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Clinical Cancer Research Vol. 5, 855-863, April 1999
© 1999 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Evaluation of 5-Aminosalicylic Acid (5-ASA) for Cancer Chemoprevention: Lack of Efficacy against Nascent Adenomatous Polyps in the ApcMin Mouse1

Steve R. Ritland, Jonathan A. Leighton, Rhoda Elison Hirsch, Jason D. Morrow, Amy L. Weaver and Sandra J. Gendler2

Departments of Biochemistry and Molecular Biology [S. R. R., S. J. G.], Gastroenterology [J. A. L.], and Biostatistics [A. L. W.], Mayo Clinic Scottsdale, Scottsdale, Arizona 85259; Departments of Medicine (Division of Hematology) and Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461 [R. E. H.]; and Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232 [J. D. M.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent experimental and epidemiological evidence suggests that nonsteroidal anti-inflammatory drugs (NSAIDs) are effective in the prevention of colorectal cancer. However, the toxicity associated with the long-term use of most classical NSAIDs has limited their usefulness for the purpose of cancer chemoprevention. Inflammatory bowel disease (IBD) patients, in particular, are sensitive to the adverse side effects of NSAIDs, and these patients also have an increased risk for the development of intestinal cancer. 5-Aminosalicylic acid (5-ASA) is an anti-inflammatory drug commonly used in the treatment of IBD and may provide protection against the development of colorectal cancer in these patients. To directly evaluate the ability of 5-ASA to suppress intestinal tumors, we studied several formulations of 5-ASA (free acid, sulfasalazine, and Pentasa) at multiple oral dosage levels [500, 2400, 4800, and 9600 parts/million (ppm)] in the adenomatous polyposis coli (Apc) mouse model of multiple intestinal neoplasia (Min). Although the ApcMin mouse is not a model of colitis-associated neoplasia, it is, nonetheless, a useful model for assessing the ability of anti-inflammatory agents to prevent tumor formation in a genetically preinitiated population of cells. We used a study design in which drug was provided ad libitum through the diet beginning at the time of weaning (28 days of age) until 100 days of age. We included 200 ppm of piroxicam and 160 ppm of sulindac as positive controls, and the negative control was AIN-93G diet alone. Treatment with either piroxicam or sulindac produced statistically significant reductions in intestinal tumor multiplicity (95% and 83% reductions in tumor number, respectively; P < 0.001 versus controls). By contrast, none of the 5-ASA drug formulations or dosage levels produced consistent dose-progressive changes in polyp number, distribution, or size, despite high luminal and serum concentrations of 5-ASA and its primary metabolite N-acetyl-5-ASA. Thus, 5-ASA does not seem to possess direct chemosuppressive activity against the development of nascent intestinal adenomas in the ApcMin mouse. However, because intestinal tumor development in the ApcMin mouse is driven by a germline mutation in the Apc gene rather than by chronic inflammation, we caution that these findings do not definitively exclude the possibility that 5-ASA may exert a chemopreventive effect in human IBD patients.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The term chemoprevention refers to the use of natural or synthetic chemical agents to reverse, suppress, or delay the process of carcinogenesis (1) . Chemoprevention may be a particularly useful strategy in the management of patients at increased risk for the development of specific cancers based on inborn genetic susceptibility, the presence of cancer-associated disease, or other known risk factors. Successful implementation of chemoprevention will rely on the accurate identification of high-risk patients and the development of safe and effective drugs suitable for use in these specific populations.

IBD,3 including Crohn’s disease and ulcerative colitis, is a group of chronic inflammatory conditions known to be associated with an increased risk for the development of intestinal cancer (reviewed in Ref. 2 ). IBD patients have a lifetime relative risk of 3–30-fold for the development of colorectal cancer, influenced in part by the duration and anatomical extent of the disease (3 , 4) . Cancer prevention has become an increasingly important consideration in IBD because therapies for the other symptoms of colitis have improved. Present cancer management techniques in IBD patients include surveillance colonoscopy and/or colectomy (5) . Because of the risk, expense, and sampling error of endoscopic surveillance, this is not an ideal approach to cancer control. Therefore, the development of safe and effective chemopreventive measures for reducing the risk of colorectal cancer would be of substantial benefit to IBD patients.

The chemopreventive efficacy of NSAIDs against intestinal tumors has been well established. Many studies over the past decade have provided compelling evidence that metabolism of arachidonic acid through the COX pathway is an important biochemical process in the growth and survival of intestinal tumor cells (reviewed in Ref. 6 ). Indeed, specific pharmacological inhibition or genetic ablation of the inducible form of COX, COX-2, can prevent intestinal tumors from developing and can even cause regression of existing adenomatous polyps in both humans and rodents (7 , 8) . It has been postulated that this antitumor effect is mediated through reduction in PG production, most notably PGE2, which is frequently overexpressed in intestinal tumor tissues (9 , 10) . More recent studies have indicated that some NSAIDs may exert their chemoprotective effect through a PG-independent apoptotic mechanism (11, 12, 13) .

Although COX-inhibiting NSAIDs have been shown to be effective in preventing the development of intestinal tumors in both animals and humans, the long-term use of these drugs is associated with significant toxicity (reviewed in Ref. 14 ). Because NSAIDs have been reported to specifically aggravate the symptoms of colitis (15) , their sustained use for the purpose of cancer chemoprevention is relatively contraindicated in IBD patients. Thus, there is a need to identify alternative chemopreventive agents that are more appropriate for use in IBD patients as well as other NSAID-intolerant, high-risk cancer groups.

One candidate chemoprevention drug that has specific relevance to IBD patients is 5-ASA (mesalamine), an anti-inflammatory drug that has been used extensively in the treatment of IBD for more than 50 years and is well tolerated by most patients (16 , 17) . Several retrospective correlative studies have suggested that the long-term use of 5-ASA in IBD patients may significantly reduce the risk for the development of colorectal cancer (18 , 19) . In support of this hypothesis, several small preclinical studies using the 5-ASA formulations olsalazine and balsalazide in azoxymethane-induced rat colon tumors and in the ApcMin mouse, respectively, have reported a chemoprotective benefit for 5-ASA agents (20 , 21) . In contrast, several other studies have failed to show a significant antiproliferative effect of 5-ASA against HT-29, SW480, and DLD-1 colon cancer cells in culture (22) or against azoxymethane-induced rat colon tumors (23) . Finally, one prospective study in the dimethylhydrazine rat model suggested that 5-ASA may have a dose-dependent effect on intestinal tumor development, with some concentrations of the drug producing a cocarcinogenic effect (24) .

Many of the 5-ASA formulations presently in clinical use are specifically designed to minimize systemic absorption and to achieve optimum delivery of the biologically active 5-ASA moiety to the distal small intestine and colon. Thus, relatively high concentrations of free 5-ASA can be achieved in the intestinal lumen without producing systemic exposure and subsequent toxicity. This type of target tissue selectivity is a desirable feature for chemopreventive agents and is a significant advantage when considering drug delivery methods to the mucosal surface of the gut. 5-ASA is known to possess a variety of pharmacological activities, including modulation of eicosanoid metabolism through both the COX and lipoxygenase pathways, modulation of immune cell activity, and antioxidant activity (reviewed in Ref. 25 ). However, despite the long clinical and experimental experience with this drug, the therapeutically relevant mechanism(s) of action for 5-ASA in the treatment of colitis remains unknown.

To further explore the potential of 5-ASA for the prevention of intestinal tumors in a controlled, preclinical trial, we evaluated three formulations of 5-ASA (free acid, Pentasa, and SASP) at multiple oral dosage levels (500, 2400, 4800, and 9600 ppm) in the ApcMin mouse. The ApcMin mouse is a genetic model of intestinal neoplasia and harbors a germline mutation at codon 850 of the Apc gene (26 , 27) . Inbred C57BL6/J-ApcMin mice develop multiple spontaneous intestinal adenomas throughout the intestinal tract (particularly in the small bowel) by 100 days of age, and these tumors rarely become invasive or metastatic. C57BL6/J-ApcMin mice have an average life span of 130–150 days of age. Because the ApcMin tumor model has genetic relevancy to human polyposis (including the human familial adenomatous polyposis syndrome), it has served as a useful experimental tool to determine the in vivo efficacy of candidate chemopreventive agents (reviewed in Ref. 28 ). Although the ApcMin mouse is not a model of colitis, it, nonetheless, shares a degree of genetic similarity with colitis-associated intestinal cancers, which frequently show APC mutations as well (29, 30, 31) . In the present study, we measured tumor multiplicity, size, and distribution as experimental end points and we included the NSAIDs sulindac and piroxicam as positive controls.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice and Drug Treatment Protocol.
Inbred B6-ApcMin mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The ApcMin system is described in detail elsewhere (reviewed in Ref. 32 ). Mice were screened for the Min/+ genotype using a previously described PCR assay (33) ; they were systematically assigned to drug treatment and control groups to normalize the distribution of males and females and to avoid the clustering of mice from a single litter. Animals were housed in an American Association for the Accreditation of Laboratory Animal Care-approved specific pathogen-free facility at a density of three mice/cage. Mice were monitored for health every 3 days and were weighed weekly. Food and water were freely available at all times. Beginning at the time of weaning (28 days of age) until 100 days of age, two different methods of drug delivery were used. In the first method (the drug in water study), drug was dissolved in buffered drinking water, and the mice were fed a diet of Richmond rodent diet #5010 (PMI Nutrition International, St. Louis, MO). Drug was dissolved in 5 mM sodium phosphate (pH 7.0) immediately before use, and cage bottles were replaced every 2 days. In the second method of drug delivery (the drug in diet study), mice were fed either AIN-93G powdered rodent diet alone (Dyets, Inc., Bethlehem, PA) or AIN-93G plus drug using food containers supplied by Dyets, Inc. All unused AIN-93G diet was stored at +4°C for a period not exceeding 3 months. Fresh AIN-93G diet was provided every 3 days, and diet plus drug was prepared immediately before use. Formulation was performed by grinding the drugs (excluding Pentasa) to a fine powder using a mortar and pestle, mixing in a small amount (~50 g) of powdered AIN-93G diet to act as a carrier, and then thoroughly stirring the mixture into the bulk AIN-93G diet under a flow hood. Previous studies have demonstrated that piroxicam and sulindac remain stable in the AIN dietary mixture for at least a week (34 , 35) , and the stability of the 5-ASA formulations in diet was verified by front-face fluorometry (described below).

Drugs.
Two nonisoform-selective NSAIDs (200 ppm of piroxicam and 160 ppm of sulindac; Sigma Chemical Co., St. Louis, MO) were used as positive controls in this study. Three 5-ASA formulations [free acid (Sigma Chemical Co.); SASP (Rugby, Norcross, GA); and Pentasa (Hoechst Marion Roussel, Kansas City, MO] were assayed at multiple oral dosage levels (500, 2400, 4800, and 9600 ppm). Finally, one 4-ASA formulation (sodium salt; Sigma Chemical Co.) was assayed at 500 ppm. In this feeding study, drug concentration (ppm in diet) can be converted to approximate dosage (mg/kg/day) using the formula mg/kg/day = ppm/5. This conversion is based on the determination that an adult ApcMin mouse eats an average of 0.2 grams of AIN-93G diet/gram body weight/day. Note that the drug concentrations used in this study corresponded to a dosage range of 100–1920 mg/kg/day.

Drug Level Assays.
The intraluminal and serum concentrations of 5-ASA and N-acetyl-5-ASA were measured using front-face fluorometry. This quantitative assay for 5-ASA is described in detail elsewhere (36) . Briefly, serum samples and cleared supernatants of ileal and colonic contents were collected and immediately snap frozen in liquid nitrogen at the time of necropsy. Fluorometric measurements were made by excitation at 310 nm, which produced emission maxima at 475 and 440 nm for 5-ASA and N-acetyl-5-ASA, respectively. A standard curve was generated using dilutions of pure drug in control serum samples. In addition to the ex vivo fluorometric assay, it was also possible to visualize the distribution of the drug in situ using UV epifluorescence. The intestinal tract was removed from euthanized mice and illuminated with a UV source (360 nm). Free 5-ASA could be visualized in the intact intestinal tract (macroscopically) and in the intestinal mucosa (microscopically) as a blue-green fluorescent signal on UV excitation.

Tumor Multiplicity.
Mice were sacrificed by CO2 inhalation in accordance with present NIH guidelines. Necropsy was performed, and the entire gastrointestinal tract was removed for dissection. The stomach and the cecum were omitted from the analysis due to their low tumor incidence. The small intestine was divided into three segments of approximately equal length (i.e., proximal, middle, and distal small intestine), and the colon was left intact. All four intestinal segments were completely dissected using bibulous paper as a support. Each segment was opened longitudinally using iris scissors and then washed extensively with PBS to remove intestinal contents. Tissues were fixed in methacarn (60% methanol, 30% chloroform, and 10% acetic acid), then rinsed in 70% ethanol. Tumor enumeration was performed using a Leica MZ3 stereo dissecting microscope with darkfield transillumination (which substantially improved tumor contrast). All intestinal tissues were analyzed while mounted on a calibrated stage micrometer and were photographed in their entirety at x6.3 magnification using Kodak 160T 35-mm film. The smallest tumors scored by this method were 0.2 mm in diameter, and the site of each tumor was recorded to facilitate distribution analysis.

Tumor Size.
Tumor size (area in mm2) was measured using NIH Image (version 1.61) analysis software. Photomicrographs of intestinal segments were scanned at 600 dpi, using a Polaroid SprintScan, and saved in the TIFF file format. The freehand selection tool of NIH Image was used to define and mark the edges of individual tumors, and the area of each tumor was then determined using the wand measurement tool. The stage micrometer included in each photomicrograph was used to calibrate the software to record measurements in mm2. Data were exported to Microsoft Excel (version 5.0) and tabulated for statistical analysis.

PG Assays.
Gas chromatography/mass spectroscopy was used to measure the levels of PGE2 in the small intestine and colon. Briefly, intestinal tissue samples were snap frozen and stored at -80°C until being processed for eicosanoids analysis. Frozen tissue was pulverized in liquid nitrogen, and PGs were extracted by resuspending the tissue in 2 ml of ice cold methanol containing indomethacin (10-5M). Tissue debris was removed by centrifugation. [2H4]PGE2 (2.4 hg; Cayman Chemical, Ann Arbor MI) was then added to the methanol fraction, and the volume increased to 20 ml with H2O (pH 3). PGE2 in the sample was then purified, derivatized, and subsequently quantified using gas chromatography/negative ion chemical ionization mass spectrometry, as described (37) .

Statistics.
Statistical comparisons of tumor multiplicity and tumor size were performed using Wilcoxon rank-sum or two-sample t tests. All probability tests were two-tailed, and all Ps < 0.05 were considered significant. Variance is expressed as the SE of the mean. Statistical power was calculated using two-sample t tests, assuming a type I error level of 5%, equal variances, a two-sided alternative hypothesis, and pooled SD for each drug delivery method.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
This study compared the chemopreventive efficacy of three different formulations of 5-ASA (free acid, Pentasa, and SASP) with two NSAIDs (sulindac and piroxicam) known to have antitumor activity in the ApcMin mouse model of intestinal neoplasia. Although both sulindac and piroxicam produced statistically significant reductions in intestinal tumor number and size (at dosage levels of 160 and 200 ppm, respectively), none of the 5-ASA drugs or dosage levels produced consistent, dose-progressive effects on tumor number, size, or distribution.

The experimental groups used in this study are summarized in Table 1Citation . A total of 119 ApcMin mice were evaluated in this study (25 mice on the control diets, 22 mice on 160 ppm sulindac or 200 ppm piroxicam, and 72 mice on several dosage levels of the 5-ASA formulations). In each group, drug treatment began at 28 days of age and the mice were sacrificed at 94–112 days of age. After sacrifice, the intestines were dissected and analyzed for tumor number, size, and distribution. Tumor number and distribution were evaluated for the entire length of the small and large intestines, whereas the computer-based analysis of tumor size was restricted to the ileum (the site of peak tumor incidence in the ApcMin mouse). Each of the 5-ASA drug formulations used in this study released free 5-ASA in the ileum (as determined by in situ fluorescence, described below).


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  		Table 1 Study groups used in 5-ASA chemoprevention trial

 
Two different oral drug delivery methods were used in this study (drug in water and drug in AIN-93G diet), and each method included its own control groups (see Table 1Citation ). The control mice fed an AIN-93G diet had significantly fewer intestinal tumors than the control mice fed the Richmond rodent diet #5010 (53.1 ± 5.4 tumors versus 84.0 ± 7.9 tumors, respectively; P < 0.01). Similar dietary effects on intestinal tumor development have been previously reported in the closely related Apc ({delta} 716) knockout mouse (8) . Because of this dietary difference, the data were separated into two groups for analysis. Fig. 1Citation illustrates the comparison of intestinal tumor multiplicity and tumor size in the drug in water study. Fig. 1ACitation compares intestinal tumor multiplicity (average ± SE of the mean) in control mice versus mice treated with piroxicam (200 ppm), sulindac (160 ppm), 5-ASA (500 ppm), or 4-ASA (500 ppm). Only the piroxicam and sulindac treatment groups showed statistically significant reductions in tumor number versus the control group (61% and 68% reductions, respectively; P < 0.01), whereas both 5-ASA and 4-ASA showed no significant effect. Fig. 1BCitation compares ileum tumor size in these same mice. Again, only the piroxicam and sulindac treatment groups showed statistically significant reductions in tumor size versus the control group (P < 0.01), whereas both 5-ASA and 4-ASA showed no significant effect.



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  		Fig. 1. Drug in water study: effect of drug treatment on intestinal tumor multiplicity and size in B6-ApcMin mice. *, statistical significance (P < 0.05); Bars, the SE of the mean. A, histogram of tumor multiplicity shows that treatment with the NSAIDs piroxicam (200 ppm) or sulindac (160 ppm) produced statistically significant reductions in tumor number versus control mice (P < 0.01 for both drugs), whereas treatment with 5-ASA (500 ppm) or 4-ASA (500 ppm) produced no significant effect. B, histogram of tumor size in the ileum of B6-ApcMin mice shows that treatment with the NSAIDs piroxicam (200 ppm) or sulindac (160 ppm) produced statistically significant reductions in tumor size versus control mice (P < 0.05 and P < 0.01, respectively), whereas treatment with 5-ASA (500 ppm) or 4-ASA (500 ppm) produced no significant effect.

 
Fig. 2Citation illustrates the results of the drug in the AIN-93G diet study comparing multiple dosage levels of 5-ASA, Pentasa, and SASP with piroxicam (200 ppm) and control mice. Once again, only the piroxicam treatment group showed a statistically significant reduction in tumor number versus the control group (Fig. 2A)Citation . In contrast, several of the treatment groups showed statistically significant changes in tumor size versus controls. Specifically, piroxicam (200 ppm), 5-ASA (2400 ppm), Pentasa (4800 ppm), and SASP (9600 ppm) each showed significant reductions in tumor size, whereas Pentasa (2400 ppm) showed a significant increase (Fig. 2B)Citation . None of the changes in tumor size seen in the mesalamine treatment groups were dose-progressive.



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  		Fig. 2. Drug in diet study: effect of drug treatment on intestinal tumor multiplicity and size in B6-ApcMin mice. *, statistical significance (P < 0.05); Bars, the SE of the mean. A, histogram of tumor multiplicity shows that none of the 5-ASA formulations (5-ASA free acid, Pentasa, or SASP) produced statistically significant effects on intestinal tumor number at the dosage levels assayed, whereas treatment with piroxicam (200 ppm) resulted in >90% reduction in tumor number. B, histogram of tumor size in the ileum of B6-ApcMin mice shows that treatment with piroxicam (200 ppm) resulted in a significant reduction in tumor size (61.4% reduction; P < 0.01). For the 5-ASA formulations (5-ASA free acid, Pentasa, or SASP), individual dosage levels produced statistically significant effects on tumor size, but no single agent produced a consistent dose-progressive effect on intestinal tumor size.

 
Because SASP and Pentasa do not release significant amounts of 5-ASA until reaching the distal small intestine and colon, we evaluated the possibility that there was a regional effect on tumor multiplicity in these treatment groups. Tumor distribution was determined by dividing the intestinal tract of each animal into 12 segments of approximately equal length (measured after tissue fixation) and by recording the number of tumors/segment. Fig. 3Citation illustrates that the pattern of tumor distribution for mice treated with 5-ASA, Pentasa, or SASP was similar to that of the control mice. As expected, none of the 5-ASA formulations or dosage levels assayed caused intestinal ulceration or acute toxicity, and there was no statistically significant difference in body weight between any of the 5-ASA treatment groups and controls. In contrast, treatment with 200 ppm piroxicam or 160 ppm sulindac produced superficial ulceration throughout the intestinal tract.



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  		Fig. 3. Effect of drug treatment on intestinal tumor distribution in B6-ApcMin mice. The regional pattern of tumor multiplicity is shown for AIN-93G control mice and for each of the three mesalamine treatment groups [free 5-ASA (21 mice), Pentasa (17 mice), and SASP (10 mice)]. The data shown represents the average tumor number for each experimental group. No statistically significant effect on tumor distribution was found for any of the 5-ASA dosage levels versus the AIN-93G control group.

 
We used front-face fluorometry to determine the approximate serum and tissue drug concentrations achieved through this oral delivery method. As expected, mice treated with 5-ASA (free acid) showed higher levels of serum 5-ASA than did animals treated with Pentasa (average of 161.3 µg/ml and 38.1 µg/ml, respectively, at the 9600 ppm dosage level). Because 5-ASA is thought to act through a topical rather than systemic mechanism, we also measured the intraluminal concentration of 5-ASA. Mice treated with Pentasa showed the highest intestinal levels of 5-ASA (average of 98.1 µg/ml in the small intestine and 632.7 µg/ml in the colon for the 9600 ppm dosage level). The fluorometric assays also revealed that the emission wavelengths varied from 438–460 nm, suggesting that there was a mixture of 5-ASA and N-acetyl-5-ASA present in each of the samples assayed. In addition to the in vitro fluorometric assay for 5-ASA, we also performed in situ drug detection. We used UV illumination (360 nm) to directly visualize the tissue distribution of 5-ASA and acetyl-5-ASA during necropsy. Because of the small difference in fluorescent emission wavelength for 5-ASA (475 nm) versus acetyl-5-ASA (440 nm), this in situ assay does not readily distinguish between the two forms. Fig. 4Citation , A and B, show the fluorescent detection of 5-ASA in the serum and intestines of drug-treated mice and illustrate the regional pattern of release for free 5-ASA from the three drug formulations studied. Fig. 5Citation shows the fluorescent detection of 5-ASA in intestinal tissues and demonstrates that 5-ASA was present in the tumor tissues of drug-treated animals. Intracellular fluorescence was also seen in histological sections of drug-treated animals, and similar results were obtained for animals treated with free 5-ASA, Pentasa, or SASP (data not shown).



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  		Fig. 4. Fluorescent detection of 5-ASA in vivo. A, 5-ASA was detected in the serum of drug-treated mice by UV excitation (360 nm), producing a characteristic blue-green emission at 440–460 nm. The highest level of serum fluorescence was observed in mice treated with 5-ASA (free acid), and the lowest level was in mice treated with SASP. Mice fed the control diet showed a very low level of serum autofluorescence. B, visualization of 5-ASA in the intestinal tract. UV excitation of excised intestines revealed the distribution of free 5-ASA for each of the drug formulations assayed.

 


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  		Fig. 5. Fluorescent detection of 5-ASA in situ. A, 5-ASA detection in the small bowel of a mouse at necropsy. S, stomach; C, cecum; arrowhead, region of 5-ASA fluorescence in the small bowel. B, 5-ASA colocalizing with colon tumor tissue. Arrowheads, position of the colon tumor.

 
The effect of 5-ASA on eicosanoid production in the gut was evaluated by gas chromatography/mass spectroscopy. We measured the levels of PGE2 in ileum and colon tissues from control mice and mice treated with 200 ppm piroxicam or 9600 ppm Pentasa (two animals/group). As expected, piroxicam treatment resulted in a marked reduction in intestinal PGE2 levels (95.3% reduction compared with control mice), whereas treatment with Pentasa produced a more modest effect (42.7% reduction versus controls).

Finally, we retrospectively calculated statistical power for these assays using two-sample t tests, assuming a type I error level of 5%, equal variances, a two-sided alternative hypothesis, and pooled SD for each drug delivery method (24.5 and 27.5, respectively). For the mice having the water delivery method, the study had 87–98% power to detect a 50% reduction in the number of tumors among the treated mice as compared with the control mice (n = 12), depending on the number of mice in the treated groups (range, 6–12). The study had considerably less power (24–64%) to detect a 50% reduction in the number of tumors among the treated mice having the diet delivery method compared with the control mice (n = 14), depending on the number of mice in the treated groups (range, 3–12).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The increased risk for the development of intestinal cancer in patients suffering from Crohn’s disease or ulcerative colitis remains a significant problem in the medical management of IBD. The purpose of this study was to evaluate several formulations and dosage levels of 5-ASA, a drug commonly used in the treatment of IBD, for direct antitumor activity against adenomatous polyps in the ApcMin mouse model of intestinal neoplasia. The major finding of this study is that 5-ASA was not effective at reducing intestinal tumor number or size in a consistent, dose-responsive manner in this model system. This is in contrast to the NSAIDs sulindac and piroxicam, which both caused marked reductions in intestinal tumor number and size and served as positive controls for our assay system.

To evaluate the chemopreventive activity of 5-ASA in a controlled, preclinical trial, we initially considered several mouse models of colitis-associated neoplasia. Although a number of such models exist, including targeted knockouts of IL-10, keratin (K8), N-cadherin (NCAD{Delta}), and G{alpha}i2, each of these models is associated with a long and variable tumor latency period, undefined tumor etiology, and incomplete penetrance of the tumor phenotype (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41) . Because of the difficulties of designing and interpreting chemoprevention studies in these colitis-associated tumor models, we chose to evaluate the antitumor activity of 5-ASA in the ApcMin mouse, a well-characterized model of Apc-mediated intestinal tumorigenesis. The ApcMin mouse model offers the advantages of defined genetic etiology, complete penetrance of the tumor phenotype, rapid tumor kinetics, and proven usefulness as a chemoprevention model. However, because the ApcMin mouse does not develop IBD, the scope of this investigation is restricted to the evaluation of 5-ASA for direct chemopreventive activity against Apc-mediated intestinal tumorigenesis.

Although the etiology of IBD-associated colorectal cancer may differ from familial adenomatous polyposis and sporadic colon cancer in several important ways (reviewed in Ref. 2 ), it has been reported that both sporadic and IBD-associated colorectal cancer are sensitive to suppression by NSAIDs and that they share a common profile of genetic mutations, including loss of APC function (29, 30, 31) . Intestinal tumors arising in ApcMin mouse also undergo inactivating mutations of Apc and up-regulation of Cox-2, molecular events that are frequently associated with the development of human colon cancer, as well (42 , 43) . Thus, although the ApcMin mouse is not a model of colitis-associated intestinal cancer, it, nonetheless, shares some of the key genetic features of tumorigenesis in this disease and may have important predictive value for selecting chemopreventive agents effective against Apc-mediated forms of intestinal cancer.

Another important consideration in the design of this study was the selection of appropriate 5-ASA agents. We evaluated three different formulations of 5-ASA in this study based on their distinct pharmacokinetic properties and based on the biological characteristics of the ApcMin mouse. Because tumors develop throughout the intestinal tract of ApcMin mice (both small and large bowel), we selected drug formulations that would provide release of free 5-ASA to different regions of the gut. Fig. 4BCitation illustrates the intestinal distribution of 5-ASA for each of the formulations studied. The first formulation, 5-ASA free acid, was selected to provide drug delivery to the proximal small bowel. Although free 5-ASA is known to be highly absorbed after oral delivery (~90% absorbed in rats; Ref. 44 ), fluorescent detection of the drug at necropsy revealed that intraluminal 5-ASA was present throughout the small intestine and, to a lesser extent, the colon. The second drug formulation, Pentasa, was chosen to provide delayed release in the distal small bowel and colon. Pentasa is provided as methycellulose-coated microbeads, which gradually dissolve after ingestion. Release of free 5-ASA in Pentasa-treated mice began in the stomach and peaked in the distal small bowel and colon (systemic absorption is 20–30% in humans; Ref. 45 ). The third formulation, SASP, was chosen to provide delivery of 5-ASA to the colon. SASP is a chemical compound of 5-ASA and sulfapyridine and undergoes bacterial cleavage in the gut, which results in the release of free 5-ASA in the distal small bowel and colon (systemic absorption is approximately 20% in humans; Ref. 25 ). Finally, we assayed one oral dosage level of 4-ASA (para-aminosalicylic acid), which is closely related to 5-ASA and has also been shown to be effective in the treatment of IBD (46) .

To determine whether there was a dose-dependent effect on tumor suppression, we evaluated multiple dosage levels of each 5-ASA formulation. The lowest dosage level assayed, 500 ppm, approximates the clinically relevant dosage level used in the treatment of human IBD patients (60–80 mg/kg/day). The highest dosage level assayed, 9600 ppm, was selected based on the absence of lethality at this dosage point in previous rodent toxicology studies (47) . Finally, 2400 ppm and 4800 ppm provided midpoint dosage levels for establishing a dose-response relationship. We found that individual dosage points of Pentasa and SASP reached statistical significance in their effect on tumor size, but not on tumor multiplicity. Our experimental observation that there was no consistent, dose-progressive effect on intestinal tumor number, size, or distribution for any of the 5-ASA formulations assayed suggests that this drug does not have potent chemopreventive efficacy across a broad range of concentrations. However, due to the relatively small number of animals assayed in each group, we cannot rule out the possibility that 5-ASA exerts weak chemosuppressive activity in this model.

5-ASA agents are known to cause modulation of eicosanoid production in the gut (48) , and this has been one of the postulated mechanisms of action of 5-ASA in IBD, in which a wide variety of inflammatory factors are thought to play an important etiological role (9 , 50) . It is interesting to note that 9600 ppm of Pentasa caused a substantial (42.7%) decrease in PGE2 levels in the intestine versus control animals, whereas it had no significant effect on tumor multiplicity or average tumor size. In contrast, 200 ppm of piroxicam decreased PGE2 levels by >95% and resulted in a >90% reduction in tumor multiplicity and a 61% decrease in residual tumor size (P < 0.01 versus controls). Although it is possible that there may be a threshold of efficacy for chemopreventive agents acting through a COX-mediated pathway, it is, nonetheless, surprising that 600 ppm of Pentasa significantly modulated gross intestinal PGE2 levels without affecting tumor number or size, perhaps reflecting a differential tumor/stroma tissue selectivity for the action of Pentasa.

One caveat with the use of the ApcMin mouse for the evaluation of chemopreventive agents is the timing of tumor initiation versus the timing of drug treatment. Specifically, because this chemoprevention study used an oral delivery route, drug treatment did not begin until the time of weaning (i.e., when young mice converted to a diet of solid food at 28 days of age). However, the majority of tumor initiation in ApcMin mice is thought to take place before 21 days of age (28) . Thus, drugs that exert their chemoprotective effect during the process of tumor initiation (but not during tumor promotion or progression) might not be expected to produce a measurable antitumor effect in this postinitiation assay system. This is an important consideration for interpreting the negative findings of this 5-ASA chemoprevention trial and for reconciling these results with some of the previous correlative clinical studies, which have shown a chemoprotective benefit from the long-term use of 5-ASA in IBD patients. The possibility that 5-ASA is effective for the prevention of intestinal tumors, but that it exerts its effect very early in the process of carcinogenesis, cannot be excluded by this study. Furthermore, because 5-ASA undergoes metabolic conversion to what is believed to be a biologically inactive form (N-acetyl-5-ASA; Refs. 25 and 51 ), it is not feasible to achieve in utero or postpartum drug delivery through dosing of the pregnant mothers.

In summary, these data demonstrate that 5-ASA does not induce measurable tumoristasis or cause intestinal tumor regression in the ApcMin mouse, nor does it seem to exert a cocarcinogenic effect in the gut. However, because of the limitations of this in vivo assay system, we restrict our interpretation of these data to conclude that although 5-ASA does not exert a direct antitumor effect on preexisting adenomatous tumors, it may act at the level of tumor initiation or it may provide an indirect chemoprotective benefit through the long-term suppression of inflammation. Definitive resolution of this issue will require further experimentation using models of inflammatory carcinogenesis.


   ACKNOWLEDGMENTS
 
We thank Suresh Sarivarayan and members of the Mayo Clinic Scottsdale transgenic animal facility for support in maintaining the ApcMin mouse colony used in this study. We are also grateful to Anita Jennings for histology and to Dr. Thomas Lidner for pathology consultations.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by NIH Grants HL-38655 and HL-58247 (to R. E. H.) and by the Mayo Foundation for Medical Education and Research (to S. J. G., S. R. R.). Back

2 To whom requests for reprints should be addressed, at Mayo Clinic Scottsdale, 13400 East Shea Boulevard., Department of Biochemistry and Molecular Biology, Scottsdale, AZ 85259. E-mail: gendler.sandra{at}mayo.edu Back

3 The abbreviations used are: IBD, inflammatory bowel disease; 5-ASA, 5-aminosalicylic acid; SASP, sulfasalazine; NSAID, nonsteroidal anti-inflammatory drug; COX, cyclooxygenase; Apc, adenomatous polyposis coli; Min, multiple intestinal neoplasia; B6, C57BL/6J; PG, prostaglandin; PGE2, PG E2; ppm, parts/million. Back

Received 8/ 7/98; revised 1/14/99; accepted 1/15/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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