E-7869 (R-flurbiprofen) inhibits progression of prostate cancer in the TRAMP mouse.

E-7869 (R-flurbiprofen) is a single enantiomer of a racemic nonsteroidal anti-inflammatory drug. E-7869 does not inhibit either cyclooxygenase-1 or cyclooxygenase-2. We used the transgenic adenocarcinoma mouse prostate (TRAMP) mouse, a prostate cancer model, to evaluate the effect of this drug on prostate cancer progression. Sixty 12-week-old male TRAMP mice were placed randomly into five groups. The animals were treated by daily oral gavage with vehicle (1% carboxymethylcellulose) or E-7869 for 18-weeks. During the course of the study, two diets were used. Three groups (vehicle, 15-mg/kg, and 20-mg/kg drug treatments) received a Teklad diet containing 2.4% saturated fat [a high saturated fat (HSF) diet], and two groups (vehicle and 20 mg/kg drug treatment) received an AIN-93G diet containing 1.05% saturated fat [a low saturated fat (LSF) diet]. At necropsy, the urogenital system and periaortic lymph nodes were removed and weighed. The prostate lobes, seminal vesicles, lungs, and periaortic lymph nodes were preserved and sectioned for histological evaluation. The lung and periaortic lymph nodes were graded as to the presence (+) or absence (-) of metastasis; the urogenital tissues were graded on a 1-6 scale for degree of neoplasia/carcinoma. For both diets, the urogenital wet weights and lymph node wet weights in the 20-mg/kg treatment groups were significantly lower as compared to vehicle control groups. In addition, treatment with 20 mg/kg E-7869 in the LSF diet group resulted in a significantly lower primary tumor incidence (P < 0.05) and reduced incidence of metastasis. In this treatment group, the reduced incidence of metastasis was not statistically significant because the LSF diet itself resulted in a remarkably lower incidence of metastasis in the vehicle control group (10% LSF versus 40% HSF). Treatment with 20 mg/kg E-7869 on the HSF diet resulted in a significantly lower incidence of metastasis (P < 0.05) and a reduction in the primary tumor incidence. These results suggest that E-7869 is a promising chemopreventive and treatment for human prostate cancer.

Treatment and survival study in the C57BL/6J-APC(Min)/+(Min) mouse with R-flurbiprofen.

Our previous studies with the mouse model of familial adenomatous polyposis (FAP), C57BL/6J-APC(Min)/+ or Min mouse, demonstrated the optimal dose for adenoma reduction with R-flurbiprofen was 10 mg/kg/day as an undivided dose. Divided doses exhibited no increased efficaciousness. This study examines 10 mg/kg R-flurbiprofen daily (qd) on survival as well as a second daily (q.o.d.) schedule and compares it with sulindac sulfone. The q.o.d. schedule at 10 mg/kg was equally efficacious as qd treatment at the same dose. For the q.o.d. group, tumor number decreased similarly (p<0.01); while body weight gain (p<0.01), hematocrit and average tumor area (both, p<0.05) were improved compared with qd treatment. Treatment with R-flurbiprofen (10 mg/kg/day) increased survival significantly (p=0.0004, log-rank) compared to vehicle treated animals. Major biological endpoints (hematocrit, weight gain, tumor number, average and total area [99% reduction]) were significantly improved in treated animals (p<0.01). Sulindac sulfone treatment (50 mg/kg/day) of the Min mouse produced no significant biological benefit. The dose schedule study suggests that for tumor reduction it is necessary to attain a threshold drug-level but not necessarily sustain it over 24 hrs (pharmacodynamic t1/2 >> pharmacokinetic t1/2). During the period of administration R-flurbiprofen dramatically prolongs survival for the mouse model of the human disease, FAP.

Mechanism of enhancement of intestinal ulcerogenicity of S-aryl propionic acids by their R-enantiomers in the rat.

We previously observed a marked increase in gastrointestinal toxicity of rac-flurbiprofen compared to the therapeutically equivalent dose of the S enantiomer. This paper quantitates these observations and examines the mechanism by which this paradoxical toxicity occurs. We have evaluated the ulcer scores, mucosal neutrophil infiltration, by immunostaining of CD11/18 antigen, and mucosal neutrophil activity by myeloperoxidase measurement at two dose levels of (R)-, (S)-, and rac-flurbiprofen, administered over 30 days. Dose-response for intestinal ulcer production was observed for rac- and (S)-flurbiprofen; animals given (R)-flurbiprofen exhibited no ulcers. Yet rac-flurbiprofen proved to be twice as ulcerogenic as (S)-flurbiprofen. The mechanism of the exacerbation of gastrointestinal toxicity of (S)-flurbiprofen by the noncyclooxygenase inhibiting (R)-flurbiprofen is believed to be associated with its effect on ICAM-1 up-regulation. This is followed by neutrophil adhesiveness to ICAM-1 via the LFA-1 antigen on its surface and the extravasation of neutrophils into the tissue. We also examined the effect of high dose (R)-flurbiprofen vs vehicle over 15 days in animals in which ulcers had been produced by treatment with (S)-flurbiprofen for the previous 15 days. (R)-flurbiprofen did not sustain induced ulcers. The results of this study suggest that human studies be conducted to determine if enhanced gastrointestinal toxicity occurs in man. This is at issue since rac compounds of this class are available over the counter and others may be introduced.

R-flurbiprofen chemoprevention and treatment of intestinal adenomas in the APC(Min)/+ mouse model: implications for prophylaxis and treatment of colon cancer.

We used the C57BL/6J-APC(Min)/+ mouse (Min mouse) to evaluate the chemopreventive effects of R-flurbiprofen (R-FB), the noncyclooxygenase-inhibiting enantiomer of FB. Weanling Min mice were administered 6 weeks of oral treatment with R-FB using 2.5-25 mg/kg of R-FB once per day (q.d.), 2.5-10 mg/kg of R-FB twice per day (b.i.d.), and 5 mg/kg of R-FB b.i.d. challenged with a high saturated fat diet. At necropsy we determined tumor and ulcer numbers, tumor size, and plasma levels of R- and S-FB. A linear dose response was observed from 2.5 to 10 mg/kg of R-FB, regardless of whether the drug was administered as a single or divided dose. Reductions in tumor number were significant (P < or = 0.02) for doses of R-FB from 2.5 to 25 mg/kg/day. A dose of 5 mg/kg R-FB b.i.d. was able to overcome the doubling in tumor number associated with the high saturated fat diet. At 20 and 25 mg/kg/day R-FB, we obtained the maximum response with up to 90% inhibition of total tumor number. At these doses, however, there was toxicity and animal deaths. This toxicity was associated with ulceration, presumably resulting from the in vivo epimerization of R- to S-FB that occurs in the mouse. Thus, we evaluated the oral pharmacokinetics of R-FB and its conversion to S-FB in wild-type mice. These kinetics experiments revealed inversion rates of 7.3 and 11.0% for the 2.5 and 10 mg/kg R-FB doses, respectively. S-FB administered alone (0.5 and 2.0 mg/kg q.d.), in doses mimicking the concentrations of S-FB associated with the R to S epimerization of the doses of R-FB used in our experiments, had little or no antitumor efficacy (P > 0.05). Thus, we conclude that R-FB itself, not the S-FB resulting from epimerization in the mouse, inhibits adenoma formation in the Min mouse. In humans, where there is no R to S epimerization, it is possible that larger doses of R-FB can be used without causing cyclooxygenase inhibition and its resulting ulcerogenicity and other side effects. To assess the effect of R-FB on established adenomas, we allowed 40 Min mice to remain untreated until 70 days of age (the time of necropsy in the previous experiments) and then treated them for an additional 42 days with 10 mg/kg R-FB q.d. or 5 mg/kg R-FB b.i.d.. Both drug-treated groups demonstrated tumor numbers significantly less than that of the vehicle control (P < 0.01). Our results suggest that prophylaxis and treatment trials of R-FB should be extended to humans.

Endogenous natriuretic factors 7: biospecificity of a natriuretic gamma-tocopherol metabolite LLU-alpha.

The structural elucidation and mechanism of action of a potential component, LLU-alpha, of what is possibly a multifactorial complex known as "natriuretic hormone" was recently reported [Wechter, W.J. et al. (1996a) Proc. Natl. Acad. Sci. U.S.A. 93: 6002-6007]. "Natriuretic hormone," a long-sought factor, is believed to regulate extracellular fluid volume and consequently be pathomimetic for hypertension, cirrhosis, congestive heart failure and other volume expanded states. The studies reported herein further characterize LLU-alpha. The precursor of the endogenous LLU-alpha was demonstrated to be gamma-tocopherol by radiolabeling studies. The pharmacokinetics of infused rac-LLU-alpha proved to be biphasic (half-lives: 12 min and 6 h). Specificity of the inhibition of the 70 pS potassium channel of the thick ascending limb of the loop of Henle was examined with the natural S-enantiomer being the most potent known inhibitor whereas the analogous alpha-tocopherol metabolite, rac-5-Me-LLU-alpha, showed no inhibition. Rac-LLU-alpha does not inhibit two isozymes of the Na+/K+-ATPase. LLU-alpha is natriuretic acting via inhibition of the 70 pS potassium channel and not Na+/K+-ATPase, the assumed mechanism of action of the "natriuretic hormone." LLU-alpha, a metabolite of a vitamin, if it were found to play a role in the regulation of extracellular fluid volume, would be the second example of a vitamin acting as a precursor for a hormone. Of considerable interest is the fact that this manuscript reports the first biological activity of gamma-tocopherol, a member of the vitamin E complex.

Antiproliferative effects of the enantiomers of flurbiprofen.

Nonsteroidal antiinflammatory drugs (NSAIDs) are recognized for inhibiting growth of colon tumors in animal models, and for reducing the risk of colon cancer in humans. The mechanisms involved have not been established, but are thought to be related to reduced prostaglandin biosynthesis. The present study investigates the effect of COX-inhibiting and non-COX-inhibiting enantiomers of flurbiprofen on rat colonocyte proliferation. Intestinal ulceration was used as a surrogate indicator of COX inhibition. Sprague Dawley rats were treated orally with 6.3 mg/kg of R- or s-flurbiprofen or vehicle. Colonocyte labeling index and small bowel ulcer index were measured. R-flurbiprofen and S-flurbiprofen significantly reduced colonocyte labeling index, by 34% and 23% respectively, compared with vehicle. R-flurbiprofen caused minimal ulcer formation (4.48 mm2) compared with S-flurbiprofen (94.4 mm2). These findings suggest that R-flurbiprofen-mediated control of colonocyte proliferation is independent of prostaglandin biosynthesis.

Endogenous natriuretic factors. 5. Synthesis and biological activity of a natriuretic metabolite of diltiazem and its derivatives.

In our search for endogenous natriuretic factors from human uremic urine, we have previously identified a new metabolite of the drug diltiazem (Murray et al. Life Sci. 1995, 57, 2145-2161). The structure of this metabolite, (+)-(2S,3S)-3-hydroxy-5-(2-hydroxyethyl)-2,3-dihydro-2-(4-methoxyphenyl) -1,5-benzothiazepin-4(5H)-one (LLU-beta1; 2), was proved by unequivocal synthesis from a diltiazem synthon. The synthetic material also proved to be natriuretic as had the urinary isolate. Given the acetylation at C-3 in diltiazem, the 3-monoacetate (8) and diacetate (3) derivatives of 2 were prepared. The 4-nor-keto (6) derivative of 2 was also synthesized. Only the parent 2 induced natriuresis over a range of doses without accompanying kaliuretic activity at some doses.