracil; Oxali: oxaliplatin; bars depict the percentage of apoptotic cells) .

Claims

Claims

1. What is claimed: the use of sulindac and/or its metabolite in combination with cytostatic drugs as adjuvant treatment for colon cancer in humans.

2. The method of claim 1, wherein the cytostatic is selected from a group including: 5-fluorouracil, capecitabine, oxaliplatin, leucovorin and irinotecan and/or their combinations.

3. The method of claim 1 or 2, wherein 5-fluorouracil is the cytostatic drug.

4. The method of claim 1 or 2, wherein oxaliplatin is the cytostatic drug.