JP2003514017A - Use of NSAIDs for the treatment of pancreatic cancer - Google Patents

Abstract

  (57) [Summary] The present invention provides a method for treating pancreatic cancer comprising the use of non-steroidal anti-inflammatory drugs (NSAIDs), especially sulindac or an analog thereof.

Description

Detailed Description of the Invention

[0001] Background Pancreatic cancer invention occupies lung, ranks second only to colon cancer and breast cancer as the cause of death due to the most common cancer (1). Pancreatic cancer is more common in men, with men between the ages of 60 and 70 being the most at risk. The cause of pancreatic cancer is unknown.

The most common symptoms are weight loss, abdominal pain and jaundice. Weight loss is usually severe and its cause is not completely understood. The average reduction is about 25 pounds. Jaundice occurs when the cancer blocks the common bile duct. Survival of pancreatic cancer is low. Pancreatic cancer often has spread (metastasized) to other parts of the body by the time a malignant tumor is identified. Life expectancy is more than 6 months from the time of diagnosis.

[0003] Tumors often cannot be removed surgically because they affect life-threatening tissue that cannot be removed or have spread to distant sites. Chemotherapy and radiation therapy can be used for tumors, but these treatments often do not work.

The number of non-steroidal anti-inflammatory drugs (NSAIDs) has increased to the point of forming a separate classification. Available in the U.S. in addition to aspirin, NSAIDs include sodium meclofenamic acid, oxyphenbutazone, phenylbutazone, indomethacin, piroxicam, sulindac and tolmethine, mefenamic acid and zomepirac for analgesia and arthritis and nociception for the treatment of arthritis. It includes ibuprofen, fenoprofen and naproxen, which are both extinction. Ibuprofen, mefenamic acid and naproxen are also used to manage dysmenorrhea.

The clinical utility of NSAIDs is limited due to many side effects. Phenylbutazone is associated with liver necrosis and granulomatous hepatitis, and sulindac, indomethacin, ibuprofen and naproxen are associated with hepatitis and cholestasis. A transient rise in serum aminotransferases, especially alanine aminotransferase, has been reported. All of these drugs, including aspirin, inhibit cyclooxygenase, which in turn inhibits the synthesis of prostaglandins that help regulate glomerular filtration rate and renal sodium and water excretion. For this reason, NSAIDs lead to fluid retention, reduced sodium excretion,
May cause hyperkalemia, oliguria and anuria. Also, all of these drugs can cause peptic ulcers. See Remington's Pharmaceutical Sciences , Mack Pub. Co., Easton, PA (18th ed., 1990) 1115-1122.

There is much literature on the effects of NSAIDs on cancer, especially colon cancer. For example, HA Weiss et al., Scand J. Gastroent. , 31 ,, 137 (1996) (suppl. 2
20) and Shiff et al . , Exp. Cell Res. , 222 , 179 (1996). More recently, B. Bellosillo et al., Blood , 92 , 1406 (1998) showed that aspirin and salicylate reduce the viability of B cell CLL cells in vitro, while indomethacin, ketoralac and NS-. 398 reports no reduction. Sulindac is being studied in a combination therapy for the treatment of colon cancer. HM Verheul et al . , Brit. J. Cancer , 79 , 114 (1999);
See FA Sinicrope et al . , Clin. Cancer Res. , 2 , 37 (1996); and M. Mooghen et al., J. Pathol. , 156 , 394 (1988).

CP Duffy et al. , Eur. J. Cancer , 34 , 1250 (1998) reported that the cytotoxic effect of certain chemotherapeutic agents was increased when combined with certain non-steroidal anti-inflammatory agents. This effect observed on human lung cancer cells and human leukemia cells was highly specific and unpredictable. That is, some combinations of NSAIDs and drugs were effective, and some were ineffective. The only conclusion drawn is
The effect was not due to the cyclooxygenase inhibitory activity of NSAID.

The Duffy group filed a PCT application in October 1997 for a combination of a “MRP substrate” that can be an anticancer drug and an NSAID that enhances the efficacy of the anticancer drug.
I went on the 24th of the month. The NSAIDs listed in the claims are acemethacin, indomethacin, sulindac, sulindac sulfide, sulindac sulfone, tolmetin and zomepirac. Naproxen and piroxicam were reported to be inactive.

Therefore, there is a continuing need for methods to suppress cancer and enhance the efficacy of anti-cancer drugs with relatively non-toxic drugs. SUMMARY OF THE INVENTION In one aspect, the present invention provides a mammal afflicted with pancreatic cancer in an amount effective to inhibit the viability of said pancreatic cancer cells in said mammal, preferably sulindac ((Z) -5- Fluoro-2-methyl-1-[[4- (methylsulfinyl) phenyl] methylene] -1H-indene-3
-Acetic acid) or an analogue thereof, preferably a COX-2 inhibitor, is provided for the treatment of pancreatic cancer. The invention also provides a method of sensitizing human pancreatic cancer cells to a chemotherapeutic agent comprising contacting the cells with an effective sensitizing amount of NSAID, preferably sulindac, or an analogue thereof. Thus, the present invention provides for the treatment of pancreatic cancer in which an effective amount of NSAID, preferably sulindac or an analogue thereof, is administered to a patient suffering from pancreatic cancer and being treated with a chemotherapeutic (“anti-neoplastic”) agent. Therapeutic methods for the treatment of humans or other mammals.

Sulindac is preferably administered with one or more chemotherapeutic agents effective against pancreatic cancer, such as gemcitabine or 5-FU.

Methods for assessing the ability of sulindac to sensitize pancreatic cancer cells to chemotherapeutic agents are also provided. The assay method comprises: (a) isolating a first portion of pancreatic cancer cells from a human cancer patient; (b) measuring their viability; (c)
Administering sulindac or the analog thereof to the patient; (d) isolating a second portion of pancreatic cancer cells from the patient; (e) determining the viability of the second portion of pancreatic cancer cells. And (f) comparing the viability measured in step (e) with the viability measured in step (b).
The reduced viability in e) indicates that the cells were sensitized to the chemotherapeutic agent.

[0012] As in the case where the pancreatic cancer cells are derived from the blood of a mammal affected by pancreatic cancer, steps (b) and (e) are preferably performed in the presence of a chemotherapeutic agent.

This allows for the rapid assessment of cancer patients who will be or are being treated for pancreatic cancer and to determine whether they will benefit from the combination of chemotherapy with the administration of Sulindac or its analogs. it can.

The term “sulindac” and analogs of sulindac, as used herein, include metabolites such as sulindac sulfone, sulindac sulfide and pharmaceutically acceptable salts thereof. Brief Description of the Drawings Figure 1. Photocopy of a representative immunoblot of pancreatic adenocarcinoma and corresponding normal tissue.
Lysates were prepared from tumor (T) specimens obtained from 6 patients, including 3 with normal (N) tissue (sample numbers correspond to those listed in Table 1). Lysates were analyzed by immunoblotting with specific COX-2, COX-1, p21 ^ras and actin antibodies as indicated. The positive control (+) for the COX-2 immunoblot is a cell lysate prepared from Raw 264.7, a mouse macrophage cell line treated with lipopolysaccharide (LPS). The negative control (-) is a colon cancer cell line HCT1 that expresses neither COX-1 nor COX-2.
16

FIG. 2. COX-2 expression rate in patient samples. COX-2 expression rate values are plotted for all tumor samples indicated by filled circles in Table 1 and normal tissues indicated by open circles. Mean, median and range are shown. COX-2 in pancreatic tumor / normal tissue set
The expression rate is shown in the inset in the figure (n = 11). The corresponding tumor value indicated by a black circle and the normal value indicated by a white circle are connected by a line. The difference in COX-2 expression between tumor samples and normal samples is
It was judged to be statistically significant (P = 0.004).

FIG. Expression of COX-2 in pancreatic tumor cell lines. A) COX-2 expression in a human pancreatic cell line detected by immunoblot analysis. The K-ras mutation status of each cell line is also shown. B) Activation of cell line BxPC-3 after treatment with MEK inhibitor PD98059 or DMSO for 10 hours
Expression levels of MAP kinase and COX-2. Phosphorylation-active Erk 1/2 Map kinase was detected with a phosphate-specific Map kinase antibody. C) COX in three hamster pancreatic cell lines
-2 expression comparison. D27 is a non-malignant parental cell line from which D27 / K-ras and B12 / 13
A transformation system was obtained. After probing with COX-2, the blot was stripped and re-probed for actin, showing comparable protein loading. D) Inducibility of COX-2 expression. Serum starved cells were stimulated with 10% FCS for the times indicated, followed by cell lysis. COX-2 expression was measured by immunoblot analysis in lysates prepared from proliferating cells (Gr), serum starved cells (0h) and FCS stimulated cells.

FIG. Effect of COX inhibitors on proliferation of pancreatic tumor cell lines. Cell line B shown with black bars
xPC-3 and PaCa-2 indicated by the shaded bar were plated in the presence of DMSO (control) or the indicated concentrations of sulindac (A), indomethacin (B) or NS-398 (C). On day 3, cells in two wells were counted and expressed as a percentage of the number of cells growing in the presence of DMSO. Mean values +/- standard deviation from at least two independent experiments are shown.

FIG. 5. Production of prostaglandin E ₂ . A) PGE ₂ levels in pancreatic tumor cell lines. Exponentially growing cells were incubated with 15 μM arachidonic acid in serum-free medium for 1 hour, after which the level of PGE ₂ in the culture supernatant was measured by enzyme immunoassay. PGE ₂ production was normalized to protein concentration. Results shown are the mean +/- standard deviation from two independent experiments. B) Effect of COX inhibitor on PGE ₂ production. BxPC-3 cell line shows DMSO or two concentrations of COX inhibitors (100 μM sulindac, 10 μM indomethacin and 10 μM NS-398 as black bars, 250 μM sulindac, 100 μM indomethacin and 50 μM NS-398 The cells were lysed after incubation for 24 hours (indicated by stippled bars), and intracellular PGE ₂ was measured by enzyme immunoassay. Shows the PGE ₂ inhibition ratio by COX inhibitor. Data are from at least 2 independent experiments.

FIG. 6 is a graph showing the effect of the combination of sulindac and gemcitabine on the proliferation of pancreatic tumor cell line BxPC.

FIG. 7 is a graph showing the effect of the combination of sulindac and gemcitabine on the proliferation of the pancreatic tumor cell line PaCa-2. DETAILED DESCRIPTION OF THE INVENTION Due to the difficulty of achieving early diagnosis and the aggressive nature of pancreatic cancer, the survival rate of patients with pancreatic cancer is low. Since there are few options for the treatment of pancreatic cancer, it is important to identify potential targets for drug therapy. To gain more insight into pancreatic tumorigenesis, pancreatic tumors have been analyzed at the molecular level to detect genetic damage. Mutations that activate in the K-ras gene have been detected in up to 90% of pancreatic carcinomas, suggesting that activation of the Ras pathway is important in pancreatic cancer development (2). Currently, experimental chemotherapy strategies for patients with pancreatic cancer include drugs targeted to the Ras signal transduction pathway.

A large body of evidence suggests that the enzymes cyclooxygenase (COX), specifically COX-2, may be potential chemotherapeutic targets. For example, epidemiological studies have shown that long-term use of aspirin or other nonsteroidal anti-inflammatory drugs (NSAIDs) can reduce colon cancer risk by 40-50% (3). NSAIDs can also inhibit chemically induced colon carcinoma in animal model systems (4). N
Since SAID is known to inhibit cyclooxygenase (COX), a key enzyme in the conversion of arachidonate to prostaglandins and other eicosanoids, these studies add to the known role of COX in inflammation. And may play a role in carcinogenesis. COX-1 and COX-2
Two isomers of COX have been identified. COX-1 is constitutively expressed, and COX-
2 is induced by mitogenic stimuli such as serum, phorbol ester and growth factors (5,6). Recently, COX-2 expression has been shown to be elevated in several human cancers, suggesting that the presence of COX-2 is correlated with cancer development (
7-11). Further studies linking COX-2 directly to carcinogenesis include observations that human colon cancer cells expressing COX-2 enhance invasiveness (12) and COX-2 expressed in colon epithelial cells inhibits apoptosis. The observation (13) of doing is included. It has also been shown that COX-2 expression in colon cancer cells promotes angiogenesis of co-cultured endothelial cells by stimulating the production of angiogenic factors (14). In addition, a mouse model of human familial adenomatous polyposis (FAP), an inherited disease that leads to colorectal cancer, provided direct genetic evidence linking COX-2 to colorectal neoplasia . That is, this system was found to reduce the number of intestinal polyps in which COX-2 gene knockout and specific COX-2 inhibitors are formed (15).

The presence of tumorigenic Ras has been associated with induction of COX-2 expression in intestinal and mammary epithelial cells and non-small cell lung cancer cell lines of H-ras transformed rats (16-18).
). To our knowledge, the association between tumorigenic Ras and COX-2 expression has not been investigated in vivo. Due to the high frequency of activating mutations in the K-ras gene in pancreatic tumors, we should be able to investigate the relationship between tumorigenic Ras and COX-2 expression in vivo. In this study, the level of COX-2 protein in primary human pancreatic adenocarcinoma was evaluated. The inventors further investigated whether COX-2 expression correlates with K-ras mutation status in pancreatic tumors and pancreatic cancer cell lines. Given the data demonstrating high levels of COX-2 protein in primary pancreatic tumors and cell lines, we found that the COX inhibitors sulindac, indomethacin and NS-398 cell growth in human pancreatic tumor cell lines. And the effect on prostaglandin E ₂ production was tested.

Cyclooxygenase-2 (COX-2) expression was upregulated in several human cancers and was also directly linked to carcinogenesis. To investigate the role of COX-2 in pancreatic cancer, we immunoblot COX-2 protein expression in primary human pancreatic adenocarcinoma (n = 23) and paired normal adjacent tissues (n = 11). Evaluated by analysis. COX-2 expression was found to be much higher in pancreatic tumor specimens than in normal pancreatic tissue. To investigate whether the high levels of COX-2 protein observed in pancreatic tumors correlate with the presence of tumorigenic K-ras, we examined K-ras in a subset of tumors and corresponding normal tissues. The mutation status was measured. The presence of oncogenic K-ras did not correlate with the level of COX-2 protein expressed in the pancreatic adenocarcinomas analyzed.
These observations were also confirmed in a panel of human pancreatic tumor cell lines. further,
In the pancreatic tumor cell line (BxPC-3) that expresses the highest levels of COX-2, COX-2 expression is
It was shown to be independent of Erk 1/2 Map kinase activation. The lack of correlation between COX-2 and tumorigenic K-ras expression indicates that Ras activation causes COX- in pancreatic tumor cells.
2 could not be sufficient to induce expression, and abnormal activation of signal pathways other than Ras resulted in COX-
2 suggesting that it may be necessary to upregulate expression. We also found that the COX inhibitors sulindac, indomethacin and NS-398
We report that it inhibited cell proliferation in both -2 positive (BxPC-3) and COX-2 negative (PaCa-2) pancreatic tumor cell lines. However, indomethacin and NS-3
The inhibition of cell proliferation by 98 was much greater in the BxPC-3 cell line than in the PaCa-2 cell line (P = 0.004 and P <0.001 respectively). Furthermore, these three COX inhibitors reduced the levels of prostaglandin E ₂ (PGE ₂ ) in the BxPC-3 cell line. Taken together, our data suggest that COX-2 plays an important role in pancreatic tumorigenesis and therefore may be a promising chemotherapeutic target for pancreatic cancer treatment.

Sulindac ((Z) -5-fluoro-2-methyl-1-[[4- (methylsulfinyl) phenyl]]
Methylene] -1H-indene-3-acetic acid) has the following chemical formula and is available as Clinoril® from Merck.

[0025]

[Chemical 1]

Sulindac is an indene anti-inflammatory agent shown in the acute and long-term relief of signs and symptoms of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, acute shoulder pain and acute gouty arthritis. Sulindac also has analgesic and antipyretic properties. Although the exact mechanism of its action is unknown, it is believed that sulfide metabolites may inhibit prostaglandin synthesis. Sulindac is absorbed approximately 90% when administered orally. Peak plasma levels are reached in fasting patients in about 2 hours and in 3-4 hours when administered with food. The average half-life of sulindac is 7.8 hours and the average half-life of sulfide metabolites is 16.4 hours. Sulindac is a functional class IV arthritis (incapacity, almost or completely bedridden, or wheelchair condition; little or no self-care), acute asthma attacks, urticaria suddenly caused by aspirin or other nonsteroidal anti-inflammatory drugs. Contraindicated in patients with rash or rhinitis and in drug-sensitive patients.

The synthesis of sulindac and its analogs (including metabolites and salts) are described, for example, in US Pat. Nos. 3,654,349 and 3,647,858 and Fed. Proc. , 31 , 377 (1
972); Drug. Metab. Disps. , 1 , 721 (1972).

For example, analogs of Sulindac include substituted indenylacetic acids of formula (I).

[0029]

[Chemical 2]

[0030]   In the formula,

Embedded image is an aryl or heteroaryl such as 1-3, N, O and

And / or a 5-, 6- or 7-membered heteroaryl ring containing S atoms; R ₁ may be hydrogen, lower (C ₁ -C ₄ ) alkyl or halogenated lower alkyl; R ₂ may be hydrogen or (C ₁ -C ₆ ) alkyl; R ₃ , R ₄ , R ₅ and R ₆ are each hydrogen, alkyl, (C ₂ -C ₅ ) acyloxy, alkoxy, nitro. , Amino, acylamino, alkylamino, dialkylamino, dialkylaminoalkyl, sufamyl, alkythio
), Mercapto, hydroxy, hydroxyalkyl, alkylsulfonyl, halogen, cyano, carboxyl, alkoxycarbonyl, carbamide, halogenoalkyl, cycloalkyl or cycloalkoxy; R ₇ may be alkylsulfinyl or alkylsulfonyl. Well; R ₈ may be hydrogen, halogen, hydroxy, alkoxy or haloalkyl; M is hydroxy, lower alkoxy, substituted lower alkoxy, amino, alkylamino, dialkylamino, N-morpholino, hydroxyalkylamino ,
It may be an OMe group in which polyhydroxyalkylamino, dialkylaminoalkylamino, aminoalkylamino and Me are cations; and pharmaceutically acceptable salts thereof.

Alkyl is preferably lower alkyl, ie (C ₁ -C ₄ ) alkyl.
The acyl is preferably (C ₂ -C ₄ ) acyl.

Other NSAIDs, including indomethacin and NS-398, also inhibit proliferation of pancreatic tumor cell lines as described below and can be used in the methods of the invention either alone or preferably in combination with sulindac.

Gemcitabine (Gemcitabine HCl or Gemzar®) is 2′-deoxy-2 ′, 2′-difluorocytidine-HCl (B-isomer), an antitumor nucleoside analogue. Can be administered by injection or infusion at a dose of about 500-4000 mg / m ² / week for up to 7 weeks / cycle for the treatment of localized or metastatic pancreatic cancer (adenocarcinoma of the pancreas). It can also be administered with other anti-cancer agents such as 5-FU, see PDR (53rd ed., 1999), pp. 1578-1582.

Pancreatic tumor cells Bx with Sulindac or NS-398 alone and in combination with gemcitabine
The effect of PC-3 and PaCa-2 on proliferation was examined. Treatment with this drug combination inhibited proliferation of both cell lines to a greater extent than either compound alone. It has been found that also the active ingredient of feverfew, which has anti-inflammatory properties, pasenolide also inhibits the proliferation of PaCa-2 cells and, when combined with gemcitabine, exhibits a greater inhibitory effect than either compound alone. Pasenolide treatment inhibited the DNA binding activity of NF- (B. These results suggest that anti-inflammatory drugs may enhance the efficacy of gemcitabine against pancreatic tumors.

The prophylactic or therapeutic dose of sulindac, its analogs or combinations thereof in the acute or chronic therapeutic management of cancer or pancreatic cancer depends on the solid tumor to be treated, the chemotherapeutic agent used or other anti-cancer therapies and administration. It depends on the stage of the cancer, such as the pathway. The dose and perhaps dose frequency, will also depend on the age, weight and response of the individual patient. In general, the total daily dose range for sulindac and its analogs for the conditions described herein is from about 50 mg to about 25 in single or divided doses.
It is 00 mg. Preferably, the daily dose should be from about 100 mg to about 1500 mg in single or divided doses, most preferably from about 150 to 500 mg per day. In managing the patient for treatment, the treatment may be initiated at a lower dose and increased with the patient's global response. It is further recommended that infants, children, patients over the age of 65 and patients with impaired renal or hepatic function be given a low dose initially and titrated based on overall response and blood levels. . It may be necessary to use dosages outside these limits in some cases. Moreover, the clinician or treating physician will know how and when to interrupt, adjust, or terminate the treatment depending on the response of the individual patient. The terms "effective amount" or "effective sensitizing amount" are included in the dosage and frequency schedule above.

Any suitable route of administration may be employed for providing the patient with an effective dosage of sulindac. For example, oral, rectal, parenteral (subcutaneous, intravenous, intramuscular), intrathecal, transdermal, etc. dosage forms may be used. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, patches and the like. Sulindac may be administered before, at the same time as, or after the application of chemotherapy, or continuously during the whole or one period of chemotherapy, ie daily. Sulindac may be combined with the same carrier or vehicle that is used to deliver the anti-cancer chemotherapeutic agent.

Thus, the compounds of the present invention may be administered systemically, eg orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft enveloped gelatin capsules, compressed into tablets, or incorporated directly into the food of the patient's diet. For oral therapeutic administration, the active compound may be used in the form of tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like, which may be taken in combination with one or more excipients. Such compositions and preparations should contain at least 0.1% of active compound. The proportion of the composition and formulation may, of course, be varied and may conveniently be between about 2% and about 60% by weight of the given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

Tablets, troches, pills, capsules and the like may include: Binders such as tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, alginic acid and the like; wetting agents such as magnesium stearate; and Sweetening agents such as sucrose, fructose, lactose or aspartame or flavoring agents such as peppermint, wintergreen oil or cherry flavor may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as vegetable oil or polyethylene glycol. A variety of other materials may be present as a coating or to modify the physical form of a solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound such as sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any substance used to prepare any unit dosage form must be pharmaceutically acceptable and substantially non-toxic in the amounts used. In addition, the active compound may be incorporated into sustained-release preparations or devices.

The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water and optionally mixed with a nontoxic surfactant. Dispersions can also be prepared with glycerol, liquid polyethylene glycols, triacetin and mixtures thereof and oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

A pharmaceutical dosage form suitable for injection or infusion is suitable for the extemporaneous preparation of sterile aqueous solutions or dispersions or sterile injectable or infusible solutions or dispersions, optionally with the active ingredient encapsulated in liposomes. It may be a sterile powder containing. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion solvent containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters and suitable mixtures thereof. For example, formation of liposomes, in the case of dispersions, maintenance of the required particle size, or use of surfactants can maintain suitable fluidity. Prevention of the action of microorganisms can be achieved by using various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol,
It can be carried out with sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example sugars, buffers or sodium chloride. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, by which the active ingredient and any additional desired ingredients present in the pre-sterilized solution are added. To produce powder.

Useful dosages of compounds of Sulindac and its analogs can be determined by comparing their in vitro activity with their in vivo activity in animal models. Methods of extrapolating effective doses in mice and other animals to humans are known in the art; see, eg, US Pat. No. 4,938,949.

The invention is illustrated with reference to the following detailed examples using the following materials and methods.

1. Patient Samples Indiana University Tissu
e Procurement Laboratory) and National Cancer Institute
Obtained a conserved organization from the Cooperative Human Tissue Network (CHTN). A total of 23 primary human pancreatic cancer specimens were analyzed in this study. A corresponding pair of normal adjacent tissues was obtained from 11 of the patients. Patients were selected on the basis of no prior history of chemotherapy. Tissue is surgically excised and frozen in liquid nitrogen within 1 hour, then-
Stored at 80 ° C. Paraffin sections were prepared from a subset of specimens. All tumor specimens used in this study were examined by a pathologist and classified as primary pancreatic adenocarcinoma. For this study, the Institutional Review Board (IRB)
) Approval was obtained.

2. Immunoblot RIPA lysis buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1 mM Na ₃ VO ₄ , 20 mM beta-glycerophosphate, 1 mM sodium fluoride, pepstatin ( Rapid homogenization with 1 μg / ml), aprotinin (20 μg / ml), and leupeptin (1 μg / ml) 13,00
Lysates were clarified by centrifugation at 0xg for 10 minutes, then boiled in sample buffer. The protein concentration was measured using a BCA protein quantification kit (Pierce). Equal amount of total protein in 10% gel (Novex) SDS-
It was decomposed by PAGE and transferred to Immobilon P membrane (MSI). Primary antibody COX-1 (C-20
, Santa Cruz Biotechnology, COX-2
(C20, Santa Cruz Biotechnology), p
21 ^ras (pan-ras Ab-3, Oncogene Science), actin (C-11, which recognizes a wide range of actin isoforms, Santa Cruz Biotechnology) or phosphate-specific Map kinase antibody(
Blots were probed using New England Biolabs as recommended by the manufacturer. ECL (Amersham)
Bands were visualized by and quantified by densitometry. The amount of COX-2 was expressed as a percentage of the positive control (3 μg cell lysate prepared from lipopolysaccharide (LPS) stimulated mouse macrophage cell line Raw 264.7). The specific recognition of COX-1 and COX-2 by the COX-1 and COX-2 antibodies, respectively, was confirmed by peptide inhibition experiments (
Data not shown).

3. Immunohistochemistry Using the avidin-biotin complex method (19), immunochemical tissue staining was performed on a 5 μm formalin-fixed paraffin-embedded section. A primary polyclonal antibody was used for evaluation of COX-2 expression (Oxford Biomedical Research, Inc., Oxford, MI; dilution 1: 100). 3,
3'-Diaminobenzidine (DAB) was used as the chromogen and 0.2% methyl green was used as the counterstain. Positive and negative controls were run in parallel with appropriate results.

4. K-ras mutation analysis Tissue lysis buffer (50 mM Tris pH 8.0, 100 mM EDTA
Genomic DNA was prepared by overnight incubation at 55 ° C. with 100 mM NaCl, 1% SDS, 0.5 mg / ml proteinase K). RNAse (0.1 mg / ml) was added, and 37
Incubation was continued for 2 hours at ° C. The sample was then extracted with phenol, phenol: chloroform and ethanol precipitated. Resuspended genomic DNA (0.5
μg) of K-ras exon 1 was amplified by PCR (5 'primer = 5'-ATGACTGAAT
ATAAACTTGT-3 '(SEQ ID NO: 1); 3'primer = 5'-CTCTATTGTTGGATCATATT-
3 ') (SEQ ID NO: 2) (20). A mutation at K-ras codon 12 of the PCR amplification product was detected by dot blot hybridization using a K-ras mutation-specific oligonucleotide (Oncogene Research Products) (21). The K-ras codon was determined by sequencing the purified PCR amplification product.
Mutations at 13 were detected.

5. Statistical Analysis A nonparametric signed rank test determined the presence of a statistically significant increase in COX-2 protein between cancer specimens and corresponding normal adjacent tissues. BxPC in the presence of COX inhibitors using two-way analysis of variance (ANOVA)
The difference in the average value of cell growth rates between the -3 cell line and the PaCa-2 cell line was examined.

6. Cell line Human pancreatic tumor cell line (AsPC-1, BxPC-3, Capan-1, Capan-2, HPAF
-II, Hs766T, PaCa-2 and PANC-1) American Type Culture Collection (Ame
rican Type Culture Collection (ATCC, Rockville, MD)) and cultured as recommended. Hamster pancreatic cell lines (D27, D27 / K-ras, B12 / 13) were cultured as described above (22,23). For inhibitor studies, BxPC-3 cells were treated with Map Kinase Kinase (MEK) inhibitor PD98059 (40 μM) or DMSO for 10 hours. Lysates were prepared and analyzed as above.

7. Cell Proliferation Cells were double plated in 6-well plates in the presence of DMSO, Sulindac (Sigma), Indomethacin (Sigma), or NS-398 (Biomol). . On day 3 cells were trypsinized, stained with trypan blue and counted using a hemocytometer. Cell proliferation was measured by averaging the cell numbers and expressed as a percentage of the DMSO control sample cell number.

8. Prostaglandin E ₂ assay cells were plated in 12 well plates. On day 3, the medium was aspirated and replaced with serum-free medium with 15 μM arachidonic acid and after 1 hour PGE ₂ was determined by enzyme immunoassay (Biotrak, Amersham) as recommended by the manufacturer. The culture supernatant was assayed.
The amount of PGE ₂ produced was normalized to protein concentration. Cells were plated in 12-well plates in the presence of COX inhibitors for 24 hours, lysed and quantified by enzyme immunoassay (Biotrak) as recommended by the manufacturer to determine intracellular PGE ₂ The level was measured. To measure intracellular PGE ₂ levels, cells were not preincubated with arachidonic acid prior to the assay.

9. Abbreviations COX, cyclooxygenase; Erk 1/2 MAP kinase (Erk, MAPK)
, Extracellular signal-regulated kinase 1 and 2 mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; NSAIDS, nonsteroidal anti-inflammatory drug; PGE ₂ , prostaglandin E _2, PMA, phorbol 12-myristate 13 -acetate. Example 1 COX-2 expression in human pancreatic adenocarcinoma Primary human pancreatic adenocarcinoma (n = 23) and paired normal adjacent tissues (n = 11) were examined for COX-2 protein expression by immunoblot analysis. Representative immunoblots of lysates prepared from 6 patients, including 3 with paired normal adjacent tissues, are shown in FIG. An undetectable level of COX-2 protein was observed in each normal sample. In contrast, COX-2 protein expression in pancreatic tumor tissue ranged from undetectable (Sample # 21) to low / moderate (Samples # 12, 14, 20) and high levels (Samples # 9, 22). It was COX-1 protein was observed in both pancreatic tumors and normal tissues, but the level of expression was variable and not consistently high in tumor specimens (FIG. 1). Similar levels of p21 ^ras and actin expression were found in both tumors and corresponding normal tissues (
(Fig. 1).

The COX-2 expression rate was measured for all tissue samples by performing densitometric analysis and calculated based on the positive control set at 100% (Table 1 and the graph in FIG. 2). The positive control for this study was the lipopolysaccharide (LPS) -stimulated mouse macrophage cell line Raw 264.7, which was previously shown to induce COX-2 expression (22). Extensive COX-2 expression was observed in pancreatic adenocarcinoma (0-93%), whereas COX-2 expression was very narrow in normal tissues (0-4.3%). Both mean and median COX-2 expression were higher in tumor samples, suggesting higher COX-2 expression in pancreatic adenocarcinomas than in normal tissues. COX-2 between pancreatic tumor and corresponding normal tissue
The difference in expression was judged to be statistically significant (P = 0.004) (Fig. 2, inset in the figure). COX-2 positive and negative samples were defined as samples with a COX-2 expression rate higher or lower than 5%, respectively. This corresponds closely with visual detection on immunoblots. By these criteria, 6 out of 11 tumor samples in the tissue set (55%
) Was COX-2 positive. Similarly, 13 of the 23 tumor samples analyzed (5
6%) were COX-2 positive, in contrast all normal tissue samples (n = 11) were COX-2 negative.

Immunohistochemical staining of pancreatic tumor specimens showed that COX-2 expression was restricted to carcinoma cells and was undetectable in the stromal compartment of the tumor. Example 2 COX-2 Expression and K-ras Mutations in Pancreatic Tumors and Cell Lines To determine whether COX-2 expression levels are correlated with tumor K-ras mutation status, genomic DNA from a subset of tissue specimens. Allele-specific hybridization of the isolated and PCR-amplified K-ras exon 1 product resulted in Kr at codon 12
The presence of as mutations was screened. Subsequently, the samples without mutation at codon 12 were sequenced to examine the presence of K-ras mutation at codon 13 (Table 1). All normal tissues analyzed were wild type at codon 12 (GGT = Gly) and codon 13 (GGC = Gly). Of the 13 pancreatic cancer samples analyzed, one sample had a mutation at codon 13 and 10 samples had a mutation at codon 12, corresponding to a K-ras mutation frequency of 85%. There did not appear to be a direct correlation between the K-ras mutation and the extent of COX-2 protein expression. For example, expressing high levels of COX-2 protein,
Some samples had a mutation in as (ie, tumor samples # 9, 16 and 22), while other samples with mutated K-ras expressed little or no COX-2 protein (ie, Tumor samples # 3, 17, 18, 19 and 21).

Similarly, no direct correlation was observed between activated K-ras expression and COX-2 in a panel of human pancreatic adenocarcinoma cell lines with known K-ras mutation status. (twenty five
, 26). Both the frequency and variability of the amount of COX-2 expressed in the pancreatic tumor cell lines reflected the results of our study in primary pancreatic adenocarcinoma. Of the eight human pancreatic tumor cell lines analyzed, only three of the seven cell lines expressing tumorigenic K-ras had detectable levels of COX-2 protein (Capan-1, Capan-2 And HPAF-
II) is shown (FIG. 4A). High levels of COX-2 protein were also observed in the wild-type K-ras-expressing cell line BxPC-3, which shows high levels of Ras-independent Raf activity (26). Map
Treatment of the BxPC-3 cell line with the kinase kinase (MEK) inhibitor PD98059 significantly reduced the levels of active phosphorylated Erk1 / 2 Map kinase, but did not affect the amount of COX-2 synthesized, and COX- 2 suggests that expression is independent of Erk1 / 2 activation (
FIG. 4B). Taken together, our results suggest that activation of the Ras pathway is not sufficient to mediate the upregulation of COX-2 in pancreatic tumor cells. We also compared the levels of COX-2 expression in three hamster pancreatic cell lines. The D27 / K-ras and B12 / 13 transformed cell lines were derived from the non-malignant hamster pancreatic dust cell line D27 by transfection with oncogenic K-ras or in vitro treatment with chemical carcinogens, respectively. (22, 23). D27 / K-
Both the ras and B12 / 13 cell lines harbored tumorigenic K-ras, but only B12 / 13 cells showed higher levels of COX-2 protein compared to the D27 parental line (FIG. 4C). These results confirm our conclusions that Ras activation alone is not sufficient to upregulate COX-2 expression in pancreatic cancer cells, and further events that occur after exposure to chemical carcinogens Suggests that it may be necessary.

To determine if it was possible to induce COX-2 expression in human pancreatic cancer cell lines, four cell lines were serum starved and subsequently treated with 10% FCS for various times (FIG. 4D). . COX-2 was still detectable after serum starvation in COX-2-positive BXPC-3 and Capan-1 cell lines, albeit at lower levels than in exponentially growing cells. . After FCS stimulation, COX-2 expression could be induced in these cell lines. In contrast, in both the COX-2 negative cell lines AsPC-1 and PaCa-2,
COX-2 expression could not be induced by serum treatment. Under these treatment conditions, Erk1 / 2 was activated (unpublished observation) and Erk1 / 2 activation was not sufficient to induce COX-2 expression in COX-2-negative pancreatic tumor cells. Also shown here. Similar results were observed when the cell lines were treated with the tumor promoting agent PMA (
Unpublished observations). Example 3 Treatment of Pancreatic Tumor Cell Lines with Cyclooxygenase Inhibitors COX-2-Positive Human Pancreatic Tumor Cell Lines BxPC-3 and COX-2-Negative Cell Lines PaCa-2 CO
Treated with X inhibitors Sulindac, indomethacin, or NS-398. Sulindac and indomethacin are non-selective COX inhibitors that inhibit both COX-1 and COX-2 (27), and NS-398 is a more specific inhibitor of COX-2 (28). After treatment for 3 days, the effect of COX inhibitors on cell proliferation was measured (Fig. 5). All three inhibitors were found to dose dependently suppress cell proliferation in both pancreatic tumor cell lines. However, indomethacin and NS-398 are more sensitive to COX-2 than the PaCa-2 cell line.
It was found to inhibit cell proliferation to a greater extent in the expressing cell line BxPC-3 (P = 0.004 and P <0.001 respectively). No significant difference in inhibition of cell proliferation was observed in the two cell lines treated with Sulindac (P = 0.333).

To assess the functional activity of COX-2 in human pancreatic tumor cell lines, prostaglandin E ₂ (PGE ₂ ) production was measured by enzyme immunoassay (FIG. 6A). PGE ₂ production
It was increased in BxPC-3, Capan-1, Capan-2 and HPAF-II cell lines and correlated with increased levels of COX-2 expressed in these cell lines. In contrast, levels of PGE ₂ that were barely detectable in the COX-2 negative pancreatic cell line were detected. To determine the effect of COX inhibitors on PGE ₂ production, COx-2-positive cell line BxPC-3 was added to two different concentrations of sulindac (100, 250 μM), indomethacin (10, 100 μM), or NS-. 398 (1
After incubating at 0, 50 μM) for 24 hours, intracellular PGE ₂ levels were measured (FIG. 6B). Since intracellular levels should be more sensitive to changes in response to inhibitor treatment, intracellular PGE ₂ was measured rather than PGE ₂ secreted into growth medium. Various COX
Two concentrations of inhibitor is evaluated by the cell proliferation assay (FIG. 5), the concentration of the higher corresponds to an IC ₅₀ value of COX inhibitors. In contrast, both lower concentrations of NS-398 and indomethacin inhibited PGE ₂ production by 75% and 95%, respectively, whereas the lower concentration of sulindac did not affect PgE ₂ levels. Still, the higher one
All three inhibitors at IC ₅₀ concentrations substantially suppressed intracellular PGE ₂ levels in the BxPC-3 cell line, ie 86% to 98%. These data suggest that COX inhibitors may exert their effect, at least in part, on the COX-2 positive BxPC-3 cell line by reducing PGE ₂ production. However, in COX-2 negative cell lines, the effects of COX inhibitors may be mediated by a mechanism independent of COX and PGE ₂ . Example 4 Synergistic effect of the combination of sulindac and gemcitabine BxPC-3 or PaCa-2 cells were plated on day 0. Sulindac (sul, microM), gemcitabine (Gem, nM), or a combination of compounds as shown in Figures 6-7 was added the next day (day 1). Cells were trypsinized on day 3 and counted manually. Two points were assigned for each experiment. The cell number was expressed as a viability based on the number of cells present in the medium + solvent control sample, which was set to 100%. The viability of samples of Sulindac alone is also shown (Gem, 0 nM).

These data indicate that the combination of sulindac and gemcitabine is more effective than either compound alone in pancreatic tumor cells.

In resting cells, COX-2 expression is usually undetectable but can be rapidly induced by mitogenic stimulants and anti-inflammatory agents (5,6,29). Recent studies have shown that COX-2 expression is upregulated in various human cancers including colon, lung, stomach, pancreas and esophagus (7-11). In this study, high levels of COX-2 protein were expressed in human pancreatic tumors, whereas increased levels of COX-2 protein were associated with pancreatic neoplasia, whereas levels were almost undetectable in paired normal pancreatic tissues. We report that it suggests a correlation. Our results corroborate a recent report showing COX-2 RNA and protein up-regulation in pancreatic tumors and COX-2 localization in malignant epithelial cells (11). Earlier studies have shown that type II phospholipase A2 expression, which catalyzes the release of arachidonic acid from membrane phospholipids, is higher in ductal adenocarcinoma of the pancreas than in normal pancreatic tissue (30).
. Furthermore, hamster N-nitrosobis (2-oxopropyl) amine (BOP) -initiated pancreatic tumor development was inhibited by the administration of two prostaglandin synthesis inhibitors, phenylbutazone and indomethacin (31). ). Taken together with our observations in vivo and in vitro, these studies show that increased prostaglandin production due to increased expression of COX-2 is a key event in the multi-step progression towards pancreatic tumorigenesis. Recent studies have suggested that Ras activation may induce COX-2 expression in several systems. Activated tumorigenic H-ras was inducibly expressed in Rat-1 fibroblasts with a concomitant increase in COX-2 expression and PGE ₂ production (32
). In this particular cell line, PD98059, a specific mitogen-activated protein kinase kinase (MEK) inhibitor, was found to suppress COX-2 induction by tumorigenic Ras, leading to Ras-dependent induction of COX-2. It suggests that Erk1 / 2 activation is required. Similarly, COX-2 and prostaglandin E ₂ in the high level is detected at Ras transformed mammary epithelial cells (C57 / MG) cells (17). In human non-small cell lung cancer (NSCLC) cell lines expressing tumorigenic K-Ras, constitutively high expression of cytosolic phospholipase A ₂ and COX-2 compared to NSCLC lines without Ras mutations An increase in PGE ₂ production was mediated (18). In this study, we addressed the question of whether tumorigenic K-Ras expression correlates with increased COX-2 expression in primary human pancreatic adenocarcinomas. We found that the presence of a mutation at codon 12 or 13 of the K-ras gene resulted in detectable levels of C
It was found that it did not correlate with the expression of OX-2 protein. The lack of COX-2 expression in a subset of tumors with tumorigenic Ras may be due to down-regulation of Erk1 / 2 activity in pancreatic carcinoma (26). In addition, two pancreatic tumor samples that showed high levels of activated Erk1 / 2 (Samples # 4 and 21, data not shown) also detected low levels of COX-2 in this study, COX only by Erk1 / 2 activation
Suggesting that it is not sufficient to induce -2 expression. The results of these studies suggest that in the tumor environment, the presence of tumorigenic K-ras does not directly lead to increased COX-2 expression in pancreatic cancer.

Similar conclusions were reached in an analysis of pancreatic cancer cell lines that were examined because they were representative of a population of cells that were homogenous to the heterogeneous primary tumor tissue. Despite activation of the K-ras mutation in 7 of the 8 cell lines, only 3 of the cell lines with the mutant K-ras expressed detectable amounts of COX-2 protein Met. COX-2 expression was also high in the wild-type K-ras BxPC-3 cell line with high levels of active Raf (26).
. COX-2 was constitutively expressed in four COX-2 positive cell lines, whereas in contrast COX-2 negative cell lines could not induce COX-2 expression by serum or PMA, It was suggested that COX-2 expression was interfered with. COX-2 expression was not reduced in the BxPC-3 cell line after treatment with the MEK inhibitor PD98059, and COX-2 induction was Ras in this cell line.
It was suggested to be unrelated to pathway activation. Differential expression of COX-2 protein in transformed hamster pancreatic cell lines generated by either transfection with tumorigenic Ras or treatment with chemical carcinogens results in Ras activation.
It was further shown that it was not sufficient to mediate the induction of COX-2 expression. In addition to Ras, activation of other signaling pathways may cooperate to determine the extent of COX-2 expression in cancer cells. Such a pathway may involve the p38 mitogen-activated protein kinase reported to regulate induction of COX-2 in lipopolysaccharide-treated human monocytes (33). Furthermore, in human vascular endothelial cells, NF- (B p65 transcription factor was found to mediate the induction of COX-2 in response to hypoxia (34). The specific pathways that are activated may depend on the cell type and stimulus. To determine which signaling pathway functions in pancreatic tumor cells Requires experimentation.

In both COX-2 positive (BxPC-3) and negative (PaCa-2) cell lines, cell growth was inhibited by treatment with the COX inhibitors sulindac, indomethacin or NS-398.
However, the cell line BxPC-3 was compared to indomethacin and NS compared to the PaCa-2 cell line.
Very high sensitivity to growth inhibition by -398 suggests that these two compounds are more selective for COX-2 expressing cells than sulindac. In addition, Bx
In the PC-3 cell line, IC ₅₀ concentrations of COX inhibitors substantially reduced intracellular PGE ₂ levels. There was no detectable PGE _{2 in} non-COX-2 expressing cell lines. These data, COX inhibitors, suggesting that it exerts its inhibitory effect by either pancreatic tumor cell line of COX / PGE ₂ dependent and -independent pathways. NS-398 is COX-2
It has been previously shown to inhibit cell proliferation by inducing apoptosis independently of (35). Similarly, sulindac sulfone, a metabolite of sulindac that does not inhibit COX activity, was found to inhibit colon carcinogenesis without reducing prostaglandin levels in a rat model (36). further,
Cyclooxygenase null embryo fibrobla
In sts), NSAID transformation and anti-cell proliferation and anti-tumor effects were recently shown to be independent of COX expression (37).

The detection of high levels of COX-2 in various human cancers and the chemopreventive effect of NSAIDs in colon cancer has shown that COX-2 has an important role in the carcinogenic phenomenon. The biological consequences of COX-2 upregulation have been reported, including inhibition of apoptosis (13), increased metastatic potential (12) and accelerated angiogenesis (14). These events may contribute to cell transformation and tumor progression. COX-2 expression
COX-2 was identified as a new target for chemotherapy because it was significantly higher in 55% of the patients' pancreatic tumor samples analyzed. These results showing the ability of COX inhibitors to inhibit pancreatic tumor cell growth and PGE ₂ production in vitro indicate that NSAIDs may be effective in treating patients with pancreatic cancer who currently have few treatment options. There is. C
OX-2 expression is also useful as a prognostic or diagnostic tool. References: 1. Landis et al., CA Cancer J. Clin. , 48 , 6-29 (1998). 2. Almoguera et al., Cell , 53 , 549-54 (1988). 3. Toon ( Thun), MJ, Cancer Metastasis Rev. , 13 , 269-77 (1994).
4. Giardiello et al. , Eur. J. Cancer , 31A , 1071-6 (1995). 5. DeWitt et al . , Arch. Biochem. Biophys. , 306 , 94-102 (1993).
. 6. Hamasaki et al . , Arch. Biochem. Biophys. , 304 , 226-34 (1993). 7. Eberhart et al., Gastroenterology , 107 , 1183-8 (1994). 8. Ristimaki et al. Cancer Res. , 57 , 1276-80 (1997). 9. Zimmermann et al., Cancer Res. , 59 , 198-204 (1999). 10. Wolff et al., Cancer Res. , 58 , 4997. -5001 (1998). 11. Tucker et al., Cancer Res. , 59 , 987-90 (1999). 12. Tsuji et al., Proc. Natl. Acad. Sci. USA , 94 , 3336-40 (1997). . 13. Tsuji et al., Cell , 83 , 493-501 (1995). 14. Tsuji et al., Cell , 93 , 705-16 (1998). 15. Oshima et al., Cell , 87 , 803-9 (1996) ). 16. Sheng et al., Gastroenterology , 113 , 1883-91 (1997). 17. Subbaramaiah et al., Cancer Res. , 56 , 4424-9 (1996). 18. Heasley et al. J. Biol. Chem. , 272 , 14501-4 (1997). 19. Hsu et al., J. Histochem. Cytochem. , 29 , 577-80 (1981). 20. Sumi et al., Pancreas , 9 , 657. -61 (1994). 21. Koni Shi et al., Cancer , 69 , 2293-99 (1992). 22. Mangold et al., Carcinogenesis , 15 , 1979-84 (1994). 23. Stayrook et al., Anticancer Res. , 18 , 823 -8 (1998). 24. Riese et al., J. Leukoc. Biol. , 55 , 476-82 (1994). 25. Berrozpe et al . , Int. J. Cancer , 58 , 185-91 ( 1994). 26. Yip-Schneider et al . , Int. J. Oncol. , 15 , 271-9 (
1999). 27. Meade et al., J. Biol. Chem. , 268 , 6610-4 (1993). 28. Futaki et al., Prostaglandins , 47 , 55-9 (1994). 29. Lee et al. , J. Biol. Chem. , 267 , 25934-8 (1992). 30. Kiyohara et al . , Int. J. Pancreatol. , 13 , 49-57 (1993). 31. Takahashi et al., Carcinogenesis , 11 , 393-5 (1990). 32. Sheng et al., J. Biol. Chem. , 273 , 22120-7 (1998). 33. Dean et al., J. Biol. Chem. , 274 , 264-9 (1999). ). 34. Schmedtje et al., J. Biol. Chem. , 272 , 601-8 (1997). 35. Elder et al . , Clin. Cancer Res. , 3 , 1679-83 (1997). 36. . Piazza et al., Cancer Res. , 57 , 2909-15 (1997). 37. Zhang et al., J. Exp. Med. , 190 , 451-59 (1999).

[0064]

[Table 1]

A Total of 23 pancreatic adenocarcinomas were obtained and numbered as indicated. Paired normal adjacent tissues (N) were also obtained from 11 patients. b COX-2 immunoblot was subjected to densitometric analysis to determine the COX-2 expression rate, which was expressed using the positive control set at 100% as a standard. c Slides prepared from paraffin sections were visualized by hematoxylin / eosin staining to determine the percentage of cancer. d K-ras mutation status at codon 12 was determined by allele-specific hybridization of K-ras exon 1 PCR amplification products generated from genomic DNA isolated from patient samples. Codon 13 by sequencing the K-ras exon 1 PCR amplification product
Mutations were measured.

[Sequence list]

[Brief description of drawings]

FIG. 1 Photograph of a representative immunoblot of pancreatic adenocarcinoma and paired normal tissues.

FIG. 2 is a diagram showing the COX-2 expression rate in patient samples.

FIG. 3 shows the expression of COX-2 in a pancreatic tumor cell line. 3A is COX-2 expression in human pancreatic cell line detected by immunoblot analysis, 3B is cell line BxP
Expression levels of activated MAP kinase and COX-2 after treatment of C-3 with the MEK inhibitor PD98059 or DMSO for 10 hours, 3C comparing COX-2 expression in 3 hamster pancreatic cell lines, 3D COX-. 2 is a diagram showing the inducibility of expression.

FIG. 4 shows the effect of COX inhibitors on the proliferation of pancreatic tumor cell lines.

FIG. 5 shows the production of prostaglandin E ₂ . 5A shows the effect of COX inhibitors on PGE ₂ levels in pancreatic tumor cell lines, and 5B shows the production of PGE ₂ .

FIG. 6 is a graph showing the effect of the combination of sulindac and gemcitabine on the proliferation of pancreatic tumor cell line BxPC.

FIG. 7 is a graph showing the effect of the combination of sulindac and gemcitabine on the proliferation of pancreatic tumor cell line PaCa-2.

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] October 9, 2001 (2001.10.9)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE, TR), OA (BF , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, G M, KE, LS, MW, MZ, SD, SL, SZ, TZ , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, B Z, CA, CH, CN, CR, CU, CZ, DE, DK , DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, J P, KE, KG, KP, KR, KZ, LC, LK, LR , LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, R O, RU, SD, SE, SG, SI, SK, SL, TJ , TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (71) Applicant Sweeney, Christopher, Je             I.             46202 Indiana,             Indianapolis, West Walna             Street, Apartment Bi             -. 725 (71) Applicant Ip-Schneider, Michelle, Tee             -.             46033 Indiana, United States of America,             Carmel, Red Oak Lane 75 (71) Applicant Crowell, Pamela, El.             46033 Indiana, United States of America,             Carmel, Charing Crossroad             12715 (72) Inventor Marshall, Mark, Stephen             46033 Indiana, United States of America,             Carmel and spruce coat 1519 (72) Inventor Sweeney, Christopher, J.             I.             46202 Indiana,             Indianapolis, West Walna             Street, Apartment Bi             -. 725 (72) Inventor Ip-Schneider, Michelle, Tee             -.             46033 Indiana, United States of America,             Carmel, Red Oak Lane 75 (72) Inventor Crowell, Pamela, El.             46033 Indiana, United States of America,             Carmel, Charing Crossroad             12715 F term (reference) 4C084 AA17 MA52 NA14 ZB261                 4C086 AA01 AA02 EA17 MA01 MA04                       MA52 ZB26                 4C206 AA01 AA02 DA23 MA01 MA04                       MA72 NA14 ZB26

Claims (11)

[Claims] 1. A method of reducing the viability of pancreatic cancer cells, comprising contacting the cancer cells with an effective amount of NSAIDs. 2. A method of sensitizing a mammalian pancreatic cancer cell to a chemotherapeutic agent comprising contacting the cell with an effective sensitizing amount of NSAID. 3. The method according to claim 1 or 2, wherein the NSAID is a COX-2 inhibitor sulindac or an analog thereof. 4. The cancer cell of the mammal is a human cancer cell.
The method described in. 5. The method of claim 3, wherein the sulindac or analog thereof is administered to a human cancer patient. 6. The method of claim 5, wherein the cancer patient is being treated with a chemotherapeutic agent. 7. The method of claim 6, wherein the chemotherapeutic agent is gemcitabine. 8. The method of claim 3, wherein the sulindac or analog thereof is administered orally. 9. (a) Isolating a first portion of pancreatic cancer cells from a human pancreatic cancer patient; (b) measuring their viability; (c) sulindac or an analogue thereof. Administering to the patient; (d) isolating a second portion of pancreatic cancer cells from the patient; (e) measuring viability of the second portion of pancreatic cancer cells; (F) comparing the viability measured in step (e) with the viability measured in step (b), wherein the decrease in viability in step (e) causes the cells to become sensitive to the chemotherapeutic agent. A method of assessing the ability of a COX-2 inhibitor, sulindac, or an analogue thereof to sensitize pancreatic cancer cells to a chemotherapeutic agent, which has been shown to be produced. 10. The method of claim 9, wherein steps (b) and (e) are performed in the presence of the chemotherapeutic agent. 11. The method according to claim 10, wherein the chemotherapeutic agent is gemcitabine and / or 5-FU.