Prevention of Chemically-Induced Urinary Bladder Cancers by Naproxen: Protocols to Reduce Gastric Toxicity in Humans Do Not Alter Preventive Efficacy

Abstract

The COX inhibitors (NSAIDs/Coxibs) are a major focus for the chemoprevention of cancer. The COX-2 specific inhibitors have progressed to clinical trials, and have shown preventive efficacy in colon and skin cancers. However, they have significant adverse cardiovascular (CV) effects. Certain NSAIDs (e.g., naproxen (NPX)] have a good cardiac profile, but can cause gastric toxicity. The present studies examined protocols to reduce this toxicity of NPX. Female Fischer-344 rats were treated weekly with the urinary bladder specific carcinogen hydroxybutyl(butyl)nitrosamine (OH-BBN) for 8 weeks. Rats were dosed daily with NPX (40 mg/Kg BW/day, gavage) or with the proton pump inhibitor omeprazole (4.0 mg/Kg BW/day) either singly or in combination beginning 2 weeks after the final OH-BBN. OH-BBN treated rats, 96% developed urinary bladder cancers. While omeprazole alone was ineffective (97% cancers), NPX alone or combined with omeprazole prevented cancers; yielding 27 and 35% cancers, respectively. In a separate study, OH-BBN treated rats were administered NPX: (A) daily, (B) 1 week daily NPX/1wk vehicle, (C) 3 weeks daily NPX/3 week vehicle, or (D) daily vehicle beginning 2 weeks after last OH-BBN treatment. In the intermittent dosing study, protocol A, B, C and D resulted in palpable cancers in 27%, 22%, 19% and 96% of rats (P<0.01). Short-term NPX treatment increased apoptosis, but did not alter proliferation in the urinary bladder cancers. Two different protocols which should decrease the gastric toxicity of NSAIDs in humans did not alter chemopreventive efficacy. This should encourage the use of NSAIDs (e.g. NPX) in clinical prevention trials.

Introduction

The NSAIDs have been a major focus in the field of chemoprevention for more than 25 years. Initially evaluated were the non-selective NSAIDs; particularly piroxicam (1) which was highly effective in preclinical models, but is now infrequently employed clinically. Approximately 20 years ago, it was shown that two different enzymes perform cyclooxygenation of arachidonic acid and were designated COX-1 and COX-2 (2). COX-1 is a constitutively expressed enzyme found in a wide variety of tissues. Although COX-2 is preferentially expressed in lymphoid cells, it can be induced by a wide variety of stimuli in many cell types. Interestingly, COX-2 has proven to be a direct target for cancer prevention (3–5). Specific inhibitors of COX-2 were synthesized and in pre-clinical studies were highly effective in the prevention of various types of cancer (6–8). Clinical trials of the COX-2 inhibitors were effective in clinical prevention trials of colon adenomas and squamous cell skin cancer (9,10). However, placebo controlled adenoma trials of rofecoxib at the standard dose and celecoxib at doses higher than the standard human dose increased the incidence of adverse CV events (11). Therefore, there were significant efforts to synthesize agents with lower CV risks than the COX-2 inhibitors and certain standard NSAIDs (e.g., diclofenac). Based on epidemiologic data, it appeared that certain NSAIDs (e.g., NPX) had lower CV risk than others (12); leading us to explore the chemopreventive efficacy of NPX in the present studies.

The second major toxicity associated with NSAIDs is gastric toxicity; particularly the induction of ulcers and potential life threatening bleeds which occurs in a very limited number of patients (13). We examined two different regimens to reduce the potential gastric toxicity of NPX. First, NPX was combined with the proton pump inhibitor omeprazole since this approach has been shown clinically to substantially reduce upper GI toxicity (14, 15). The second approach was to employ intermittent dosing with the NSAID on the expectation that it would facilitate recovery from any gastric damage associated with NPX treatment

The OH-BBN induced urinary bladder cancers are highly invasive and appear histologically similar to human transitional cell carcinoma (TCC) (16). The tumors appear by array analysis to have strong overlap both at the pathway level and at the specific gene level to invasive human bladder cancer (17,18). The model has been shown to be highly sensitive to the preventive activity of a variety of NSAIDs, as well as various EGFr inhibitors (16). Interestingly, the efficacy of these two classes of agents is mediated by blocking tumor progression from micro-carcinomas to the development of palpable invasive cancers, and not by blocking the formation of premalignant lesions (16).

The effects of these altered regimens to reduce gastric toxicity in humans on the efficacy of NPX in the OH-BBN urinary bladder cancer model were examined. These studies showed: (1) NPX administered by gavage either daily or intermittently 1 week on/1 week off, or 3 weeks on/3 weeks off, all similarly inhibited urinary bladder cancer formation 73–82% (P<.005); (2) The half-life of NPX in the rat (roughly 3.0 hours) is much shorter than the half-life in the human; (3) omeprazole, which by itself did not decrease bladder cancer formation, did not interfere with the efficacy of NPX; (4) Although NPX failed to alter proliferation related biomarkers, it did increase levels of the apoptotic proteins Caspase 3 and Caspase 7.

Materials and Methods

Reagents

NPX and omeprazole were obtained from Sigma Chemical Company (St. Louis, MO). The carcinogen OH-BBN was purchased from TCI America (Portland, OR). Female Fischer-344 rats were obtained from Harlan Sprague-Dawley, Inc (Indianapolis, IN) at 4 weeks of age. Diets were purchased from Teklad (Harlan Teklad, Madison, WI) and were provided ad libitum.

Urinary Bladder Model

The OH-BBN-induced urinary bladder cancer model was performed as previously described (16,19). Beginning at 56 days of age, rats (30 per group) were treated twice a week with 150 mg OH-BBN/gavage for 8 weeks. The rats were weighed 1x/week, and palpated for urinary bladder tumors 2x/week. Intragastric administration of NPX and/or omeprazole was initiated 2 weeks after the final OH-BBN treatment. The vehicle for NPX was saline and for omeprazole the vehicle was polyethylene glycol 400: ethanol (90:10, v/v). The gavage volume was 0.5 ml/rat, and animals were treated 7x/week. Beginning 2 months after the last dose of OH-BBN, rats were checked weekly for the development of palpable bladder tumors. The studies were terminated seven months after the last OH-BBN treatment. At necropsy, urinary bladders with associated lesions were excised and weighed. Percent incidence and the percent of rats with large tumors >200 mg bladder weight were recorded as endpoints for cancer prevention. Statistical analysis of bladder weights were performed by the Wilcoxon Rank Test and the final tumor incidence was analyzed by the Fischer’s Exact Test (16).

PK of NPX in Female Fischer-344 Rats

At 60 days of age, rats were administered NPX (40 mg/Kg BW) in saline by gavage either once or for 14 consecutive days. At time points 0, 1, 2, 4, 8 and 24 hours, blood (N=4) was obtained. The serum was frozen in liquid nitrogen and stored at −80°C. The serum was sent to Midwest Research Institute for quantitative analysis of NPX levels. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitation of NPX in rat serum was employed. Rat serum samples (50 µL) were fortified with NPX across the concentration range. Ketoprofen was added as an internal standard (I7). The samples were extracted via protein precipitation prior to analysis. Metabolities were separated by HPLC on a Phenomenex luna C18 column 5 µm Mobile Phase A: Ammonium Acetate Buffer, pH 4.0: Mobile Phase B: 100% Acetonitrile Gradient Type: Isocratic, 25% A/75% B Flow rate: 1.0 mL/min: Run time: 8.0 min. The resulting samples derived from HPLC separation were analyzed on an Applied Biosystems API-4000 Q Trap Mass Spectrometer. The LC-MS procedure employed is based on the method of Elsinghorst et al., across a range of 40 to 1,000 ng/mL (20).

Determination of Biomarkers in Urinary Bladder Cancers Treated Short Term with NPX

Female Fischer-344 rats with urinary bladder cancers induced with OH-BBN were treated intragastrically with 40 mg/kg NPX for 5 days or with the vehicle. At the time of sacrifice, bladder cancers were quickly removed and processed for IHC. Slides were prepared for TUNEL assay and immunofluorescence microscopy. Apoptosis was determined either by the TUNEL assay, using the DeadEnd Colorimetric TUNEL System (Promega, Madison, WI) according to the manufacturer’s instructions or alternatively stained for Caspase 3 or Caspase 7. Briefly, after deparaffinization and rehydration, the tissue sections were pretreated with 20 µg/ml proteinase K solution for 10 min at room temperature. Thereafter, slides were rinsed in PBS and incubated with TUNEL reaction mixture for 1 h at 37°C in a humidified chamber. Slides were then washed with PBS followed by a stop solution for 10 min at room temperature. The slides were counterstained with DAPI under glass coverslips. The stained tissue was examined at 200× magnification 9 using a Nikon Eclipse TE2000-E Confocal microscope. The specific antibodies employed immuno-fluorescence microscopy. Slides were baked at 60°C for 2 h, deparaffinized with xylene and rehydrated through a graded alcohol bath. 10 mM Sodium citrate buffer (pH 6.0) was used for antigen retrieval by heating slides and buffer for 10 min in the microwave followed by 20 min of ambient cooling. Slides were washed with DI water followed by 1xPBS. For immunofluorescence, specimens were blocked in 5% donkey serum-1xPBS- 0.3%Triton (PBST) buffer for ~1 h. The primary antibody was prepared according to the manufacturer’s instructions and left on overnight at 4°C. Slides were washed in 1xPBS followed by 2 h of appropriate secondary antibody incubation prepared according to the manufacturer’s instructions. Slides were washed with 1xPBS followed high salt PBS (23.38g NaCl in 1 Liter 1XPBS). Fluoro-Gel II with Dapi was used to mount glass coverslips and then sealed using clear nail polish. All stained tissues were examined at 200× magnification using a Nikon Eclipse TE2000-E Confocal microscope. Antibodies Employed: Primaries: p-Akt (Ser473) Rb - Cell Signaling: 4060S, Lot #14 p53 Total Mo – Cell Signaling: 2524, Lot #4 p-AMPK (Tyr172) Rb – Santa Cruz: sc-33524, Lot #C1011 p-p53 (Ser392) Gt – Santa Cruz: sc-7997, Lot #L1809 p-mTOR (Ser2448) Rb – Cell Signaling: 5536S, Lot #1 p-mTOR (Ser2448) Rb – Cell Signaling: 5536S, Lot #1; caspase 3 – Cell Signaling: 9664, Lot #18; caspase 7 – Imgenex: IMG 5702. The TUNEL assay was determined, using the DeadEnd Colorimetric TUNEL System (Promega, Madison, WI) according to the manufacturer’s instructions.

Statistical Analysis

All quantitative results are expressed as mean values ± S.D. Statistically significant differences were obtained using the Student’s t test or by one-way ANOVA. A p < 0.05 value was considered to be statistically significant.

RESULTS

Effect of Intermittent Dosing with NPX in OH-BBN Treated Rats

We had previously found that daily dosing with NPX (40 mg/kg), which is roughly the HED (human equivalent dose), prevented the development of urinary bladder cancers in OH-BBN treated rats. OH-BBN was administered for 8 weeks starting at 8 weeks of age. Two weeks after the last OH-BBN dosing, rats were administered: NPX daily (40 mg/kg BW/day, by gavage) (Group A); 1 week daily NPX/1week vehicle (Group B); 3 week daily NPX/3 week vehicle (Group C) or only vehicle (Group D). At the end of the study, the number of rats with large urinary bladder cancers (>200 mg) were: (A), 8/30; (B), 6/29; (C), 5/28; (D), 24/25 (Fig. 1) (P<0.01 for all three treatment groups vs vehicle control). Tumor development monitored by palpation (Fig.1) shows the increased latency and lower final incidence of the various treatment groups vs vehicle treatment. Measurement of the weights of the urinary bladder plus cancers showed that the final weights were higher in vehicle controls than in any of the three NPX treatment groups (P<.025) (Supplementary Fig. 1A). In contrast, none of the three treatment groups differed significantly from one another.

Figure 1. The effects of NPX, omeprazole or the combination on development of urinary bladder cancer in OH-BBN-treated rats.

Incidence of rats with urinary bladder cancers with final weights >200 mg (n=30) administered either NPX (40/mg/Kg BW/day), omeprazole (4 mg/Kg BW/day), combination of these two agents, or vehicle beginning 2 weeks after the final OH-BBN administration. In addition, there were 10 rats administered rosiglitazone (10 mg/kg BW/day) by gavage. Rats were palpated weekly for tumor development beginning 8 weeks after the final dose of OHBBN. Only those rats whose final bladder weights were >200 mg are included.

Effects of Combining Omeprazole and NPX

Starting at 2 weeks after the last dose of OH-BBN, rats were administered on a daily basis: (A) omeprazole (4 mg/kg BW), (B) NPX (40 mg/kg BW), (C) omeprazole + NPX, or (D) vehicle. Groups A, B, C, and D had large bladder cancers in 28/29, 8/30, 10/29 and 24/25 of the rats (Fig. 2) at the end of the study. Thus, NPX alone or NPX with omeprazole were similarly effective in reducing the incidence and increasing the latency of large bladder cancer formation. The efficacy of NPX alone or NPX plus omeprazole is also reflected in the final weights of the tumors (Supplementary Fig. 1B). This showed that final bladder weights (bladder + cancers) of animals treated with NPX alone or NPX plus omeprazole were significantly different from control (P<.025), but not different from one another. Interestingly, while omeprazole by itself did not alter the latency or final incidence of palpable cancers, the cancers that did develop in the rats were larger in size than tumors in rats given the vehicle alone; although it failed to reach statistical significance (P>.05). For reasons unrelated to the present study, we treated 10 rats with the known bladder tumor promoter rosiglitazone. As can be seen in Fig. 2, rosiglitazone greatly decreased tumor latency in this study.

Figure 2. The effects of intermittent dosing regimens with NPX on development of urinary bladder cancers in OH-BBN-treated rats.

Incidence of rats with bladder cancers with final weights >200 mg (n=30) administered NPX (40/mg/Kg BW/day) either daily, one week on/one week off, 3 weeks on/3 weeks off, or vehicle beginning 2 weeks after the final OH-BBN administration.

Phamacokinetics of NPX (Table 1)

TABLE 1.

Pharmacokinetics of NPX in the Rat

Time Point (Units)	t1/2 hr	Tmax hr	Cmax ng/ml	AUC hrxng/ml	Vd L/kg	CI L/hr/kg
24 hour	3.03	2	71,280	686,277	0.255	.058
14 Day	3.28	2	66,580	847,418	0.22	.049

Hunan t1/2 is approximately 16 hours

Serum levels of NPX were determined in rats treated with NPX (40 mg/Kg BW), by gavage, for either 1 day or 14 days. The half-life of NPX was approximately 3 hours at either time point. The Cmax was roughly 300 µM, although levels greater than 100 uM were probably achieved for less than 6 hours following gavage treatment. A graph of the PK data is presented in Supplementary Fig 2.

Effects on Potential Biomarkers

The effects of limited treatment with NPX (7 days) on a variety of potential biomarkers were examined. NPX increased levels of three apoptosis related biomarkers (Caspase 3 and 7, TUNEL). In contrast, it failed to alter Ki67 levels (Fig. 3). Additional biomarkers related to the AKT/PI3K/MTor pathway were also examined (Supplementary Fig. 3). Although it did slightly, but significantly, decrease levels of phosphorylated AKT (S473) and phosphorylation of PI3K, the relative levels of inhibition were quite limited.

Figure 3. Effects of limited treatment with NPX on biomarker expression.

Control rats developing palpable bladder cancers were treated for 5 days with NPX or vehicle. Animals were euthanized and bladder cancers were fixed in Zamboni’s. Using procedures described in Materials and Methods, IHC was performed on vehicle treated tumors and short-term NPX treated cancers. Five vehicle control cancers and 5 bladder cancers of rats treated short-term with NPX were examined. A) Effects of NPX on Ki67 labeling of bladder cancers. Results are based on counting at least 1000 cells for each of the tumors. No significant difference was observed.

B) Effects of NPX on staining of Caspase 3 and 7 in tumors. Results are based on counting at least 1000 cells for each of the cancers. Significant differences in staining of both Caspase 3 and 7 were observed.

Discussion

The NSAIDs and the Coxibs are highly effective in inhibiting the development of OH-BBN induced invasive urinary bladder cancers (16). The OH-BBN-induced bladder cancers have significant overlap with human invasive bladder cancer at the gene expression level (17,18). In prior studies, we presented our rationale that a bladder plus cancers that was greater than 200 mg was likely to be identified by palpation (16). Using this endpoint, we had previously shown that NPX and other NSAIDs/Coxibs were effective when administered beginning 3 weeks or even 3 months after the final OH-BBN administration. In the late intervention, microscopic lesions already existed in the majority of rats at the time of initial treatment (16). This result implies that the major effect of the agents is on the progression of cancers from the microscopic to the palpable size.

We have examined the efficacy of NPX in multiple animal models because it had the best clinical CV profile of any of the NSAIDs besides aspirin. Our prior data found that NPX was highly effective in colon, bladder and skin tumor models (16,19–21). However, NPX (like most non-selective NSAIDs including aspirin) causes gastric toxicity in humans (12–14). While the likelihood of a fatal hemorrhage is relatively low, the incidence of ulceration is significant; particularly with chronic and long-term dosing. We, therefore, investigated protocols that might reduce the gastric toxicity of NPX. The first approach is quite obvious and entailed combining NPX with omeprazole. This combination of NPX plus a PPI inhibitor is used clinically for persons with arthritis who require an NSAID for pain, but who are at cardiac risk (14,15). As shown in Fig. 2, omeprazole by itself minimally affected the development of palpable cancers while NPX reduced urinary bladder cancer incidence by approximately 75% (P<0.01). Most importantly, the combination of NPX plus omeprazole was as effective as NPX alone. When the weights of the cancers (Supplementary Fig. 1B) were evaluated, NPX alone and NPX + omeprazole decreased the weight of the cancers relative to the controls. Interestingly, although omeprazole did not decrease the latency of large urinary bladder cancers relative to OH-BBN alone, it did increase the final weights of the resulting cancers; although not significantly (P>0.05). For unrelated reasons, we included ten rats that were treated with OH-BBN and a PPAR gamma agonist rosiglitazone in this study. We had previously shown that this agent promoted bladder cancers in this animal model (22). The main point is that while rosiglitazone clearly decreased tumor latency in this model, omeprazole did not. These results imply that this combination of NPX + omeprazole is highly effective and, based on clinical data, likely to decrease gastric toxicity. The gastric toxicity cannot be modeled in the animal since gavage dosing of NPX at 40 mg/Kg BW even for many months failed to induce significant gastric lesions. In fact, doses of NPX up to 3× higher similarly failed to induce significant gastric toxicity in rats. We, therefore, feel that performing a toxicity study at doses far in excess of the dose we employed in the study, which is roughly the HED, made little sense. Parenthetically, investigators have shown that the levels of omeprazole we employed (which are at the human equivalent dose) decreased the acidity of the stomach in rats (23).

Our second approach to reducing toxicity entailed intermittent dosing with NPX. This employed either daily dosing with NPX or intermittent dosing 1 week on/1 week off or 3 weeks on/3 weeks off. We were surprised to find that the intermittent dosing was as effective as daily dosing with the agent. Simultaneous PK studies showed that the half-life of NPX in the rat was relatively short (T_1/2), about 3 hours, in contrast to the human half-life of 15 hours. This argues that the efficacy observed with intermittent dosing is unrelated to extended serum levels. Interestingly, the PK data show that NPX and/or certain of its conjugates are overwhelmingly excreted via the urine. This implies that NPX may be particularly applicable for use in urinary bladder cancer. Piroxicam has shown therapeutic efficacy in urinary bladder cancer in dogs (24–26). The use of NPX which preferentially accumulates in urine in humans (27) might allow the use of a lower dose with more limited gastric toxicity than would be required with piroxicam. It might also suggest its use in prevention of urinary bladder cancer in humans, in which there is at least some data supporting the use of NSAIDs (28). We have recently tested intermittent dosing with NPX in a rat model of colon cancer and have found it to be highly effective (Rao C; Steele VE, Lubet RA, Unpublished Data).

Cyclooxygenase inhibitors (NSAIDs/Coxibs) can prevent cancer in preclinical models, in epidemiologic studies, and in clinical trials both as primary or secondary endpoints. The clearest evidence in humans is in colon cancer (29), esophageal cancer (30), and in squamous cell skin cancer (9). However, there is also significant evidence in various other cancers (based on epidemiologic data as well as preclinical data). Regarding urinary bladder cancer (28), there is some, albeit sometimes conflicting, epidemiologic evidence. Furthermore, there is clear efficacy of standard NSAIDs and Coxibs in the treatment of bladder cancer in dogs and rodents, and a hint of efficacy in certain clinical trials (31). We have done extensive work with this class of agents in the OH-BBN-induced model of bladder cancer in rats (7,16,32).

Our prior studies have shown that various NSAIDs/Coxibs (NPX, NO-NPX, sulindac, celecoxib at their HEDs and aspirin at doses in substantial excess of its HED) are effective in blocking the development of large palpable cancers even when initiated after animals have pre-invasive microscopic cancers (16). One of the great hurdles to employing NSAIDs/Coxibs in a preventive setting (despite clear efficacy) is potential toxicities. Gastric toxicity is quite clear with a variety of NSAIDs and aspirin, while CV toxicity is clearer with Coxibs and certain NSAIDs (e.g., diclofinac). In fact, lower doses of aspirin are cardio protective, while NPX has the best CV profile of the standard NSAIDs (33). Presumably, the positive CV effects of low dose aspirin, and perhaps NPX, are due to their ability to inhibit COX-1 activity and decrease thromboxane levels. Aspirin achieves this by being a suicide substrate for COX-1, while NPX is a long lived non-specific inhibitor that inhibits both COX-1 and COX-2. NPX is recommended for persons with arthritis who need an NSAID, but have higher CV risk. It is administered together with a PPI inhibitor, often omeprazole. In fact, the PPI inhibitors have been shown to substantially reduce the gastric toxicity associated with NSAIDs by 50–75% in most studies (14,15). The finding that the PPIs do not inhibit the anti-arthritic effects of NSAIDs made us optimistic that they were not likely to interfere with the preventive efficacy of this NSAID. The rationale being that the anti-arthritic effects of NPX are likely driven by COX-2 inhibition. Since this is also likely to be a primary mechanism of cancer prevention as well (3,4,5), we felt that omeprazole was not likely to inhibit the efficacy of NPX. As shown in Fig. 2, omeprazole at its HED did not alter bladder cancer formation. In contrast, NPX at its HED (40 mg/Kg BW/day) or the combination of NPX and OMPZ at the HEDs were highly effective in the prevention of bladder tumors. Importantly, the addition of omeprazole or esomeprazole has been shown to substantially reduce any gastric toxicity associated with NPX treatment (14,15,34). Parenthetically, the combination of esomeprazole and NPX is currently available as a fixed dose product in the marketplace, and is approved for use in arthritis.

Alternatively intermittent NPX appears totally effective. The concept from the GI perspective is that intermittent dosing may allow for recovery of any gastric lesions, due to regeneration of COX activity as well as natural repair mechanisms. However, co-therapy with a PPI is thought to be a safer GI risk reducing strategy since there is demonstrated clinical efficacy (14,15). As described above, we cannot test the ability of intermittent dosing to reduce gastric toxicity in rats since gavage dosing of NPX (40 mg/Kg BW/day) for multiple weeks does not cause significant gastric toxicity. Given the relatively short half-life of NPX in the rat, we are not sure why the intermittent dosing appears to be so effective. The efficacy of intermittent dosing with either NPX or high dose aspirin in a colon model of carcinogenesis was recently observed (C.V. Rao, V.E. Steele and R.A. Lubet Unpublished Data).

In an attempt to determine the mechanism of action of NPX, we examined its effects on a variety of potential biomarkers. The basic approach is similar to the pre-surgical model of biomarkers employed in humans (35). Persons with early stage cancer are exposed to potential agents for a limited time period prior to surgery. We have routinely used this approach in a breast cancer model employing either Ki67 or gene arrays to perform the analysis (36). Our laboratory used such an approach in the OH-BBN bladder model to investigate the efficacy of the EGFr inhibitor Iressa; and found alterations of a wide variety of biomarkers and genes (37). We hoped since NSAIDs in general (NPX in particular) were effective during the later stages of tumor progression, we might observe alterations in a variety of biomarkers. As shown in Fig. 3, we observed no effects on Ki67 staining. We did, however, observe significant increases in the apoptotic biomarkers TUNEL and Caspases 3 and 7. Furthermore, we observed differences in AKT phosphorylation and PI3K phosphorylation (Supplementary Fig. 3). However, the magnitude of the changes in AKT and PI3K phosphorylation are limited, and would be difficult to use as a biomarker clinically. The changes observed in caspases and in AKT and PI3K phosphorylation are in agreement with our recent cell culture studies employing NPX (38). Since rats were treated for five days with NPX it may be that all of the observed changes are secondary to alterations in prostaglandins following COX inhibition.

The most important question associated with these studies deal with whether the results presented are likely to reduce adverse events in human. Regarding upper GI toxicity, omeprazole has clearly demonstrated a striking ability to decrease GI bleeding by 50–75% in multiple clinical studies (13,15). There is no reason to think that it would not be effective in a prevention study. The prior findings in arthritic patients that omeprazole reduces gastric toxicity without reducing efficacy of NPX is particularly important since we feel that the anti-arthritic effects of NPX are related to inhibition of COX enzymes; which is the same determinant of its preventive activity (3,4,5). The clinical question regarding potential trials should focus on baseline GI risk. If one precludes persons with prior bleeds or who are greater than 70 years of age (or with major GI co-morbidity), than one might expect to achieve a low incidence of clinically significant upper GI events that would be further reduced by a PPI; e.g., omeprazole. An alternative is to use intermittent dosing with an NSAID. Certainly one could expect that dosing for 3 to 4 weeks on and 3 to 4 weeks off in humans would decrease gastric toxicity. This presumably would allow sufficient time for significant repair of any erosion which might arise. However, since GI toxicity can occur, even short-term combinations with a NSAID and PPI inhibitor which have demonstrable clinical efficacy may be preferable. Alternative methods to reduce gastric toxicity of NSAIDs is the use of NO or H2S analogs (32). However, these are not nearly as advanced clinically and the agents are likely to be more expensive than omeprazole or NPX which are off patent.

One question relevant to clinical trials with NSAIDs is the use of aspirin. There has been substantial enthusiasm for employing low dose aspirin as a cancer preventive (39). There are a few purely practical problems associated with trying to perform aspirin trials (e.g., the exact dose to employ; ≤100 mg vs ≥ 325); how does one randomize the high numbers of persons taking low dose aspirin; and the fact that persons on even low dose aspirin may have significant upper GI adverse effects. In addition, there are a few results which make a comparison of aspirin with a standard NSAID of great interest: (1) animal data from various organs (colon, bladder, skin) show aspirin is routinely ineffective at doses which correspond to human doses of 100 or 325 mg (3) while a wide variety of NSAIDs and Coxibs are highly effective at their HEDs. These animal models predicted the high clinical efficacy of Coxibs and the combination of NSAIDs plus DFMO in colon and skin (9,10,40). (2) there is clinical trial data implying potentially greater efficacy of NSAIDs vs low dose aspirin. Thus, celecoxib resulted in roughly a 45% decrease in total colon adenomas and a 55–65% decrease in advanced adenomas (10), and were similarly effective in individuals taking low dose aspirin. The combination of sulindac plus DFMO resulted in a 65% decrease in total adenomas (40), and an even higher inhibition of advanced adenomas. The inhibition observed in these trials is far greater than that of low to moderate doses of aspirin resulting in a roughly 20–25% decrease in total adenomas and a 35–40% decrease in advanced adenomas. Finally, the efficacy of NSAIDs or coxibs in treatment of urinary bladder cancer in dogs and cats encourages examination of more standard NSAIDs. These studies indirectly argue for the potential of a human trial comparing the efficacy of aspirin with more standard NSAIDs (e.g., NPX vs aspirin).