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BACKGROUND A clinical assay was implemented to perform next-generation sequencing (NGS) of genes commonly mutated in multiple cancer types. This report describes the feasibility and diagnostic yield of this assay in 381 consecutive patients with non–small cell lung cancer (NSCLC). METHODS Clinical targeted sequencing of 23 genes was performed with DNA from formalin-fixed, paraffin-embedded (FFPE) tumor tissue. The assay used Agilent SureSelect hybrid capture followed by Illumina HiSeq 2000, MiSeq, or HiSeq 2500 sequencing in a College of American Pathologists–accredited, Clinical Laboratory Improvement Amendments–certified laboratory. Single-nucleotide variants and insertion/deletion events were reported. This assay was performed before methods were developed to detect rearrangements by NGS. RESULTS Two hundred nine of all requisitioned samples (55%) were successfully sequenced. The most common reason for not performing the sequencing was an insufficient quantity of tissue available in the blocks (29%). Excisional, endoscopic, and core biopsy specimens were sufficient for testing in 95%, 66%, and 40% of the cases, respectively. The median turnaround time (TAT) in the pathology laboratory was 21 days, and there was a trend of an improved TAT with more rapid sequencing platforms. Sequencing yielded a mean coverage of 1318×. Potentially actionable mutations (ie, predictive or prognostic) were identified in 46% of 209 samples and were most commonly found in KRAS (28%), epidermal growth factor receptor (14%), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (4%), phosphatase and tensin homolog (1%), and BRAF (1%). Five percent of the samples had multiple actionable mutations. A targeted therapy was instituted on the basis of NGS in 11% of the sequenced patients or in 6% of all patients. CONCLUSIONS NGS-based diagnostics are feasible in NSCLC and provide clinically relevant information from readily available FFPE tissue. The sample type is associated with the probability of successful testing. Cancer 2015;121:631–639. © 2014 American Cancer Society.

Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (≥ 1000× average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4-100.0 for sensitivity and 94.2-100.0 for specificity) or whole-genome sequencing (95% CI = 89.1-100.0 for sensitivity and 99.9-100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2-100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of ≥ 15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management.

Non-steroidal anti-inflammatory drugs (NSAIDs) specifically inhibit cyclooxygenase (COX) activity and are widely used as anti-arthritics, post-surgical analgesics, and for the relief of acute musculoskeletal pain. Recent studies suggest that non-specific NSAIDs, which inhibit both COX-1 and COX-2 isoforms, delay bone healing. The objectives of this study were 2-fold; first, to measure the relative changes in the normal expression of COX-1 and COX-2 mRNAs over a 42 day period of fracture healing and second, to compare the effects of a commonly used non-specific NSAID, ketorolac, with a COX-2 specific NSAID, Parecoxib (a pro-drug of valdecoxib), on this process. Simple, closed, transverse fractures were generated in femora of male Sprague-Dawley rats weighing approximately 450 g each. Total RNA was prepared from the calluses obtained prior to fracture and at 1, 3, 5, 7, 10, 14, 21, 35 and 42 days post-fracture and levels of COX-1 and COX-2 mRNA were measured using real time PCR. While the relative levels of COX-1 mRNA remained constant over a 21-day period, COX-2 mRNA levels showed peak expression during the first 14 days of healing and returned to basal levels by day 21. Mechanical properties of the calluses were then assessed at 21 and 35 days post-fracture in untreated animals and animals treated with either ketorolac or high or low dose parecoxib. At both 21 and 35 days after fracture, calluses in the group treated with the ketorolac showed a significant reduction in mechanical strength and stiffness when compared with controls (p

Prostaglandin E(2) (PGE(2)) is the major prostaglandin produced both centrally and in the periphery in models of acute and chronic inflammation, and its formation in both locations is blocked by cyclooxygenase-2 (COX-2) inhibitors such as celecoxib. In animal models of inflammation, PGE(2) inhibition in the brain may occur secondarily to a peripheral action by inhibiting local PG formation that elicits increased firing of pain fibers and consequent activation of PG synthesis in the central nervous system (CNS). Celecoxib was studied in the kainate-induced seizure model in the rat, a model of direct central prostaglandin induction, to determine whether it can act directly in the CNS. In the kainate-treated rat brain there was increased PGE(2), PGF(2alpha), and PGD(2) production, with COX activity and PGE(2) formation increased about 7-fold over normal. We quantitated mRNA levels for enzymes involved in the prostaglandin biosynthetic pathways and found that both COX-2 and PGE synthase (PGEs) mRNA levels were increased in the brain; no changes were found for expression of COX-1 or PGD synthase mRNA. By Western blot analysis, COX-2 and PGEs were induced in total brain, hippocampus, and cortex, but not in the spinal cord. Immunohistological studies showed that COX-2 protein expression was enhanced in neurons. Dexamethasone treatment reduced the expression of both COX-2 and PGEs in kainate-treated animals. Celecoxib reduced the elevated PGE(2) levels in brain of kainate-treated rats and inhibited induced COX activity, demonstrating the ability of this compound to act on COX-2 in CNS. Doses of celecoxib that inhibited brain COX-2 were lower than those needed for anti-inflammatory activity in adjuvant arthritis, demonstrating a potent direct central action of the compound.

A clinical assay was implemented to perform next-generation sequencing (NGS) of genes commonly mutated in multiple cancer types. This report describes the feasibility and diagnostic yield of this assay in 381 consecutive patients with non-small cell lung cancer (NSCLC).Clinical targeted sequencing of 23 genes was performed with DNA from formalin-fixed, paraffin-embedded (FFPE) tumor tissue. The assay used Agilent SureSelect hybrid capture followed by Illumina HiSeq 2000, MiSeq, or HiSeq 2500 sequencing in a College of American Pathologists-accredited, Clinical Laboratory Improvement Amendments-certified laboratory. Single-nucleotide variants and insertion/deletion events were reported. This assay was performed before methods were developed to detect rearrangements by NGS.Two hundred nine of all requisitioned samples (55%) were successfully sequenced. The most common reason for not performing the sequencing was an insufficient quantity of tissue available in the blocks (29%). Excisional, endoscopic, and core biopsy specimens were sufficient for testing in 95%, 66%, and 40% of the cases, respectively. The median turnaround time (TAT) in the pathology laboratory was 21 days, and there was a trend of an improved TAT with more rapid sequencing platforms. Sequencing yielded a mean coverage of 1318×. Potentially actionable mutations (ie, predictive or prognostic) were identified in 46% of 209 samples and were most commonly found in KRAS (28%), epidermal growth factor receptor (14%), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (4%), phosphatase and tensin homolog (1%), and BRAF (1%). Five percent of the samples had multiple actionable mutations. A targeted therapy was instituted on the basis of NGS in 11% of the sequenced patients or in 6% of all patients.NGS-based diagnostics are feasible in NSCLC and provide clinically relevant information from readily available FFPE tissue. The sample type is associated with the probability of successful testing.

Cyclo-oxygenase (COX) enzymes are the targets for non-steroidal anti-inflammatory drugs (NSAIDs). These drugs demonstrate a variety of inhibitory mechanisms, which include simple competitive, as well as slow binding and irreversible inhibition. In general, most NSAIDs inhibit COX-1 and −2 by similar mechanisms. A unique class of diarylheterocyclic inhibitors has been developed that is highly selective for COX-2 by virtue of distinct inhibitory mechanisms for each isoenzyme. Several of these inhibitors, with varying selectivity, have been utilized to probe the mechanisms of COX inhibition. Results from analysis of both steady-state and time-dependent inhibition were compared. A generalized mechanism for inhibition, consisting of three sequential reversible steps, can account for the various types of kinetic behaviour observed with these inhibitors.

The use of NextGen Sequencing clinically necessitates the need for informatics tools that support the complete workflow from sample accessioning to data analysis and reporting. To address this need we have developed Clinical Genomicist Workstation (CGW). CGW is a secure, n-tiered application where web browser submits requests to application servers that persist the data in a relational database. CGW is used by Washington University Genomic and Pathology Services for clinical genomic testing of many cancers. CGW has been used to accession, analyze and sign out over 409 cases since November, 2011. There are 22 ordering oncologists and 7 clinical genomicists that use the CGW. In summary, CGW a 'soup-to-nuts' solution to track, analyze, interpret, and report clinical genomic diagnostic tests.

Epidemiological and dietary studies suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) reduce the risk of colon cancer, possibly through a mechanism involving inhibition of cyclooxygenase (COX)-2, which is overexpressed in premalignant adenomatous polyps and colon cancer. Because ultraviolet light (UV) can induce COX-2 and nonspecific NSAIDs can decrease UV-induced skin cancer, we evaluated the ability of two compounds, celecoxib (a specific COX-2 inhibitor) and indomethacin (a nonspecific NSAID), to block UV-induced skin tumor development in SKH:HR-1-hrBr hairless mice. Mice fed 150 or 500 ppm celecoxib showed a dose-dependent reduction (60% and 89%, respectively) in tumor yield. Indomethacin (4ppm) reduced tumor yield by 78%. Although both acute and chronic UV exposure increased cell proliferation and edema, neither compound reduced these parameters. In contrast, UV-induced prostaglandin synthesis in the epidermis was effectively blocked by both compounds. UV-induced increases in COX-2 expression in skin were also not altered in any of the treatment groups. Similarly, tumors that constitutively express high levels of COX-2 displayed no reduction by treatment with celecoxib or indomethacin. The dramatic protective effects of celecoxib suggests that specific COX-2 inhibitors may offer a way to safely reduce the risk of skin cancer in humans.

A series of methysulfonyl or sulfonamido substituted 4,5-diaryloxazole were prepared and evaluated for their ability to inhibit the inducible form of cyclooxygenase (COX-2) in vitro and in vivo. Several unique substitution patterns were identified that led to potent and selective inhibitors of COX-2. In general, 2-trifluoromethly-4,5-diaryloxazoles substituted with a methylsulfonyl or sulfonamido group were particularly potent inhibitors. One of the more potent compounds with a selectivity for COX-2 of about 800 fold was 4b (SC-299). SC-299, a highly fluorescent molecule, may be useful for spectroscopic studies on preferential inhibitor binding to COX-2. © 1999 John Wiley & Sons, Inc. Med Res Rev, 19, No. 3, 199–208, 1999.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the formation of prostaglandins by cyclo-oxygenases (COX). The discovery of a second COX isoform (COX-2) associated with inflammation led to agents that selectively inhibit COX-2, e.g. celecoxib. We evaluated the kinetics of inhibition of celecoxib and several NSAIDs. Celecoxib displays classic competitive kinetics on COX-1 (Ki = 10-16 μM). An initial competitive interaction with COX-2 can also be discerned with celecoxib (Ki = 11-15 μM), followed by a time-dependent interaction leading to potent inhibition, characterized as inactivation (Kinact = 0.03-0.5 s-1). Half-maximal inhibition (IC50) using end-point assays reflects the competitive component on COX-1 (IC50 = 4-19 μM) and the inactivation component on COX-2 (IC50 = 0.003-0.006 μM). NSAIDs exhibit four distinct modes of COX inhibition based on kinetic behaviour: (1) competitive, e.g. ibuprofen; (2) weak binding, time-dependent, e.g. naproxen, oxicams; (3) tight binding, time-dependent, e.g. indomethacin; (4) covalent, e.g. aspirin. In addition, most NSAIDs display different kinetic behaviour for each isoform. Weakly binding inhibitors show variable behaviour in enzyme assays, with apparent inhibitory activity being markedly influenced by experimental conditions; determination of kinetic constants with this class is unreliable and IC50 values are strongly dependent on assay conditions. Although IC50 determinations are useful for structure/activity analyses, the complex and distinct mechanisms of enzyme inhibition of each COX isoform by the NSAIDs renders comparison of inhibitory activity on COX-1 and COX-2 using IC50 ratios of questionable validity.

A series of sulfonamide-substituted 4,5-diarylthiazoles was prepared via three synthetic routes as selective COX-2 inhibitors. Recently in the synthesis of selective COX-2 inhibitors we have discovered that the sulfonamide moiety is a suitable replacement for the methylsulfonyl moiety yielding compounds with activity both in vitro and in vivo.

The enzymes cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandin (PG) H2, the precursor of PGs and thromboxane. These lipid mediators play important roles in inflammation and pain and in normal physiological functions. While there are abundant data indicating that the inducible isoform, COX-2, is important in inflammation and pain, the constitutively expressed isoform, COX-1, has also been suggested to play a role in inflammatory processes. To address the latter question pharmacologically, we used a highly selective COX-1 inhibitor, SC-560 (COX-1 IC50= 0.009 μM; COX-2 IC50= 6.3 μM). SC-560 inhibited COX-1-derived platelet thromboxane B2, gastric PGE2, and dermal PGE2production, indicating that it was orally active, but did not inhibit COX-2-derived PGs in the lipopolysaccharide-induced rat air pouch. Therapeutic or prophylactic administration of SC-560 in the rat carrageenan footpad model did not affect acute inflammation or hyperalgesia at doses that markedly inhibitedin vivoCOX-1 activity. By contrast, celecoxib, a selective COX-2 inhibitor, was anti-inflammatory and analgesic in this model. Paradoxically, both SC-560 and celecoxib reduced paw PGs to equivalent levels. Increased levels of PGs were found in the cerebrospinal fluid after carrageenan injection and were markedly reduced by celecoxib, but were not affected by SC-560. These results suggest that, in addition to the role of peripherally produced PGs, there is a critical, centrally mediated neurological component to inflammatory pain that is mediated at least in part by COX-2.

A series of 3,4-diarylpyrazoles was synthesized and evaluated for their ability to selectively inhibit cyclooxygenase-2 (COX-2). A number of potent and selective inhibitors were identified and found to have oral anti-inflammatory activity in a rat carrageenan-induced foot pad edema assay.

Series of 1,2-diarylimidazoles has been synthesized and found to contain highly potent and selective inhibitors of the human COX-2 enzyme. The paper describes a short synthesis of the target 1,2-diarylimidazoles starting with aryl nitriles. Different portions of the diarylimidazole (I) were modified to establish SAR. Systematic variations of the substituents in the aryl ring B have yielded very potent (IC50 = 10-100 nm) and selective (1000-12500) inhibitors of the COX-2 enzyme. The study on the influence of substituents in the imidazole ring established that a CF3 group at position 4 gives the optimum oral activity. A number of the diarylimidazoles showed excellent inhibition in the adjuvant induced arthritis model (e.g., ED50 = 0.02 mpk for 22 and 34). The diarylimidazoles are also potent inhibitors of carrageenan-induced edema (ED50 = 9-30 mpk) and hyperalgesia (ED50 = 11-40 mpk). Several orally active diarylimidazoles show no GI toxicity in the rat and mouse up to 200 mpk.

Series of 1,2-diarylpyrroles has been synthesized and found to contain very potent and selective inhibitors of the human cyclooxygenase-2 (COX-2) enzyme. The paper describes short and practical syntheses of the target molecules utilizing the Paal-Knorr reaction. Electrophilic substitution on 1 proceeds in a regioselective fashion, and the method was used to generate a number of tetrasubstituted pyrroles. Detailed SAR on the series has been studied by modifications of the aryl rings and the substituents in the pyrrole ring. Diarylpyrrole 1 is a very potent (COX-2, IC50 = 60 nm) and selective (COX-1/COX-2 =1700) inhibitor whereas the isomeric 2 is completely inactive against COX-2. Modifications of the substituents on the fluorophenyl ring in 1 yields very potent inhibitors of COX-2 (IC50 = 40-80 nm) with excellent selectivity (1200 to2500) vs COX-1. Analog 20 containing a sulfonamide group is an excellent inhibitor of COX-2 with an IC50 of 14 nm. Tetrasubstituted pyrroles containing groups such as COCF3, SO2CF3, or CH2OAr at position 3 in the pyrrole ring give excellent inhibitors (COX-2, IC50 = 30-120 nm). In vivo testing in the carrageenan-induced paw edema model in the rat establishes that the 1,2-diarylpyrroles are orally active antiinflammatory agents. Compound 3 is the most potent inhibitor of edema with an ED50 of 4.7 mpk.

Prostaglandins (PGs) are important mediators of epithelial integrity and function in the gastrointestinal tract. Relatively little is known, however, about the mechanism by which PGs affect stem cells in the intestine during normal epithelial turnover, or during wound repair. PGs are synthesized from arachidonate by either of two cyclooxygenases, cyclooxygenase-1 (Cox-1) or cyclooxygenase-2 (Cox-2), which are present in a wide variety of mamalian cells. Cox-1 is thought to be a constitutively expressed enzyme, and the expression of Cox-2 is inducible by cytokines or other stimuli in a variety of cell types. We investigated the role of PGs in mouse intestinal stem cell survival and proliferation following radiation injury. The number of surviving crypt stem cells was determined 3.5 d after irradiation by the microcolony assay. Radiation injury induced a dose-dependent decrease in the number of surviving crypts. Indomethacin, an inhibitor of Cox-1 and Cox-2, further reduced the number of surviving crypts in irradiated mice. The indomethacin dose response for inhibition of PGE2 production and reduction of crypt survival were similar. DimethylPGE2 reversed the indomethacin-induced decrease in crypt survival. Selective Cox-2 inhibitors had no effect on crypt survival. PGE2, Cox-1 mRNA, and Cox-1 protein levels all increase in the 3 d after irradiation. Immunohistochemistry for Cox-1 demonstrated localization in epithelial cells of the crypt in the unirradiated mouse, and in the regenerating crypt epithelium in the irradiated mouse. We conclude that radiation injury results in increased Cox-1 levels in crypt stem cells and their progeny, and that PGE2 produced through Cox-1 promotes crypt stem cell survival and proliferation.

The role of prostaglandin E2 (PGE2) in the development of inflammatory symptoms and cytokine production was evaluated in vivo using a neutralizing anti-PGE2 monoclonal antibody 2B5. In carrageenan-induced paw inflammation, pretreatment of rats with 2B5 substantially prevented the development of tissue edema and hyperalgesia in affected paws. The antibody was shown to bind the majority of PGE2 produced at the inflammatory site. In adjuvant-induced arthritis, the therapeutic administration of 2B5 to arthritic rats substantially reversed edema in affected paws. Anti-PGE2 treatment also reduced paw levels of IL-6 RNA and serum IL-6 protein without modifying tumor necrosis factor RNA levels in the same tissue. In each model, the antiinflammatory efficacy of 2B5 was indistinguishable from that of the nonsteroidal antiinflammatory drug indomethacin, which blocked the production of all PGs. These results indicate that PGE2 plays a major role in tissue edema, hyperalgesia, and IL-6 production at sites of inflammation, and they suggest that selective pharmacologic modulation of PGE2 synthesis or activity may provide a useful means of mitigating the symptoms of inflammatory disease.

The NSAIDs are potent anti-inflammatory and analgesic agents. It is now believed that the NSAIDs exert their therapeutic activity through the inhibition of COX-2 at the site of inflammation. Unfortunately, these compounds are equally capable of inhibiting constitutively expressed COX-1 in tissues such as the gastrointestinal tract and kidney, which results in serious, mechanism-based toxicities that limit the drug's therapeutic utility. With the identification of selective COX-2 inhibitors, alternatives to traditional NSAID therapy should be available that will provide clinical usefulness with reduced toxicity.

A novel series of 5,6-diarylspiro[2.4]hept-5-enes was shown to provide highly potent and selective cyclooxygenase-2 (COX-2) inhibitors. A study of structure-activity relationships in this series suggests that 3,4-disubstituted phenyl analogs are generally more selective than 4-substituted phenyl analogs and that replacement of the methyl sulfone group on the 6-phenyl ring with a sulfonamide moiety results in compounds with superior in vivo pharmacological properties, although with lower COX-2 selectivity. Several compounds have been shown to possess promising pharmacological properties in adjuvant-induced arthritis and edema analgesia models. The absence of gastrointestinal (GI) toxicity at 200 mpk of several selected compounds in rats and mice corresponds well with the weak potency for inhibition of COX-1 observed in the enzyme assay. Methyl sulfone 55 and sulfonamide 24 were shown to have superior in vivo pharmacological profiles, low GI toxicity, and good oral bioavailability and duration of action.

A novel series of terphenyl methyl sulfones and sulfonamides have been shown to be highly potent and selective cyclooxygenase-2 (COX-2) inhibitors. The sulfonamide analogs 17 and 21 were found to be much more potent COX-2 inhibitors and orally active anti-inflammatory agents than the corresponding methyl sulfone analogs 16 and 20, respectively, albeit with some decrease in COX-2 selectivity. Structure-activity relationship studies have determined that incorporation of two fluorine atoms in the central phenyl group, as in 20 and 21, is extremely advantageous for both in vitro COX-2 potency and selectivity as well as in vivo activity. Several noticeable examples in the 1,2-diaryl-4,5-difluorobenzenesulfonamide series are 21a-c,k,l,n (COX-2, IC50 = 0.002-0.004 microM), in which all have in vitro COX-1/COX-2 selectivity1000. In addition, sulfonamides 21a,b,d,g,j,m,n,q were shown to have greatly enhanced oral activity with more than 90% inhibition of prostaglandin E2 production in the air pouch model of inflammation. Furthermore, sulfonamide 21b was found to be very active in the rat adjuvant-induced arthritis model (ED50 = 0.05 mg/kg) and carrageenan-induced hyperalgesia assay (ED50 = 38.7 mg/kg) with no indication of gastrointestinal toxicity in rats at doses as high as 200 mg/kg.

Prostaglandins (PGs) can be synthetized via two isoforms of cyclooxygenase (COX). COX-1 is constitutively expressed in normal tissues, and its activity represent the normal physiological output of PGs. In inflammatory states, the newly discovered COX-2 is rapidly induced, and its activity accounts for the large amounts of PGs seen in inflammation. The commercially available nonsteroidal anti-inflammatory drugs (NSAIDs) are nonselective inhibitors of both COX isoforms; therefore, they provide anti-inflammatory activity as well as side effects associated with COX-1 inhibition. Selective inhibition of COX-2 expression explains at least in part the potent anti-inflammatory activity of steroids. Anti-inflammatory activity of newly developed COX-2 inhibitors, such as NS-398 or SC-58125, suggest a new approach of inflammatory diseases with more efficacious NSAIDs essentially devoid of side effects such as stomach ulcers.

Novel 4,5-diarylspiro[2.4]hept-5-enes, 6,7-diarylspiro[3.4]oct-6-enes, and 2,3-diarylspiro[4.4]non-2-enes have been synthesized and shown to be very potent inducible cyclooxygenase (COX-2) inhibitors with inhibition (IC 50 ) in the low nanomolar range and enzyme selectivity ratios as high as four orders of magnitude. The methyl sulfone spiro[2.4]hept-5-ene 1 (SC-58451) was found to be orally active (ED 50 = 0.3 mpk) in the rat adjuvant-induced arthritis model with no gastric lesions observed at 200 mpk.

The enzyme cyclo-oxygenase catalyses the oxygenation of arachidonic acid, leading to the formation of prostaglandins. Recently two forms of cyclo-oxygenase have been described: a constitutive (COX-1) enzyme present in most cells and tissues, and an inducible (COX-2) isoenzyme observed in many cells in response to pro-inflammatory cytokines. Constitutive and inducible forms of human cyclo-oxygenase (hCOX-1 and hCOX-2) were cloned and expressed in insect cells, utilizing a baculovirus expression system. hCOX-1 had a specific activity of 18.8 mumol of O2/mg with a Km of 13.8 microM for arachidonate and Vmax. of 1500 nmol of O2/nmol of enzyme, whereas hCOX-2 had a specific activity of 12.2 mumol of O2/mg with a Km of 8.7 microM for arachidonate and a Vmax. of 1090 nmol of O2/nmol of enzyme. Indomethacin inhibited both hCOX-1 and hCOX-2, whereas NS-398 and Dup-697 selectively inhibited hCOX-2. Both NS-398 and Dup-697 exhibited time-dependent inactivation of hCOX-2, as did indomethacin on both enzymes. The competitive inhibitor of hCOX-1, mefenamic acid, also displayed competitive inhibition of hCOX-2. These results demonstrate the ability to generate selective non-steroidal anti-inflammatory drugs (NSAIDs), which could provide useful improvement therapeutically in the treatment of chronic inflammatory disease.

The interaction between nitric oxide (NO) and cyclooxygenase (COX) was studied in a rabbit model of renal inflammation, the ureteral obstructed hydronephrotic kidney (HNK). Ex vivo perfusion of the HNK but not the control kidney (e.g., unobstructed contralateral kidney, CLK), led to a time-dependent release of nitrite (NO2-), a breakdown product of NO. Stimulation of the HNK with bradykinin (BK) evoked a time-dependent increase in prostaglandin E2 (PGE2) production. NG-monomethyl-L-arginine (L-NMMA), which blocks the activity of both constitutive and inducible nitric oxide synthase (cNOS and iNOS), aminoguanidine, a recently described selective iNOS inhibitor, dexamethasone, or cycloheximide abolished the release of NO2- and attenuated the exaggerated BK-induced PGE2 production. This supports the existence of iNOS and COX-2 in the HNK. In the CLK, BK elicited release of both NO2- and PGE2 but this did not augment with time. L-NMMA but not aminoguanidine, dexamethasone, or cycloheximide attenuated NO2- and PGE2 release indicative of the presence of constitutive but not inducible NOS or COX. The current study suggests that the endogenous release of NO from cNOS in the CLK activates a constitutive COX resulting in optimal PGE2 release by BK. In addition, in the HNK, NO release from iNOS activates the induced COX resulting in markedly increased release of proinflammatory prostaglandin. The broader implication of this study is that the cyclooxygenase isozymes are potential receptor targets for nitric oxide.

We have examined the role of cyclooxygenase 2 (COX-2) in a model of inflammation in vivo. Carrageenan administration to the subcutaneous rat air pouch induces a rapid inflammatory response characterized by high levels of prostaglandins (PGs) and leukotrienes in the fluid exudate. The time course of the induction of COX-2 mRNA and protein coincided with the production of PGs in the pouch tissue and cellular infiltrate. Carrageenan-induced COX-2 immunoreactivity was localized to macrophages obtained from the fluid exudate as well as to the inner surface layer of cells within the pouch lining. Dexamethasone inhibited both COX-2 expression and PG synthesis in the fluid exudate but failed to inhibit PG synthesis in the stomach. Furthermore, NS-398, a selective COX-2 inhibitor, and indomethacin, a nonselective COX-1/COX-2 inhibitor, blocked proinflammatory PG synthesis in the air pouch. In contrast, only indomethacin blocked gastric PG and, additionally, produced gastric lesions. These results suggest that inhibitors of COX-2 are potent antiinflammatory agents which do not produce the typical side effects (e.g., gastric ulcers) associated with the nonselective, COX-1-directed antiinflammatory drugs.

Novel 4,5-diarylspiro[2.4]hept-5-enes, 6,7-diarylspiro[3.4]oct-6-enes, and 2,3-diarylspiro[4.4]non-2-enes have been synthesized and shown to be very potent inducible cyclooxygenase (COX-2) inhibitors with inhibition (IC 50 ) in the low nanomolar range and enzyme selectivity ratios as high as four orders of magnitude. The methyl sulfone spiro[2.4]hept-5-ene 1 (SC-58451) was found to be orally active (ED 50 = 0.3 mpk) in the rat adjuvant-induced arthritis model with no gastric lesions observed at 200 mpk.

A novel series of terphenyl methyl sulfones and sulfonamides have been shown to be highly potent and selective cyclooxygenase-2 (COX-2) inhibitors. The sulfonamide analogs 17 and 21 were found to be much more potent COX-2 inhibitors and orally active anti-inflammatory agents than the corresponding methyl sulfone analogs 16 and 20, respectively, albeit with some decrease in COX-2 selectivity. Structure−activity relationship studies have determined that incorporation of two fluorine atoms in the central phenyl group, as in 20 and 21, is extremely advantageous for both in vitro COX-2 potency and selectivity as well as in vivo activity. Several noticeable examples in the 1,2-diaryl-4,5-difluorobenzenesulfonamide series are 21a−c,k,l,n (COX-2, IC50 = 0.002−0.004 μM), in which all have in vitro COX-1/COX-2 selectivity >1000. In addition, sulfonamides 21a,b,d,g,j,m,n,q were shown to have greatly enhanced oral activity with more than 90% inhibition of prostaglandin E2 production in the air pouch model of inflamma...

A novel series of 5,6-diarylspiro[2.4]hept-5-enes was shown to provide highly potent and selective cyclooxygenase-2 (COX-2) inhibitors. A study of structure-activity relationships in this series suggests that 3,4-disubstituted phenyl analogs are generally more selective than 4-substituted phenyl analogs and that replacement of the methyl sulfone group on the 6-phenyl ring with a sulfonamide moiety results in compounds with superior in vivo pharmacological properties, although with lower COX-2 selectivity. Several compounds have been shown to possess promising pharmacological properties in adjuvant-induced arthritis and edema analgesia models. The absence of gastrointestinal (GI) toxicity at 200 mpk of several selected compounds in rats and mice corresponds well with the weak potency for inhibition of COX-1 observed in the enzyme assay. Methyl sulfone 55 and sulfonamide 24 were shown to have superior in vivo pharmacological profiles, low GI toxicity, and good oral bioavailability and duration of action.

Series of 1,2-diarylpyrroles has been synthesized and found to contain very potent and selective inhibitors of the human cyclooxygenase-2 (COX-2) enzyme. The paper describes short and practical syntheses of the target molecules utilizing the Paal−Knorr reaction. Electrophilic substitution on 1 proceeds in a regioselective fashion, and the method was used to generate a number of tetrasubstituted pyrroles. Detailed SAR on the series has been studied by modifications of the aryl rings and the substituents in the pyrrole ring. Diarylpyrrole 1 is a very potent (COX-2, IC50 = 60 nm) and selective (COX-1/COX-2 = >1700) inhibitor whereas the isomeric 2 is completely inactive against COX-2. Modifications of the substituents on the fluorophenyl ring in 1 yields very potent inhibitors of COX-2 (IC50 = 40−80 nm) with excellent selectivity (1200 to >2500) vs COX-1. Analog 20 containing a sulfonamide group is an excellent inhibitor of COX-2 with an IC50 of 14 nm. Tetrasubstituted pyrroles containing groups such as COCF...

Series of 1,2-diarylimidazoles has been synthesized and found to contain highly potent and selective inhibitors of the human COX-2 enzyme. The paper describes a short synthesis of the target 1,2-diarylimidazoles starting with aryl nitriles. Different portions of the diarylimidazole (I) were modified to establish SAR. Systematic variations of the substituents in the aryl ring B have yielded very potent (IC50 = 10−100 nm) and selective (1000−12500) inhibitors of the COX-2 enzyme. The study on the influence of substituents in the imidazole ring established that a CF3 group at position 4 gives the optimum oral activity. A number of the diarylimidazoles showed excellent inhibition in the adjuvant induced arthritis model (e.g., ED50 = 0.02 mpk for 22 and 34). The diarylimidazoles are also potent inhibitors of carrageenan-induced edema (ED50 = 9−30 mpk) and hyperalgesia (ED50 = 11−40 mpk). Several orally active diarylimidazoles show no GI toxicity in the rat and mouse up to 200 mpk.

A series of sulfonamide-substituted 4,5-diarylthiazoles was prepared via three synthetic routes as selective COX-2 inhibitors. Recently in the synthesis of selective COX-2 inhibitors we have discovered that the sulfonamide moiety is a suitable replacement for the methylsulfonyl moiety yielding compounds with activity both in vitro and in vivo.

A series of novel sulfone substituted 4,5-diarylthiazoles have been synthesized and evaluated for their inhibition of the two isoforms of human cyclooxygenase (COX-1 and COX-2). This series displays exceptionally selective COX-2 inhibition.

Evidence from tissue culture studies suggests that glial cells are the principal source of prostaglandins in the brain. We have used immunohistochemistry, Western blot analysis, and enzyme activity assays to localize cyclooxygenase (COX), the enzyme responsible for the conversion of arachidonic acid to prostaglandins, in situ in the normal ovine brain. We observed very few immunoreactive glial cells. In contrast, an extensive distribution of COX-like immunoreactive (ir) neuronal cell bodies and dendrites and a corresponding pattern of COX enzyme activity were observed. COXir neurons were most abundant in forebrain sites involved in complex, integrative functions and autonomic regulation such as the cerebral cortex, hippocampus, amygdala, bed nucleus of the stria terminalis, substantia innominata, dorsomedial nucleus of the hypothalamus, and tuberomammillary nucleus. Moderate populations were observed in other regions of the central nervous system implicated in sensory afferent processing, including the dorsal column nuclei, spinal trigeminal nucleus, and superior colliculus, and in structures involved in autonomic regulation, such as the nucleus of the solitary tract, parabrachial nucleus, and the periaqueductal gray matter. We did not observe COXir axons or terminal fields, however. Our results suggest that neurons may use prostaglandins as intracellular or perhaps paracrine, but probably not synaptic, mediators in the normal brain.

The effect of endogenous glucocorticoids on the expression of the cyclooxygenase enzyme was studied by contrasting cyclooxygenase expression and prostanoid synthesis in adrenalectomized and sham-adrenalectomized mice with or without the concurrent administration of endotoxin. Peritoneal macrophages obtained from adrenalectomized mice showed a 2- to 3-fold induction in cyclooxygenase synthesis and activity when compared to sham controls. Intravenous injection of a sublethal dose of endotoxin (5 micrograms/kg) further stimulated cyclooxygenase synthesis, resulting in a 4-fold increase in prostaglandin production. Similar cyclooxygenase induction can be achieved in macrophages obtained from normal mice but only after high doses of endotoxin (2.5 mg/kg) that are 100% lethal to adrenalectomized mice. Restoration of glucocorticoids in adrenalectomized animals with dexamethasone completely inhibited the elevated cyclooxygenase and protected these animals from endotoxin-induced death. In contrast, no signs of cyclooxygenase induction were observed in the kidneys of the adrenalectomized mice, even when treated with endotoxin. Dexamethasone did not affect the constitutive cyclooxygenase activity and prostaglandin production present in normal and adrenalectomized kidneys. These data indicate the existence of a constitutive cyclooxygenase that is normally present in most cells and tissues and is unaffected by steroids and of an inducible cyclooxygenase that is expressed only in the context of inflammation by proinflammatory cells, like macrophages, and that is under glucocorticoid regulation. Under normal physiological conditions glucocorticoids maintain tonic inhibition of inducible cyclooxygenase expression. Depletion of glucocorticoids or the presence of an inflammatory stimulus such as endotoxin causes rapid induction of this enzyme, resulting in an exacerbated inflammatory response that is often lethal.

Atriopeptin (AP) is expressed in several tissues with each tissue capable of specific differences in processing of the prohormone (pro-AP) to mature low molecular forms of the peptide. Since pro-AP has low biological activity, processing into mature AP is a critical activation event. This observation prompted us to study whether granule storage or regulated secretion of AP is essential for cleavage of mature peptide. We examined the processing of AP in adrenal medulla derived cells, using the rat pheochromocytoma cell line (PC12 cell) stably transfected with a genomic human AP DNA in the presence and absence of nerve growth factor (NGF), and also examined the mechanism of AP secretion and compared the results with those obtained using transfected chinese hamster ovary cells (CHO cells). The amount of prohormone was 5–10 fold higher than that of low molecular form of AP in the transfected PC12 cells. This ratio was essentially unchanged in differentiated PC12 cells after NGF treatment of the cells. Potassium depolarization of the transfected PC12 cells caused a 5-fold increase in AP release into the medium primarily as the intact prohormone. On the other hand, transfected CHO cells only exhibited constitutive AP release which is non-response to depolarization. These results suggest that the AP prohormone is sorted into secretory granules as the prohormone in PC12 cells and undergoes regulated release in response to depolarization indicating granule storage or release is not the critical determinant of AP prohormone cleavage.

The formation of new blood vessels by angiogenesis to provide adequate blood supply is a key requirement for the growth of many tumors. While normal blood vessels expressed the COX-1 enzyme, new angiogenic endothelial cells expressed the inducible COX-2. We evaluated the role of COX inhibitors in the mouse corneal micropocket assay in which angiogenesis is driven by the addition of a Hydron pellet containing basic fibroblast growth factor (bFGF). Neovascular areas were measured with a slit lamp five days after pellet implantation into the corneal stroma. All animals containing implants with bFGF (90 ng) developed intensive areas of neovascularization, whereas the controls implanted with the Hydron pellet alone did not. Indomethacin (a nonselective COX-1/COX-2 inhibitor) and SC-236 (a COX-2-selective inhibitor) inhibited angiogenesis in a dose-dependent manner. Importantly, the indomethacin-treated mice developed severe gastrointestinal toxicity at the efficacious dose of 3 mg/kg/day. By contrast, gastrointestinal lesions were not observed, and platelet COX-1 activity was unaffected, at anti-angiogenic doses of SC-236 (1-6 mg/kg/day). Furthermore, a COX-1-selective inhibitor, SC-560, was ineffective at doses up to 10 mg/kg, a dose that completely blocked platelet COX-1 activity in these mice. SC-236 was also effective in reducing angiogenesis driven by bFGF, vascular endothelium growth factor (VEGF), or carrageenan in the matrigel rat model. Finally, in several tumor models, SC-236 consistently and effectively inhibited tumor growth and angiogenesis. This novel antiangiogenic activity of COX-2 inhibitors indicates their potential therapeutic utility in several types of cancer.

We have studied the effect of glucocorticoids administered in vivo on the activity and synthesis of the cyclooxygenase enzyme (COX) in mice treated with or without concurrent intravenous administration of LPS. Mouse peritoneal macrophages from LPS-treated animals showed a two to three fold increase in COX activity determined by the production of PGE2 and PGI2 after stimulation of the cells with exogenous arachidonate. Dexamethasone injected simultaneously with LPS, 12 h before killing of the animal and removal of the macrophages, completely blocked the LPS-induced increase COX activity in peritoneal macrophages. The regulation observed in COX activity by LPS and dexamethasone are due primarily to changes in COX mass as determined by immunoprecipitation of [35S]methionine endogenously labeled enzyme. In contrast, the COX present in the nonadherent cells and in renal medullary microsomes obtained from the same animals, showed no significant changes between treatments. These results indicate that LPS in vivo stimulates COX synthesis in the peritoneal macrophages but not in the kidney. The effect of dexamethasone to inhibit COX synthesis is selective to the LPS-induced enzyme.

A new class of selective cyclooxygenase-2 (COX-2) inhibitors has been identified by high throughput screening. Structurally distinct from previously described selective COX-2 inhibitors, these benzopyrans contain a carboxylic acid function and CF3 functionality. The compound SC-75,416 is a representative of this class. A range if in vitro and in vivo tests were employed to characterize its potency and selectivity. Using human recombinant enzymes, this compound displays a concentration that provides 50% inhibition (IC50) of 0.25 microM for COX-2 and 49.6 microM for COX-1. A mutation of the side pocket residues in COX-2 to COX-1 had little effect on potency suggesting that these inhibitors bind in a unique manner in COX-2 distinct from COX-2 inhibiting diaryl heterocycles. Using rheumatoid arthritic synovial cells stimulated with interleukin-1beta (IL-1beta) and washed platelets the compound displayed IC50 of 3 nM and 400 nM respectively. Potency and selectivity was maintained but predictably right shifted in whole blood with IC50 of 1.4 microM for lipopolysaccharide (LPS) stimulated induction of COX-2 and >200 microM for inhibition of platelet thromboxane production. SC-75,416 is 89% bioavailable and its in vivo half life is sufficient for once a day dosing. In the rat air pouch model of inflammation, the compound inhibited PGE2 production with an effective dose that provides 50% inhibition (ED50) of 0.4 mg/kg, while sparing gastric prostaglandin E2 (PGE2) production with an ED50 of 26.5 mg/kg. In a model of acute inflammation and pain caused by carrageenan injection into the rat paw, the compound reduced edema and hyperalgesia with ED50s of 2.7 and 4 mg/kg respectively. In a chronic model of arthritis the compound demonstrated an ED50 of 0.081 mg/kg and an ED(80) of 0.38 mg/kg. In a model of neuropathic pain, SC-75,416 had good efficacy. This compound's unique chemical structure and effect on COX enzyme binding and activity as well as its potency and selectivity may prove useful in treating pain and inflammation.

Many of aspirin's therapeutic effects arise from its acetylation of cyclooxygenase-2 (COX-2), whereas its antithrombotic and ulcerogenic effects result from its acetylation of COX-1. Here, aspirin-like molecules were designed that preferentially acetylate and irreversibly inactivate COX-2. The most potent of these compounds was o -(acetoxyphenyl)hept-2-ynyl sulfide (APHS). Relative to aspirin, APHS was 60 times as reactive against COX-2 and 100 times as selective for its inhibition; it also inhibited COX-2 in cultured macrophages and colon cancer cells and in the rat air pouch in vivo. Such compounds may lead to the development of aspirin-like drugs for the treatment or prevention of immunological and proliferative diseases without gastrointestinal or hematologic side effects.

The discovery of a second isoform of cyclooxygenase (COX) led to the search for compounds that could selectively inhibit COX-2 in humans while sparing prostaglandin formation from COX-1. Celecoxib and rofecoxib were among the molecules developed from these efforts. We report here the pharmacological properties of a third selective COX-2 inhibitor, valdecoxib, which is the most potent and in vitro selective of the marketed COX-2 inhibitors that we have studied. Recombinant human COX-1 and COX-2 were used to screen for new highly potent and in vitro selective COX-2 inhibitors and compare kinetic mechanisms of binding and enzyme inhibition with other COX inhibitors. Valdecoxib potently inhibits recombinant COX-2, with an IC(50) of 0.005 microM; this compares with IC values of 0.05 microM for celecoxib, 0.5 microM for rofecoxib, and 5 microM for etoricoxib. Unique binding interactions of valdecoxib with COX-2 translate into a fast rate of inactivation of COX-2 (110,000 M/s compared with 7000 M/s for rofecoxib and 80 M/s for etoricoxib). The overall saturation binding affinity for COX-2 of valdecoxib is 2.6 nM (compared with 1.6 nM for celecoxib, 51 nM for rofecoxib, and 260 nM for etoricoxib), with a slow off-rate (t(1/2) approximately 98 min). Valdecoxib inhibits COX-1 in a competitive fashion only at very high concentrations (IC(50) = 150 microM). Collectively, these data provide a mechanistic basis for the potency and in vitro selectivity of valdecoxib for COX-2. Valdecoxib showed similar activity in the human whole-blood COX assay (COX-2 IC(50) = 0.24 microM; COX-1 IC(50) = 21.9 microM). We also determined whether this in vitro potency and selectivity translated to significant potency in vivo. In rats, valdecoxib demonstrated marked potency in acute and chronic models of inflammation (air pouch ED(50) = 0.06 mg/kg; paw edema ED(50) = 5.9 mg/kg; adjuvant arthritis ED(50) = 0.03 mg/kg). In these same animals, COX-1 was spared at doses greater than 200 mg/kg. These data provide a basis for the observed potent anti-inflammatory activity of valdecoxib in humans.

To understand the potential role of cyclooxygenase (COX) in normal and inflammatory human diseases, we characterized the expression of COX-1 and COX-2 in biopsies of osteoarthritis, atherosclerosis, and cancer. Tissues were prepared for immunohistochemistry by standard methods, and representative cases assayed via Western blot and quantitative RT-PCR. COX-2 was not detected in normal human tissues with few exceptions. Moderate to marked COX-2 was observed in the macula densa (MD) and thick ascending limb (TAL) in human fetal kidneys, but was not detected in neonatal and adult MD and TALs. Low level, constitutive COX-2 was detected in colonic epithelium, peribronchial glands, and pancreatic ductal epithelium. Low to moderate COX-2 was detected basally in the cortex, hippocampus, hypothalamus, and spinal cord, and in reproductive tissues during ovulation, implantation and labor. No COX-2 was detected in the existing vasculature in normal tissues, and was also not expressed throughout the ductus arteriosus. COX-2 was markedly induced in human tissues of osteoarthritis, atherosclerosis and cancer. COX-2 was prominently expressed in the synovium, fibrocartilage of osteophytes, and in the blood vessels in the osteoarthritic (OA) knee joint. COX-2 was also prominently detected in the macrophages/foam cells in atherosclerotic plaques, and in the endothelium overlying and immediately adjacent to the fibrofatty lesion. Moderate- to intense COX-2 expression was consistently observed in the inflammatory cells, neoplastic lesions, and blood vessels in all epithelial-derived human cancers studied. In contrast, COX-1 was relatively ubiquitously observed in both normal and pathophysiological conditions. These data collectively imply COX-2 plays an important role in mediating a variety of inflammatory diseases, and imply COX-2 inhibitors may be effective in the prevention and/or treatment of OA, heart disease, and epithelial cancers.

Two compounds (celecoxib and valdecoxib) from the diarylheterocycle class of cyclooxygenase inhibitors were radiolabeled and used to characterize their binding to cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), several single-point variants of COX-2 (Val523Ile, Tyr355Ala, Arg120Ala, Arg120Gln, Arg120Asn) and one triple-point variant of COX-2 [Val523Ile, Arg513His, Val434Ile (IHI)]. We demonstrate highly specific and saturable binding of these inhibitors to COX-2. Under the same assay conditions, little or no specific binding to COX-1 could be detected. The affinity of [(3)H]celecoxib for COX-2 (K(D) = 2.3 nM) was similar to the affinity of [(3)H]valdecoxib (K(D) = 3.2 nM). The binding to COX-2 seems to be both rapid and slowly reversible with association rates of 5.8 x 10(6)/M/min and 4.5 x 10(6)/M/min and dissociation rates of 14 x 10(-3)/min (t(1/2) = 50 min) and 7.0 x 10(-3)/min (t(1/2) = 98 min) for [(3)H]celecoxib and [(3)H]valdecoxib, respectively. These association rates increased (4- to 11-fold) when the charged arginine residue located at the entrance to the main hydrophobic channel was mutated to smaller uncharged amino acids (Arg120Ala, Arg120Gln, and Arg120Asn). Mutation of residues located within the active site of COX-2 that define a 'side pocket' (Tyr355Ala, Val523Ile, IHI) of the main channel had a greater effect on the dissociation rate than the association rate. These mutations, which modified the shape of and access to the 'side pocket', affected the binding affinity of [(3)H]valdecoxib more than that of [(3)H]celecoxib. These binding studies provide direct insight into the properties and binding constants of celecoxib and valdecoxib to COX-2.

Cyclooxygenase (COX) performs the critical initial reaction in the arachidonic metabolic cascade, leading to formation of proinflammatory prostaglandins, thromboxanes, and prostacyclins. The discovery of a second COX isoform (COX-2) associated with inflammation led to agents that selectively inhibit COX-2. Cyclooxygenase-2 inhibitors are also being developed for canine applications. To assess the compound potency on canine enzymes, canine COX-1 and COX-2 were cloned, expressed, and purified. Cyclooxygenase-1 was cloned from a canine kidney complementary DNA (cDNA) library, with 96 % sequence homology to human COX-1. Cyclooxygenase-2 was cloned from canine kidney and lipopolysaccharide-stimulated macrophage cDNA libraries, with a 93 % sequence homology to human COX-2. The arachidonic acid Michaelis constants for canine COX-1 and COX-2 were 4.8 and 6.6 micrometer, respectively, compared with 9.6 and 10.2 micrometer for ovine. Inhibition results indicated that, for all compounds tested, there was no significant difference between potencies determined for canine enzymes and those for human enzymes.