PGE-M Levels Research Articles

Eicosanoids in non-small cell lung cancer (NSCLC).

7613 Background: COX-2 is frequently upregulated in NSCLC leading to high PGE2 levels that may play a role in NSCLC pathogenesis. PGE-M, the main urine metabolite of PGE2, accurately reflects changes in systemic and intratumoral PGE2 levels [CCR 11:6634, 2005]. A ≥ 70% decline in PGE-M in NSCLC pts treated with docetaxel (D) + celecoxib (C) 400 mg bid was associated with a possible survival benefit. In an ongoing trial, study pts are prospectively selected using a change in PGE-M levels before and after C 600 mg bid which is the dose needed to effect maximum COX-2 inhibition [CCR 12:3381, 2006]. Methods: Previously treated NSCLC pts, PS 0-2, evaluable/measurable disease, adequate marrow, renal and hepatic function, no current NSAID or sulfa allergy are eligible. Screened pts with ≥ 70% decline in PGE-M receive D 75 mg/m2 or pemetrexed 500 mg/m2 q3wk + continued C 600 mg bid × 4 cycles followed by C 400 mg bid until PD or excess toxicity. Results: Through 12/0930 pts (F = 13, M = 17) enrolled. At baseline men had higher PGE-M (median: 34.7 vs. 16.9 ng/mg Cr) and lower LTE4 levels (78.6 vs. 101.4 pg/mg Cr) (LTE4 is main leukotriene metabolite). Median PGE-M levels decreased from 21.4 to 8.1 ng/mg Cr (p = 0.000) pre- and post-C; LTE4 was unchanged (97.7 to 82.3 pg/mg Cr; p = 0.92); and PGI-M decreased from 119 to 47.5 pg/mg Cr (p = 0.004). In men PGE-M decreased post C whereas LTE4 increased (78.6 to 93.4 ng/mg Cr; p = 0.017) suggesting a possible shunting to the 5-LO/leukotriene pathway. Both PGE-M and LTE4 decreased in women. 13 pts received C + chemo; 10 evaluable; 6 SD and 4 PD. Treatment was well tolerated; no cardiac toxicities noted. Conclusions: These preliminary data indicate C 600 mg bid effectively inhibits intratumoral COX-2 in > 40% NSCLC pts. The effect is greatest in current smokers and males compared to never smokers and females. Post C, LTE4 increased in males (former smokers primarily). These data may have implications for chemoprevention trials. [Lung Cancer SPORE CA90949] No significant financial relationships to disclose.

Quantification of urinary PGEm, 6-keto PGF 1α and 2,3-dinor-6-keto PGF 1α by UFLC–MS/MS before and after exercise

Eicosanoids play an important role in the evaluation of pro-inflammatory responses and in the safety and toxicity of novel therapeutic agents. This work describes a high-throughput UFLCMS/MS method for the analysis of three urinary prostanoid biomarkers of pro-inflammatory responses, tetranor PGEm, 6-keto PGF 1α and 2,3-dinor-6-keto PFG 1α. Nine male volunteers of various age and fitness level participated in this study. Six provided pre- and post-exercise samples and three provided intraday samples. Tetranor PGEm and 6-keto PGF 1α increased significantly in patients after exercise ( p < 0.017 and p < 0.029). In individual patient sets, tetranor PGEm levels increased from 1.5- to 6-fold pre- vs. post-exercise, levels of 6-keto PGF 1α increased more dramatically from 2- to 55-fold pre- vs. post-exercise. The prostanoid 2,3-dinor-6-keto PGF 1α remained unchanged post-exercise. Data was normalized to urinary creatinine concentration, which increased approximately 40% post-exercise.

Abstract 2790: Prospective study of urinary prostaglandin E2 metabolite and lung cancer risk

Abstract Cumulative evidence from both in vitro and animal studies suggests that cyclooxygenase-2 (COX-2) may be involved in the development and progression of lung cancer. Overproduction of prostaglandin E synthase and prostaglandin E2 (PGE2), one of the end products of the COX-2 pathway, has been implicated in the pathogenesis of non-small cell lung cancer. It is believed that the pro-inflammation effects of COX-2 pathway are largely mediated through PGE2. PGE2 is quickly converted to 11α-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), a major PGE2 metabolite and excreted from urine. In this study, we evaluated the association of urinary PGE-M levels and lung cancer risk. A nested case-control study was conducted within the Shanghai Women's Health Study, in which 74,942 Chinese women aged 40-70 years were recruited from 1997 to 2000 and followed up for up to 10 years. Urinary PGE-M in 192 incident lung cancer patients were compared to 192 matched controls. The baseline level of urinary PGE-M was comparable between cases and controls. Excluding 20 case-control pairs who were diagnosed with lung cancer within one year of follow-up or excluding 22 case-control pairs who ever smoked did not change the null association. No interaction was found between PGE-M and smoking or passive smoking in relation to lung cancer risk. Our data suggest that the PGE2 pathway may not play a major role in lung cancer development in non-smoking women. Further studies are being conducted to evaluate whether genetic variations in the PGE2 pathway will modify the urinary PGE-M and lung cancer association. Given the substantial laboratory evidence, further evaluation of the COX-2 pathway in lung cancer pathogenesis, including other urinary biomarkers and genetic variations, may be warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2790.

Urinary Prostaglandin E2 Metabolite and Gastric Cancer Risk in the Shanghai Women's Health Study

Chronic inflammation has been implicated in the etiology of gastric cancer. Prostaglandin E(2) (PGE(2)) is one of the major end-products of the cyclooxygenase-2 pathway, an enzyme that is an important mediator of inflammation. Using a novel method of quantifying the primary urinary metabolite of PGE(2) (PGE-M; 11alpha-hydroxy-9,15-dioxo-2,3,4,5-tetranorprostane-1,20-dioic acid), we evaluated urinary PGE-M concentrations in association with subsequent risk of development of gastric cancer in the Shanghai Women's Health Study, a large population-based prospective cohort, using a nested case-control study design. Controls were matched (1:1) to 153 gastric cancer cases by menopausal status; age, time, and date of sample collection; time interval since last meal; and availability of urine sample. Odds ratios (95% confidence intervals) were calculated using conditional logistic regression adjusted for potential confounders. Baseline urinary PGE-M levels were slightly higher among gastric cancer cases with a median of 6.4 ng/mg creatinine (interquartile range, 3.4-11.2) compared with 5.4 ng/mg creatinine among controls (interquartile range, 2.8-9.0), but this difference was not statistically significant (P = 0.34, Wilcoxon). With increasing quartiles of urinary PGE-M levels, the odds ratios (95% confidence intervals) for risk of gastric cancer increased in quartiles 2 to 4: 1.00 (0.48-2.08), 1.40 (0.67-2.91), and 1.98 (0.95-4.13), with a statistically significant test for trend (P = 0.04). The association persisted after additional adjustment for Helicobacter pylori status and was slightly strengthened among non-nonsteroidal anti-inflammatory drug users, subjects with positive H. pylori status, and for cases diagnosed within 46 months after study enrollment. Our findings suggest that higher levels of urinary PGE-M, a marker of inflammation, may be associated with gastric cancer risk.

The effect of sulindac on growth differentiation factor 15 and 13,14-dihydro-15-keto prostaglandin A2 in nipple aspirate fluid

Use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated in several large epidemiologic studies with a lower risk of epithelial cancers including breast though no randomized, placebo controlled phase III studies have been conducted to date. NSAID use was associated with a significantly lower risk of breast cancer among participants in a multiethnic cohort, though the nearly 50% reduction in risk was limited to women with estrogen receptor positive tumors; an observation previously reported by Terry et al., and supported by Gierach et al., and findings of Harris et al for cyclooxygenase-2 (COX-2) inhibitors. These findings in epidemiologic studies are supported by work in human mammary tissues and cell culture where overexpression of COX-2, one of two cyclooxygenase targets of NSAIDs, acts as an early event in the transition of normal breast cells to malignancy. Further, COX-2-associated prostaglandin E2 (PGE2) production has been shown to increase aromatase activity in mammary epithelial cells possibly explaining the observed benefit of NSAID use in hormone receptor positive disease and the recent observation of lower circulating estradiol levels among regular users of NSAIDs. Thus, there exists sufficient rationale for the use of NSAIDs for the prevention of breast cancer. While the best studied target of NSAIDs is the COX isoenzymes, the anti-tumor activities of each of the NSAID agents via 'non COX' pathways remains unknown. Sulindac, a non selective NSAID with well recognized COX-independent induction of apoptosis has remained a strong candidate for chemoprevention largely because of its broader anti-tumor activity. Growth differentiation factor 15 (GDF-15), a potent proapoptotic molecule in the TGF-β superfamily and potential tumor suppressor is upregulated by several NSAIDS independent of its activity as a COX inhibitor. Sulindac sulfone, a metabolite of sulindac and a COX-independent mediator of apoptosis, has been shown to be a potent inducer of GDF-15 at pharmacologically relevant doses whereas Celecoxib, a COX-2 specific inhibitor, has low GDF-15 induction potency even at toxic doses. Other NSAIDs such as aspirin and indomethacin as well as a number of candidate dietary chemopreventives including resveratrol, indole-3-carbinol, 3,3' Diindolylmethane (DIM), and lycopene as well as the PPAR agonists troglitazone, and 15-deoxy-Δ12,14-prostaglandin J2 all show potent induction of GDF-15 leading to the suggestion that GDF-15 might serve as a complementary molecular target to COX-2 for cancer chemoprevention and serve as a surrogate biomarker of apoptotic response. To better understand the effect of NSAIDs in the breast, we conducted a phase 1b dose study using sulindac as a representative drug of the non selective NSAIDs with known activity to induce GDF-15 and inhibit COX-2. To assess sulindac and its metabolites and their effects on a stable COX-2 derived prostaglandins 13,14-dihydro-15-keto prostaglandin A2 [PGEM], and the NSAID inducible growth differentiation factor (GDF-15) in nipple aspirate fluid (NAF), we randomized 30 women at increased risk for breast cancer to 150 mg once daily or twice daily sulindac for 6 weeks. Sulindac and sulfide were detectable in 57.7% of NAF samples with sulfone detectable in 11.6%. Sulindac was associated in a dose independent manner with a nonsignificant decrease in NAF PGEM levels (p = 0.1). Serum levels of sulindac, but not NAF sulindac, were correlated with a decrease in NAF PGEM levels (p = 0.03). GDF-15, showed a borderline significant trend towards higher levels in NAF with 300 mg daily sulindac (p = 0.07). This study suggests that COX inhibition in the breast may be achieved at 150 mg sulindac once daily while a higher daily dose is needed to exhibit activities via COX independent pathways. The study also suggests that NAF levels of PGEM and GDF-15 could be considered as drug effect biomarkers for future early phase chemoprevention trials.

A Phase II Study of Celecoxib in Combination with Paclitaxel, Carboplatin, and Radiotherapy for Patients with Inoperable Stage IIIA/B Non–Small Cell Lung Cancer

Cyclooxygenase (COX)-2 up-regulation plays an important role in the pathogenesis of lung cancer. Selective COX-2 inhibitors have promoted chemosensitivity and radiosensitivity of tumor cells in preclinical trials. In a single-institution phase II study, we sought to determine the effectiveness of concurrent chemoradiation given with celecoxib and examined biomarkers to predict response to COX-2 inhibition. Seventeen patients with stage IIIA or IIIB non-small cell lung cancer (NSCLC) were enrolled in the study. All received 400 mg celecoxib twice daily continuously while on trial in addition to concurrent chemoradiation therapy with paclitaxel and carboplatin. Celecoxib was continued until disease progression. The overall objective response rate was 42.9%, and the median overall survival time was 203 days. In contrast to nonresponders, those patients with complete and partial responses had a significant decrease in the level of urinary 11alpha-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), the major metabolite of prostaglandin E(2), after 1 week of celecoxib administration. Patients with very high levels of PGE-M before initiation of therapy also responded poorly to therapy. Serum vascular endothelial growth factor levels did not predict response or survival. The trial was terminated because it did not meet the predetermined goal of 80% overall response rate. In unselected patients, the addition of celecoxib to concurrent chemoradiotherapy with inoperable stage IIIA/B NSCLC does not improve survival. Urinary PGE-M is a promising biomarker for predicting response to COX-2 inhibition in NSCLC.

Abstract CN04-03: Phase 1b sulindac study for women at high risk for breast cancer

Abstract CN04-03 Background Regular use of non steroidal anti-inflammatory agents (NSAIDs) has been associated with reduced risk of breast cancer. The breast specific activity of each of the numerous NSAID agents and their relative potency to prevent breast cancer remains unknown. Sulindac, a non selective NSAID with pro apoptotic activity is a strong candidate for chemoprevention. Objective and Methods To assess concentrations of sulindac and its metabolites as well as their effects on known molecular markers in nipple aspirate fluid (NAF) in 15 subjects receiving 150 mg once daily and 15 subjects receiving 150 mg twice daily sulindac. NAF and serum samples were collected before and after 6 weeks of sulindac dosing. Results Sulindac and sulfide were detectable in 57.7% of NAF samples with sulfone detectable in 11.6%. There were no statistically significant differences between sulindac or sulfide levels in NAF by dose. There was a significant positive correlation between NAF sulindac and NAF sulfide levels. NAF 13,14-dihydro-15-keto prostaglandin A2 (PGEM), a stable derivative of prostaglandins, exhibited a non significant trend towards decreased levels (p =0.1). Serum levels of sulindac, but not NAF sulindac, was correlated with a decrease in NAF PGEM levels (p=0.03). The NSAID inducible protein, growth differentiation factor GDF-15, showed a borderline significant trend towards higher levels in NAF in the 300 mg daily group (p = 0.07). Serum CRP levels were decreased with sulindac exposure with the greatest decrease observed among those women with elevated CRP levels at baseline. Conclusion Sulindac and its sulfide metabolites partition to the breast and are detectable in NAF. Sulindac exposure was associated with a trend towards reduction in PGEM that was correlated with serum levels of sulindac. GDF-15 levels increased in the high dose group. NAF levels of PGEM and GDF-15 may prove useful as NSAID response biomarkers if for larger efficacy based intervention studies in breast cancer prevention. Citation Information: Cancer Prev Res 2008;1(7 Suppl):CN04-03.

Liquid chromatography–tandem mass spectrometric quantification of the dehydration product of tetranor PGE-M, the major urinary metabolite of prostaglandin E 2 in human urine

A LC–MS/MS method has been developed to analyze tetranor PGE-M, the major urinary metabolite of PGE 2, that involves the acid-catalyzed dehydration of tetranor PGE-M and its deuterated (d 6) analog followed by LC–MS/MS measurement of the dehydrated tetranor PGE-M product (tetranor PGA-M). We also report a method for quantification of creatinine in urine by LC–MS/MS to normalize tetranor PGE-M concentrations with that of urinary creatinine. These methods were used to study the effect of aspirin on urinary tetranor PGE-M levels in healthy male volunteers. Aspirin did not affect urinary creatinine concentrations but decreased urinary tetranor PGE-M concentrations by approximately 44%.

Novel Strategies for the Treatment of Lung Cancer: Modulation of Eicosanoids

Given that lung cancer remains the leading cause of cancerrelated death in the United States, one might question the value of the last three decades of basic and clinical research in this disease. Indeed, although modern platinum-based chemotherapy regimens can double the median survival of non–small-cell lung cancer (NSCLC) patients diagnosed with advanced disease, few patients survive beyond 2 years, and curative therapy remains elusive. In fact, one might further assert that most of the survival improvement beyond that achieved with supportive care alone was realized almost 20 years ago with the introduction of cisplatin into standard care, and that little has changed since that time. Fortunately, the pace of therapeutic progress appears to be accelerating, driven in large part by our improved (and improving) knowledge of lung cancer biology. This knowledge has led to the initiation of seminal studies employing so-called targeted therapy that have yielded clear survival benefits in patients with advanced NSCLC. Encouraged by the aforementioned successes, investigators continue to focus on exploiting key molecular targets that have the potential to enhance therapeutic outcome. One area of particular interest involves the eicosanoids—prostaglandins (PG), prostacyclins, thromboxanes (Tx), and leukotrienes (LT)—which are signaling molecules generated through the oxygenation of arachidonic acid. They exert complex control over many bodily systems, mainly in inflammation or immunity, and act as messengers in the CNS. Recently, a number of preclinical, clinical, and pharmacologic studies have documented the importance of eicosanoids in the development of many cancers, including NSCLC. For example, cyclooxygenase-2 (COX-2), one of two isoforms of COX that catalyzes the conversion of arachidonic acid to prostaglandin PGG2, is frequently upregulated in NSCLC. 8,9 PGG2 is subsequently reduced to PGH2, an unstable endoperoxide intermediate. Specific PG synthases metabolize PGH2 to at least five structurally related bioactive lipid molecules: PGE2, PGD2, PGF2 , PGI2, and TxA2. 10 In turn, COX-2–derived PGE2 can stimulate cellular proliferation and angiogenesis, reduce apoptosis, enhance cellular invasiveness, and inhibit immune surveillance. Each of these contributes to the pathogenesis and progression of NSCLC. Strategies designed to decrease PGE2 production are therefore thought to represent possible therapeutic options in NSCLC. In this issue of the Journal of Clinical Oncology, Edelman et al report the results of Cancer and Leukemia Group B (CALGB) trial 30203 in which patients with advanced NSCLC were randomly assigned to receive a standard platinum-based chemotherapy plus celecoxib (a specific COX-2 inhibitor) or chemotherapy with zileuton (a 5-lipoxygenase [LOX] inhibitor), or chemotherapy with both celecoxib and zileuton. LOX is a key enzyme in the production of LT known to increase cellular proliferation, increase transcriptional activity of oncogenes, and decrease apoptosis. The overall results of this randomized phase II trial were not particularly encouraging as none of the treatment regimens increased overall survival compared with historical controls treated with chemotherapy alone. Consistent with previous reports, however, patients with tumors that expressed moderate to high COX-2 levels as assessed by immunohistochemistry had a poor prognosis. Moreover, treatment with chemotherapy plus celecoxib effected a superior survival in patients with high intratumoral COX-2 expression compared with their counterparts treated with chemotherapy alone. This finding is consistent with a recent report by Chan et al, who noted that regular aspirin use reduced the risk of colorectal cancers that overexpress COX-2 but not the risk of colorectal cancers with weak or absent expression of COX-2. These data collectively suggest that cancers expressing high COX-2 levels may be “addicted” to COX-2 enzymatic activity and therefore potentially more susceptible to COX-2 inhibitors. This is an eminently testable hypothesis. For example, patients with high intratumoral COX-2 levels might be randomly assigned to receive chemotherapy plus a COX-specific inhibitor or a placebo. Another option might be to select patients using a biomarker of intratumoral COX-2 because adequate tumor samples are not always readily available in lung cancer patients. To this end, we recently developed an assay to measure urinary 11 -hydroxy-9,15dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), the major metabolite of PGE2. 16 We found that nonselective and selective inhibitors of COX-2 effectively decreased urinary PGE-M levels to a similar degree in healthy human participants and in individuals with NSCLC, indicating that a majority of PGE-M in humans, with or without an underlying malignancy, is COX-2 derived. We also found that a short course of moderately high-dose celecoxib (400 mg bid) resulted in a marked decrease in intratumoral PGE2 levels, with a concomitant decline in urinary PGE-M levels. Collectively, these data indicate that urinary PGE-M could be a useful biomarker of intratumoral COX-2 activity and PGE2 production in NSCLC. JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 26 NUMBER 6 FEBRUARY 2

Phase II study of the clinical and biological activity of cetuximab and celecoxib in patients (pts) with refractory colorectal cancer (CRC)

14516 Background: The epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) pathways play key roles in colon carcinogenesis. EGFR and COX-2 signaling can be mutually reinforcing. Celecoxib, a selective COX-2 inhibitor, inhibits the EGFR pathway through inhibition of PI3/Akt and NF-κB pathways. This study explores the clinical and biological effects of blockade of complementary pathways of the EGFR by combining cetuximab with celecoxib. Methods: Cetuximab-naive pts with refractory CRC were treated with cetuximab (400 mg/m2 loading dose followed by weekly cetuximab at 250 mg/m2) and celecoxib (200 mg orally twice daily). Patients were assessed for response every 8 weeks. Urinary PGE-M, a metabolite of PGE-2 which correlates with in vivo COX-2 activity, and serum TGF-a, a ligand that binds to EGFR, were measured to assess the biological effect of COX-2 and EGFR blockade. Results: Seventeen of a planned 40 pts accrued to this study. Currently, data is available on 12 of these patients. Three pts had to stop treatment on 1st infusion due to a grade 3 cetuximab infusion reaction. One pt had a mild infusion reaction and was successfully rechallenged after pre-medication and a slower infusion rate. By RECIST criteria, 1 pt (8.3%) achieved a partial response (PR) and 4 pts (33%) had a best response of stable disease (SD). The mean time on study for the 9 pts without grade 3 infusion reaction was 95.7 days (range 28- 175). Pts with SD or PR were on study for a mean of 135 days (range 107–175 days) and pts with progressive disease (PD) were on study for a mean of 47 days (range 28–56 days). Baseline urinary PGE-M levels in pts with SD or PR were higher than in pts with PD (30.5+17.6 vs 18.1+9.4 ng/mg Cr). Baseline serum TGF-a levels were similar between the two groups (27.1+9.6 vs 25.4+10.3 pg/ml respectively). Conclusions: In preliminary analysis, this regimen resulted in response rates similar to published response rates for single agent cetuximab. Pts with at least SD had higher baseline urinary PGE-M levels, which may serve as a marker for efficacy of this combined therapy. Data on serial TGF-a and PGE-M levels will be presented. Response data on all 17 pts will be presented. Supported by P50 CA95103 and Bristol-Myers Squibb. Celecoxib provided by Pfizer. [Table: see text]

Urine PGE-M: A Metabolite of Prostaglandin E 2 as a Potential Biomarker of Advanced Colorectal Neoplasia

The enzyme cyclooxygenase-2 is expressed in a majority of colorectal carcinomas (CRCs) and is important in prostaglandin production. We have developed an accurate method to measure the urinary metabolite of prostaglandin E(2) (PGE-M) using recently developed mass spectrometric techniques. The purpose of this pre-validation study was to determine if urinary PGE-M levels can be used as a biomarker to discriminate between healthy patients and those with colorectal disease. Urine PGE-M was assessed in a total of 228 patients with CRC, colonic adenomatous polyps, Crohn's disease, and in subjects with no endoscopically detectable disease. Thirteen rectal carcinoma patients were treated with celecoxib and urinary PGE-M was measured before and after treatment. Urine PGE-M levels were increased among healthy men compared with healthy women (median, 8.59 [interquartile range (IQR), 5.67-22.3] vs 4.25 [IQR, 2.35-6.03], P = .0027). Urine PGE-M levels among patients with Crohn's disease (median, 19.85 [IQR, 6.89-90.2]), CRC (median, 14.65 [IQR, 5.94-92.1]), or large adenomas greater than 1 cm in size (median, 18.85 [IQR, 11.9-25.6]) were significantly increased when compared with patients who had either small polyps less than 1 cm in size (median, 9.69 [IQR, 6.41-22.2]), or no polyps (median, 7.05 [IQR, 2.35-24.7]) (P = .0001). PGE-M levels decreased significantly after celecoxib treatment in patients with rectal cancer (median, 21.7 [IQR, 16.2-29.9] vs 9.14 [IQR, 7.14-13.2], P = .009). The increase in urinary PGE-M in patients with colorectal cancers and large adenomas suggests that urinary PGE-M is a potentially useful biomarker for the detection of advanced colorectal neoplasia.

Targeting Cyclooxygenase-2 in Recurrent Non–Small Cell Lung Cancer: A Phase II Trial of Celecoxib and Docetaxel

Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in prostaglandin (PG) synthesis and is overexpressed in 70% to 90% of non-small cell lung cancers (NSCLC). Preclinical studies suggest inhibition of COX-2 can enhance the cytotoxic effect of docetaxel. To test this concept clinically, we administered celecoxib (400 mg p.o. twice daily) plus docetaxel (75 mg/m(2) every 3 weeks) to a cohort of patients with recurrent, previously treated NSCLC. Patients first received single agent celecoxib for 5 to 10 days to ascertain the effectiveness of COX-2 inhibition, which was determined by measuring pre- and post-celecoxib levels of urinary 11alpha-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), the major metabolite of prostaglandin E(2) (PGE(2)). We enrolled 56 patients (35 men, 21 women; median age, 61 years). All patients had received at least one prior chemotherapy regimen. The overall response rate was 11% and median survival was 6 months, similar to that observed with docetaxel alone. Pre-celecoxib urinary PGE-M decreased from a mean level of 27.2 to 12.2 ng/mg Cr after 5 to 10 days of celecoxib (P = 0.001). When grouped by quartile, patients with the greatest proportional decline in urinary PGE-M levels experienced a longer survival compared to those with no change or an increase in PGE-M (14.8 versus 6.3 versus 5.0 months). Our data suggest that combining celecoxib with docetaxel using the doses and schedule employed does not improve survival in unselected patients with recurrent, previously treated NSCLC. However, in light of the apparent survival prolongation in the subset with a marked decline in urinary PGE-M levels, further investigation of strategies designed to decrease PGE(2) synthesis in NSCLC seems warranted.

Quantification of the major urinary metabolite of PGE 2 by a liquid chromatographic/mass spectrometric assay: determination of cyclooxygenase-specific PGE 2 synthesis in healthy humans and those with lung cancer

Prostaglandin (PG)E 2 is a major cyclooxygenase (COX) product that is important in human physiology and pathophysiology. Quantification of systemic PG production in humans is best assessed by measuring excreted urinary metabolites. Accurate and easy-to-perform assays to quantify the major urinary metabolite of PGE 2, 11α-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), do not exist. We now report the development of a robust and facile method to measure urinary PGE-M excretion in humans using stable isotope dilution techniques employing liquid chromatography/tandem mass spectrometry (LC/MS/MS). Concentrations of the metabolite in urine from healthy humans are nearly twofold greater in men than in women (10.4 ± 1.5 vs. 6.0 ± 0.7 ng/mg creatinine). Levels of PGE-M in healthy humans are suppressed significantly not only by the nonselective COX inhibitor ibuprofen but also by the COX-2 selective inhibitor rofecoxib, suggesting that the majority of PGE 2 formed in vivo is derived from COX-2. Increased COX-2 expression and increased PGE 2 production are associated with malignancy. Levels of PGE-M were found to be greatly increased in humans with unresectable non-small cell cancer of the lung, and this increase is dramatically reduced by administration of the COX-2 inhibitor celecoxib, implying that COX-2 contributes significantly to the overproduction of PGE 2. In summary, quantification of PGE-M using LC/MS/MS provides a facile and accurate method to assess PGE 2 formation in human physiological and pathophysiological processes.

Urine PGE-M as a marker of intratumoral cyclooxygenase-2 activity in non-small cell lung cancer (NSCLC)

9527 Background: Prostaglandin E2 (PGE2) is implicated in the pathogenesis of NSCLC. PGE2 levels are elevated in NSCLC often in association with overexpression of cyclooxygenase-2 (COX2). The most accurate method to assess PG production in vivo in humans is via quantification of urinary metabolites. We hypothesized that urine levels of 11α-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), the major metabolite of PGE2, could be a useful marker of tumor COX2 activity & PGE2 production. Methods: PGE-M levels were determined in urine specimens derived from a previously reported phase II trial employing celecoxib & docetaxel in recurrent NSCLC (Proc. ASCO 22:640, 2003). We employed liquid chromatography electrospray ionization MS using selected reaction monitoring. PGE-M was chemically synthesized & converted to a [2H6] methyloxime isomer for use as an internal standard. After purification, endogenous PGE-M was analyzed as a methyloxime derivative. Assay precision is ± 5%; accuracy is 92%. Results: Pts=57; RR=10.5%; MST=228 days. Urinary PGE-M levels = 11.3 ± 5.7 (mean±2 SD, n=10) & 6.4 ± 3.8 ng/mg creatinine (mean±2 SD, n=10) in normal men & women. In men & women with recurrent NSCLC, PGE-M levels were elevated 2-fold (p<0.01) & 3.1-fold above normal mean (p<0.005). PGE-M levels fell by a mean of 51% (p<0.001) & 43% (P<0.001) respectively in men & women following 5–7 days of celecoxib (400 mg p.o. bid). These data suggest that a large proportion of the PGE2 overproduction is COX2 derived. In a multivariate model accounting for sex, race, smoking history & histology we correlated PGE-M changes with clinical parameters & found a strong association with survival. Pts experiencing a reduction in urine PGE-M levels after celecoxib administration were much less likely to die than those who did not (P<0.005). Conclusion: Changes in urinary PGE-M levels following short course celecoxib may be a useful predictor of intratumoral COX2 inhibition. (Grant support: CA68485; P5090949). Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Aventis

Urine PGE-M as a marker of intratumoral cyclooxygenase-2 activity in non-small cell lung cancer (NSCLC)

9527 Background: Prostaglandin E2 (PGE2) is implicated in the pathogenesis of NSCLC. PGE2 levels are elevated in NSCLC often in association with overexpression of cyclooxygenase-2 (COX2). The most accurate method to assess PG production in vivo in humans is via quantification of urinary metabolites. We hypothesized that urine levels of 11α-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), the major metabolite of PGE2, could be a useful marker of tumor COX2 activity & PGE2 production. Methods: PGE-M levels were determined in urine specimens derived from a previously reported phase II trial employing celecoxib & docetaxel in recurrent NSCLC (Proc. ASCO 22:640, 2003). We employed liquid chromatography electrospray ionization MS using selected reaction monitoring. PGE-M was chemically synthesized & converted to a [2H6] methyloxime isomer for use as an internal standard. After purification, endogenous PGE-M was analyzed as a methyloxime derivative. Assay precision is ± 5%; accuracy is 92%. Results: Pts=57; RR=10.5%; MST=228 days. Urinary PGE-M levels = 11.3 ± 5.7 (mean±2 SD, n=10) & 6.4 ± 3.8 ng/mg creatinine (mean±2 SD, n=10) in normal men & women. In men & women with recurrent NSCLC, PGE-M levels were elevated 2-fold (p<0.01) & 3.1-fold above normal mean (p<0.005). PGE-M levels fell by a mean of 51% (p<0.001) & 43% (P<0.001) respectively in men & women following 5–7 days of celecoxib (400 mg p.o. bid). These data suggest that a large proportion of the PGE2 overproduction is COX2 derived. In a multivariate model accounting for sex, race, smoking history & histology we correlated PGE-M changes with clinical parameters & found a strong association with survival. Pts experiencing a reduction in urine PGE-M levels after celecoxib administration were much less likely to die than those who did not (P<0.005). Conclusion: Changes in urinary PGE-M levels following short course celecoxib may be a useful predictor of intratumoral COX2 inhibition. (Grant support: CA68485; P5090949). Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Aventis

Serial changes in the biophysical profile in patients undergoing cervical ripening with a controlled release PGE2 vaginal pessary.

The purpose of this study was to evaluate the effects of exogenous administration of PGE2 upon the components of the biophysical profile. The study group included 17 nulliparas at > or = 38 weeks gestation, with a Bishop score of < or = 4, requiring induction of labor. A controlled release vaginal pessary containing 10 mg of PGE2, designed to release hormones at approximately 0.8 mg per hour in vitro, was used for 12 hours of cervical ripening. The BPP was performed by the same sonographer at three intervals: prior to pessary insertion, at 6 hours, and 12 hours. None of these patients had membrane rupture or went into spontaneous labor during the ripening process. All patients subsequently required amniotomy and oxytocin. The proportion of patients scoring 2 points for fetal breathing movements decreased from 59% at baseline to 0% at 12 hours, P < 0.0005, and the proportion of patients with fetal body movements decreased from 100% at baseline to 25% at 12 hours, P < 0.0005. However, the other components of the biophysical profile were not affected. The mean maternal plasma PGE2 metabolite concentrations were 235 pg/ml, 475 pg/ml, and 466 pg/ml at 0, 6 and 12 hours, respectively, P < 0.005. In term patients, vaginal administration of the PGE2 pessary was associated with improved Bishop score over 12 hours and significant increases in maternal plasma PGEM levels at 6 hours and 12 hours. These changes were inversely related to fetal breathing and body movements.

Gas chromatography/negative ion chemical ionization triple quadrupole mass spectrometric determination and pharmacokinetics of 11α-Hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid in plasma

11α-Hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid (PGE-M) was determined in plasma. Analysis was performed using an isotope dilution assay and gas chromatography/triple quadrupole mass spectrometry (GC/MS/MS). Basal levels of PGE-M were 64.05 ± 34.17 pg/ml. After infusion of 120 μg prostaglandin E 1 (PGE 1) in six subjects in 15 min, maximum levels of PGE-M of 618.6 ± 210.3 pg/ml were reached 20–45 min after the end of infusion. 3 h after the end of the infusion, plasma levels were close to the preinfusion levels. The pharmacokinetics of PGE-M in plasma were also determined. PGE-M has a half-life of 8.96 ± 3.53 min for formation and 31.71 ± 6.39 min for elimination. In addition to PGE-M, PGE 1, 15-keto-PGE 1 and PGE 0 were also determined by GC/MS/MS.

A dystocia and caesarean section model to characterize uteroplacental prostaglandin concentrations associated with retained placenta in dairy cattle

Plasma concentrations of prostaglandin F 2a (PGF 2a, 13, 14-dihydro-15-keto-prostaglandin F 2a (PGFM), prostaglandin E 2 (PGE 2) and 13,14-dihydro-15-keto-prostaglandin E 2 (PGEM) were determined by RIA in blood samples taken from the jugular vein and the uteroplacental circulation (umbilical vein, umbilical artery and uterine vein) of 13 Holstein Friesian cows during caesarean section. According to discharge of placenta cows were divided in 2 groups. Group I (shedding of placenta within 12 hours, NRP, n=8) and Group II (retained placenta, RP, n=5). In blood samples taken from the jugular vein before surgery, no significant differences existed between groups regarding PGF 2a, PGFM, PGE 2 and PGEM. Concentrations of PGF 2a and PGFM in the uteroplacental circulation of NRP cows were significantly higher than those of RP cows (except for PGFM in the umbilical vein). For all sampling sites except the jugular vein before surgery, PGE 2 and PGEM levels of NRP cows were significantly higher compared to RP cows.

Blood Levels of Prostaglandin Metabolites (PGFM, PGEM) after Parturition in Cows with and without Retained Placenta Considering Spontaneous Calving and Dystocia

Blood samples were taken from 71 Holstein Friesian cows at 0, 3, 24 and 48 hours after parturition. Peripheral plasma concentrations of prostaglandin F2 alpha and E2 metabolites (PGFM, PGEM) were determined by radioimmunoassay. The cows were divided into 4 groups, according to type of calving (spontaneous, dystocia) and discharge of placenta. Group 1: spontaneous calving, shedding of placenta within 12 hours (SC, NRP, n = 19); group 2: spontaneous calving, retained placenta (SC, RP, n = 7); group 3: Dystocia, shedding of placenta within 12 hours (Dys, NRP, n = 32); group 4: Dystocia, retained placenta (Dys, RP, n = 13). The PGFM concentrations decreased within 3 hours after parturition only in NRP cows (groups 1 and 3, p < 0.05). Three hours after calving the animals of group 3 had considerably higher PGFM levels than those in group 4 (0.46 +/- 0.26 vs 0.26 +/- 0.11 ng/ml). Significant decreases (p < 0.05) in PGEM concentrations occurred in cows that shed the placenta within 24 hours after parturition. Cows that retained the placenta showed a significant slower decrease of the PGEM levels. It is concluded that postpartum prostaglandin levels are influenced by stress during parturition (dystocia, transportation) as well as by retained placenta.

Fetal breathing movements and prostaglandin levels in pregnancies complicated by premature rupture of the membranes.

We have previously demonstrated that maternal and fetal prostaglandin levels may be elevated in patients with pregnancies complicated by premature and prolonged rupture of the membranes (PPROM) compared to patients with intact membranes. In the fetal lamb, infusion of prostaglandin abolishes fetal breathing movements and in human pregnancies with PPROM and with poor outcome, fetal breathing movements are absent. The aim of this study was to determine fetal breathing activity in pregnancies complicated by PPROM which had elevated prostaglandin levels. One-hour ultrasound examinations were performed on nine fetuses whose mothers had had premature rupture of the membranes at 28 weeks gestation and a median of 4 days prior to the ultrasound examination. The number of fetal breathing movements (FBM) and percentage of time that each fetus spent breathing was documented and this was then related to control values. Following the completion of the ultrasound examination, cordocentesis was performed and blood sent for estimation of bicyclo PGEM levels. All of the fetuses made some breathing activity during the one-hour period, but the number of FBM varied from 1 to 181 (median 21). The percentage time that the fetuses spent breathing was much lower than that which would be expected for their gestational age, being a median of 1.3% (range less than 0.1 to 50.8%) of control values. As bicyclo PGEM levels were elevated in these nine fetuses, these data suggest that reduction in breathing activity in fetuses of pregnancies complicated by PPROM may be due to elevated prostaglandin levels.